Analysis of chromosome-sized DNA and genome typing of isolated strains ofTaylorella equigenitalis

Analysis of chromosome-sized DNA and genome typing ofTaylorella equigenitalis NCTC11184, Kentucky 188, and five strains ofT. equigenitalis isolated in Japan were carried out. The three restriction enzymes used,ApaI,NaeI andNotI, cleaved the genomic DNAs of five Japanese strains ofT. equigenitalis into relatively limited numbers of restriction fragments, which were well resolved on crossed-field gel electrophoresis (CFGE). The respective profiles after CFGE of the restriction fragments from all five strains were essentially identical to each other after digestion byApaI,NaeI orNotI. Hence it appears that these strains have a common genome type with respect to these three restriction enzymes. It was also shown that the respective profiles from these strains were essentially different from those ofT. equigenitalis NCTC11184 and those of Kentucky 188 after digestion withApaI,NaeI orNotI.Abbreviations CFGE crossed-field gel electrophoresis - FIGE field inversion gel electrophoresis - PFGE pulsed-field gel electrophoresis     

Genotyping of isolates ofTaylorella equigenitalis from thoroughbred brood mares in Japan

Profiles of the genomic DNA of 104 strains ofT. equigenitalis isolated from brood mares with contagious equine metritis in Hokkaido during the breeding seasons from 1980 to 1993, as well as those of five strains (SS28, EQ56, EQ59, EQ70 and HH139) previously isolated in Japan were examined after restriction digestion and crossed-field gel electrophoresis. These profiles were essentially identical to each other and the various isolates and strains appeared to have a common genotype, designated genotype J, with respect to two restriction enzymes,ApaI andNotI. These results suggest a common source for all these isolates obtained over the course of more than 10 years in Japan.Abbreviations CEM contagious equine metritis - CFGE crossed-field gel electrophoresis - PFGE pulsed-field gel electrophoresis     

Demonstration of Heterogeneous Genotypes of Taylorella equigenitalis Isolated from Horses in Six European Countries by Pulsed-Field Gel Electrophoresis

Forty-six isolates of Taylorella equigenitalis were analysed by pulsed-field gel electrophoresis (PFGE) after separate digestion of the genomic DNA with ApaI and with NotI. The isolates had been obtained from horses in six European countries and were classified into 18 genotypes. In Belgium, 2 genotypes were detected in 2 isolates, in England 9 among 15, in Finland 2 in 2, in France 2 among 10, in Sweden 3 among 5, and in Switzerland 3 among 12. Two English isolates and 4 French isolates gave identical PFGE profiles to those of Kentucky 188 from the United States. A common genotype was found in 5 isolates from Belgium and England and also in 10 isolates from France and Switzerland. The analysis of genomic DNA from 12 isolates of T. equigenitalis obtained from male horses in France, Sweden and Switzerland gave no evidence of a sex-related difference in the genomic DNA. Genomic DNA from 11 streptomycin (STM)-susceptible isolates obtained in Sweden and Switzerland were classified into four genotypes by PFGE. Each of the six genotypes determined among the 17 isolates from these two countries had single phenotypes for resistance or susceptibility to STM.     

Identification of Taylorella equigenitalis responsible for contagious equine metritis in equine genital swabs by direct polymerase chain reaction

A direct-PCR assay was developed for the rapid detection of Taylorella equigenitalis, a Gram-negative bacterium responsible for contagious equine metritis (CEM) in Equidae. The bacteria may be detected in equine genital swabs without need for a preliminary step of DNA extraction or bacterial isolation. Specificity was determined with 125 isolates of T. equigenitalis, 24 isolates of Taylorella asinigenitalis, five commensal bacteria of the genital tract and a facultative intracellular pathogen of foals found in large concentration in soil. Our PCR is specific and amplified a 413-bp 16S ribosomal DNA product only in all T. equigenitalis.     

Comparison of the Value of Pulsed-field Gel Electrophoresis,Random Amplified Polymorphic DNA and Amplified rDNA Restriction Analysis for Subtyping Taylorella equigenitalis

Eight strains of Taylorella equigenitalis were identified by a polymerase chain reaction using a primer pair specific to the 16S rDNA of T equigenitalis. These eight strains were chosen because they had previously been shown to represent eight distinct genotypes by pulsed-field gel electrophoresis analysis after separate digestion of the genomic DNA with ApaI or NotI. The eight strains could be classified into six or seven types by random amplified polymorphic DNA analysis using different kinds of primers. Amplified rDNA restriction analysis after separate digestion with five restriction enzymes, including AluI and MboI, of the 1,500 bp fragments of rDNA amplified by polymerase chain reaction did not discriminate the genomic variations among the eight strains of T equigenitalis. Thus, pulsed-field gel electrophoresis was shown to discriminate these eight organisms better than random amplified polymorphic DNA analysis, while amplified rDNA restriction analysis was found to be unsuitable for subtyping T equigenitalis.     

Indirect immunofluorescence test using polyclonal antibodies for the detection of Taylorella equigenitalis

Contagious equine metritis is a horse disease that causes endometrial inflammation due to Taylorella equigenitalis. Since Taylorella asinigenitalis was characterized, genital swab culture has proved to be an insufficient method for distinguishing between the two Taylorella species. Here, we developed an indirect immunofluorescence (IIF) test using polyclonal antibodies. Specificity, sensitivity, and detection limit were assessed using isolated bacteria (55 T. equigenitalis strains, 46 T. asinigenitalis strains and 18 other bacterial species), experimental and genital swabs in comparison to bacterial culture and polymerase chain reaction (PCR) testing. Our results indicated that IIF using polyclonal antibodies allows T. equigenitalis to be discriminated from T. asinigenitalis. This test constitutes a rapid, sensitive and specific tool for confirming presumptive colonies of T. equigenitalis.     

A two-step species-specific 16S rRNA PCR assay for the detection of Taylorella equigenitalis in horses

: A two-step PCR assay was developed for the molecular detection of Taylorella equigenitalis, a Gram-negative genital bacterial pathogen in horses. Two specific oligonucleotide primers (TE16SrRNABCHf [25mer] and TE16SrRNABCHr [29mer]) were designed from multiple alignments of the 16S rRNA gene loci of several closely related taxa, including T. asinigenitalis. Subsequent enhanced surveillance of 250 Thoroughbred animals failed to detect the presence of this organism directly from clinical swabs taken from the genital tract of mares and stallions. Such a molecular approach offers a sensitive and specific alternative to conventional culture techniques, and has the potential to lead to improved diagnosis and subsequent management of horses involved in breeding programmes.     

Nucleotide Sequencing and Analysis of 16S rDNA and 16S-23S rDNA Internal Spacer Region (ISR) of Taylorella equigenitalis, as an Important Pathogen for Contagious Equine Metritis (CEM)

The primer set for 16S rDNA amplified an amplicon of about 1500 bp in length for three strains of Taylorella equigenitalis (NCTC11184^T, Kentucky188 and EQ59). Sequence differences of the 16S rDNA among the six sequences, including three reference sequences, occurred at only a few nucleotide positions and thus, an extremely high sequence similarity of the 16S rDNA was first demonstrated among the six sequences. In addition, the primer set for 16S-23S rDNA internal spacer region (ISR) amplified two amplicons about 1300 bp and 1200 bp in length for the three strains. The ISRs were estimated to be about 920 bp in length for large ISR-A and about 830 bp for small ISR-B. Sequence alignment of the ISR-A and ISR-B demonstrated about 10 base differences between NCTC11184^T and EQ59 and between Kentucky188 and EQ59. However, only minor sequence differences were demonstrated between the ISR-A and ISR-B from NCTC11184^T and Kentucky188, respectively. A typical order of the intercistronic tRNAs with the 29 nucleotide spacer of 5'-16S rDNA-tRNA^Ile-tRNA^Ala-23S rDNA-3' was demonstrated in the all ISRs. The ISRs may be useful for the discrimination amongst isolates of T. equigenitalis if sequencing is employed.     

Molecular genotyping by pulsed-field gel electrophoresis of restricted genomic DNA of strains of taylorella equigenitalis isolated in Ireland and in the United States

The profiles, after digestion with ApaI or NotI and pulsed-field gel electrophoresis (PFGE), of genomic DNA from 18 strains of Taylorella equigenitalis isolated in Ireland were of three different types. The 13 strains in one of these types gave PFGE profiles identical to that of an American prototype strain, Kentucky 188, but different from strain NCTC11184^T and from a strain isolated in Japan.Eight additional strains isolated in the United States gave four distinct types of genomic PFGE profiles. All four types were different from that of T. equigenitalis NCTC11184^T or that of the Japanese strain. The profile of three strains of one type was identical to that of Kentucky 188.     

Contagious Equine Metritis Within the United States: A Review of the 2008 Outbreak

Contagious equine metritis (CEM) is a reportable foreign animal disease in the United States caused by the organism Taylorella equigenitalis. Import and export regulations regarding transport of horses into the United States from countries which either have a high prevalence of CEM or those that trade freely with countries having a high prevalence of CEM have failed to prevent the 2008 outbreak, which has not yet been traced to the original horse. It is important to recognize the clinical signs of acute CEM infections (endometritis, infertility, and abortion) to prevent further outbreak of disease. With early recognition, proper testing, and quarantine measures, the outbreaks and economic losses to the United States can be kept to a minimum. However, there are certain characteristics of the disease that are difficult to diagnose and control, such as the ability of the organism to infect an animal without creating clinical signs and the difficulty in culturing it. A review of the transmission, clinical signs, diagnostic methods, treatment, and prevention of CEM as well as a brief summary of the 2008 outbreak of CEM within the United States has been discussed in this article.     

基于流式细胞法和K-mer分析法检测沙鞭基因组大小北大核心CSCD

禾本科沙鞭属仅包含沙鞭一个种。沙鞭具有根茎繁殖、克隆整合等典型沙生植物特征,是我国内蒙古高原非固定沙地的建群种。本研究采用流式细胞术和K-mer分析方法测定沙鞭基因组大小,建立和优化以芨芨草为内参物种、青海固沙草和方穗山羊草为外参物种的二倍体植物DNA含量(DNA C值)测定体系。研究结果表明:1)沙鞭流式细胞峰值荧光是青海固沙草的2。54倍,是芨芨草的1。35倍,峰值信号远小于方穗山羊草,推测沙鞭基因组大小约为1580。10±5。02 Mb;2)K-mer分析结果表明,沙鞭基因组大小为1563。54 Mb,杂合率1。15%,重复序列比例为66。27%,基因组GC含量为43。4%,属于高杂合、高重复的复杂基因组;3)沙鞭基因组可使用PacBio平台CLR或HiFi模式进行三代测序,测序深度应不低于20×。沙鞭基因组大小的准确测定不仅补充了禾本科针茅族植物的DNA含量数据,同时也为沙鞭基因组测序、进化基因组研究、种质资源开发和利用以及遗传资源保护提供了数据参考,可为针茅族近缘物种基因组大小测定提供借鉴。     

The genome of Brucella melitensis

The genome of Brucella melitensis strain 16M was sequenced and contained 3,294,931 bp distributed over two circular chromosomes. Chromosome I was composed of 2,117,144 bp and chromosome II has 1,177,787 bp. A total of 3198 ORFs were predicted. The origins of replication of the chromosomes are similar to each other and to those of other α-proteobacteria. Housekeeping genes such as those that encode for DNA replication, protein synthesis, core metabolism, and cell-wall biosynthesis were found on both chromosomes. Genes encoding adhesins, invasins, and hemolysins were also identified.     

Analysis of mouse, rat, dog, marmoset, and human serum proteins by capillary electrophoresis: comparison with agarose gel electrophoresis

BACKGROUND: Serum protein analysis in both humans and experimental animal species has so far been carried out by labor-intensive techniques, such as agarose gel electrophoresis (AGE). OBJECTIVE: The objective of this study was to evaluate capillary electrophoresis (CE) as an alternative technique to AGE for the analysis of serum proteins from healthy animals. METHODS: Blood samples were collected into tubes without anticoagulant from 6 fasted healthy male mice, rats, dogs, marmosets, and humans. Serum proteins were separated by CE using a technique standardized for the analysis of human proteins, and the results (efficiency, resolution, and precision) were compared with those obtained through AGE. RESULTS: Compared with AGE, CE resulted in narrower peaks and more peaks. The efficiency of protein separation by CE was significantly higher for all species, and resolution (R) was significantly higher in samples from dogs. Using rat serum, intraday reproducibility was lower for all protein fractions, and interday reproducibility was lower for most peaks, compared with AGE. CONCLUSIONS: We conclude that CE is a viable alternative to AGE for the determination of protein electrophoresis in a routine veterinary clinical pathology laboratory. The minimal sample requirement (2 microL), complete automation, and quantitative results make CE an especially valuable technique for protein analysis in experimental animal models.     

鸡沙门氏菌脉冲场凝胶电泳分型研究

为应用脉冲场凝胶电泳(PFGE)从分子水平上对禽源沙门氏菌之间的差别进行分析和研究,本研究采用自动微生物鉴定仪对12株疑似鼠伤寒沙门氏菌和15株疑似鸡白痢沙门氏菌进行了鉴定.以限制性核酸内切酶Xba I对其全基因组DNA进行酶切、PFGE分型,Info Quest FP聚类分析软件对电泳结果进行分析.结果显示,共鉴定出11株鼠伤寒沙门氏菌、1株圣保罗沙门氏菌、6株阿姆斯特丹沙门氏菌、5株亚拉巴马沙门氏菌、1株鸡白痢沙门氏菌、2株纽波特沙门氏菌和2株肠炎沙门氏菌,一共分为12个PFGE型.实验结果表明,江苏地区存在有不常见的沙门氏菌血清型,同种血清型之间的基因型差别较小(相似值为0.85～1),不同血清型之间差别较大(相似值为0.45～0.70).PFGE能从分子水平上对禽源沙门氏菌的基因组DNA进行分型.     

Agarose gel electrophoresis of alkaline phosphatase isoenzymes in the serum of hyperthyroid cats

Cats with hyperthyroidism [(increased serum thyroxine (T(4))] commonly have increased serum alkaline phosphatase (ALP) activity in addition to other serum biochemical abnormalities. Serum biochemical profiles were obtained from 10 hyperthyroid cats which had increased serum ALP. Agarose gel electrophoresis of serum from these cats was performed and stained for alkaline phosphatase activity. Alkaline phosphatase activity was calculated for each of the separate bands obtained, and the results were compared to those of tissue extracts, serum from normal cats, and serum from normothyroid cats with increased serum ALP activity. The hyperthyroid cats had increased ALP activity in bands corresponding to isoenzymes originating in the liver, bone, and an unidentified tissue source.     

Effect of Antibiotic-containing Extenders on Taylorella equigenitalis Contaminated Semen

Taylorella equigenitalis is a gram-negative coccobacillus and the causative agent of a transmissible venereal disease in horses known as contagious equine metritis. Outbreaks of contagious equine metritis have been documented in various countries since 1977, with the most recent discovery in the United States in December 2008. During disease occurrences, culturing semen samples for T equigenitalis before breeding may help to prevent transmission of this disease; however, little is known about the antimicrobial activity of equine semen extenders against the organism. The purpose of this study was to investigate the infectivity levels of T equigenitalis in three equine semen extenders inoculated with known concentrations of the organism. The semen extenders used for this study included INRA 96, E-Z Mixin BF, and VMDZ. In addition, Timentin was added to INRA 96 at three different concentrations (0.5, 1.0, and 1.5 mg/mL) to investigate possible synergistic effects of antibiotic supplementation of extenders. Results were based on the visual counting of the colonies on chocolate Eugon agar plates. Both INRA 96 (with added Timentin) and VMDZ (as supplied by the manufacturer) significantly reduced the numbers of T equigenitalis isolated from semen extenders as compared with INRA 96 (as supplied by the manufacturer) or the antibiotic free E-Z Mixin BF. Our findings indicate that INRA 96 (with added Timentin) or VMDZ may significantly decrease the growth of T equigenitalis in extended semen; however, it is also important to consider the possible effects of antibiotic supplemented extenders on sperm longevity and fertility in addition to eliminating specific pathogens in semen.     

Genotyping of Ureaplasma diversum isolates using pulsed-field electrophoresis

Ureaplasma diversum has been associated with reproductive disorders in cattle and in the present study genotypic variations among U. diversum isolates obtained from the vaginal mucus of healthy cattle and sick animals were analyzed by enzymatic digestion and pulsed-field gel electrophoresis (PFGE). The influence of time and broth volume was important in obtaining sufficient cell sediment and DNA for PFGE. The method presented a high discriminatory power and satisfactory reproducibility for the analysis of detected variations among U. diversum isolates and strains. Different band profiles and wide genotypic heterogeneity were detected but no association between DNA polymorphism and sick or healthy animals could be established.     

Separation of serum glycated proteins by agarose gel electrophoresis and nitroblue tetrazolium staining in diabetic and normal cats

BACKGROUND: The total glycated protein (fructosamine) concentration in serum consists mainly of glycated albumin and lipoproteins. Measurement of fructosamine is used to diagnose and monitor diabetes mellitus in cats. OBJECTIVE: The aims of this study were to measure glycated proteins in diabetic and healthy (nondiabetic) cats using a semiquantitative technique and to determine whether measurement of any of the fractions of glycated protein could be potentially advantageous for the diagnosis and monitoring of diabetic cats. METHODS: Serum samples from 6 cats with diabetes mellitus and 10 clinically healthy adult cats were assayed for total glycated protein using a nitroblue tetrazolium (NBT) fructosamine assay. Serum proteins were separated by agarose gel electrophoresis and stained with NBT to identify individual glycated proteins within the bands. Gels were scanned by densitometry at 525 nm and the glycated protein content was calculated with reference to the total glycated protein content of the sample. RESULTS: Diabetic cats with increased total fructosamine concentrations had higher concentrations of glycated albumin and glycated alpha- and beta-lipoproteins compared with healthy cats. The concentration of glycated proteins in each of the fractions had a positive linear association with the total glycated protein content of serum, but there was large variation in the relative contributions of the 3 protein fractions to the total glycated protein concentration. CONCLUSIONS: Based on the results of this study, measurement of individual glycated fractions does not seem to offer any potential diagnostic advantage over measurement of total glycated protein (fructosamine) concentration alone. In some diabetic and healthy cats, glycated lipoproteins formed the major part of the total glycated protein, whereas in other cats albumin was the major contributor.