裸鼠巴斯德氏菌病的诊断

目的对某实验动物中心裸鼠发病死亡的原因进行诊断。方法对发病裸鼠进行病理组织学和细菌学检查以及血清学试验。结果根据临床症状、病理剖检、细菌分离培养、生化鉴定和血清凝集试验,确诊为嗜肺巴斯德氏菌感染导致的细菌性疾病。药敏试验结果表明,分离菌株对阿莫西林／克拉维酸、阿奇霉素、复方新诺明敏感,而对红霉素已经产生耐药性。结论为巴斯德氏菌病原鉴定与疾病诊断提供了科学依据。     

Evaluation of six commercial identification kits for the identification of Staphylococcus aureus isolated from bovine mastitis

AIMS: Comparison of six commercially available in human medicine well-established slide agglutination systems for the identification of Staphylococcus aureus. METHODS AND RESULTS: Slide agglutination tests were compared with the conventional tube coagulase test, biochemical identification and with the molecular identification by polymerase chain reaction (PCR) amplification of species-specific parts of the gene encoding the 23S RNA. Systems evaluated included Masta-Staph (Mast Diagnostics), Staphylase-Test (Oxoid), Staphytect-Plus (Oxoid), Staphyloslide Latex (Becton Dickinson), Slidex Staph Plus (bioMerieux) and Dry Spot Staphytect Plus (Oxoid). A total of 141 staphylococcal strains isolated from cases of bovine mastitis including 90 S. aureus, 14 Staphylococcus epidermidis, 10 Staphylococcus warneri, 13 Staphylococcus xylosus, 11 Staphylococcus haemolyticus and three other coagulase-negative staphylococci were tested with each method. Staphylococcus aureus strains were selected by macrorestriction analysis with pulsed field gel electrophoresis (PFGE). Only genetically unrelated strains were included in the study. The sensitivities and specificities of the test were as follows: Masta-Staph 86.7 and 90.1%, Staphylase-Test 78.4 and 85.1%, Staphytect-Plus 81.1 and 86.5%, Staphyloslide Latex 77.8 and 84.4%, Slidex Staph Plus 77.8 and 84.4%, Dry Spot Staphytect Plus 75.6 and 83.0%. CONCLUSIONS: The results of this evaluation suggest that the six slide agglutination methods tested can provide rapid identification of S. aureus also from bovine mastitis. The sensitivity and specificity seems to be less than those reported from human S. aureus isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the first comparative reported investigations about the applicability of different commercially available slide agglutination tests for the detection of S. aureus from bovine mastitis using PFGE selected clinical isolates.     

Identification by 16S rDNA fragment amplification and determination of genetic diversity by random amplified polymorphic DNA analysis of Pasteurella pneumotropica isolated from laboratory rodents

Pasteurella pneumotropica is an opportunistic bacterium frequently isolated from colonies of various laboratory rodents. Identification of this species, including its differentiation into two distinct biotypes (Jawetz and Heyl), is usually based on the use of conventional bacteriologic methods. In this study, a 16S rDNA fragment amplification procedure was developed for use as an alternative method for identification and differentiation of P. pneumotropica. Polymerase chain reaction (PCR) products were two distinctive fragments of 937 and 564 bp specific for biotypes Jawetz and Heyl, respectively. Specificity of PCR products could be achieved by EcoRI cleavage, leading to 596 plus 341-bp and 346 plus 218-bp fragments for each of the amplification products. Use of this procedure confirmed identification of 34 field isolates and allowed definitive identification of some strains that could not have been done by use of bacteriologic examinations. Field isolates subjected to random amplified polymorphic DNA (RAPD) analysis had high genetic diversity among biotype Jawetz strains in contrast to biotype Heyl strains. In conclusion, RAPD could represent an additional means for identification of ambiguous strains of biotype Heyl and a valuable epidemiologic tool for identification of biotype Jawetz strains of P. pneumotropica.     

Evaluation of PCR as a means of identification of Pasteurella pneumotropica.

A polymerase chain reaction with new primers (new PCR) designed from Pasteurella pneumotropica 16S rDNA as an identification system for this organism was compared with the PCR reported by Wang et al. (Wang's PCR) by using 15 bacterial reference species and 70 clinical isolates with the conventional identification system. For the 15 reference strains, both PCRs were identical. For the 70 clinical isolates, the new PCR and Wang's PCR showed consistency with the conventional system in 62.9% (44/70) and 51.4% (36/70), respectively. Twenty-six isolates were inconsistent with the conventional system and the new PCR with respect to morphology and serology. These findings suggested that the new PCR was more sensitive than Wang's PCR, and the new PCR in combination with morphology and serology is useful for P. pneumotropica identification.     

Implementation of a PCR assay of Pasteurella pneumotropica to accurately screen for contaminated laboratory mice

The authors implemented a PCR protocol to rapidly screen for Pasteurella pneumotropica and to accurately identify contaminated laboratory mice in a clinical setting. This protocol was implemented in response to a severe outbreak of P. pneumotropica in their animal facility. Although a sentinel program was in place to routinely screen for P. pneumotropica, it was inadequate for the identification of contaminated animals. As a result, several additional strains of mice were contaminated and developed clinical signs of infection. The authors implemented a screening method using PCR with reported primer pairs previously developed to identify the biotype isolates of P. pneumotropica in laboratory mice. Throat culture swabs were collected from live mice and placed in a bacterial culture. The DNA from these cultures was isolated and screened by PCR. This procedure enabled the authors to eliminate P. pneumotropica from several animal housing rooms. The assay can be easily applied in most animal facilities.     

Assessment of rpoB and 16S rRNA genes as targets for PCR-based identification of Pasteurella pneumotropica

Diagnosis of Pasteurella pneumotropica in laboratory animals relies on isolation of the organism, biochemical characterization, and, more recently, DNA-based diagnostic methods. 16S rRNA and rpoB gene sequences were examined for development of a real-time PCR assay. Partial sequencing of rpoB (456 bp) and 16S rRNA (1368 bp) of Pasteurella pneumotropica isolates identified by microbiologic and biochemical assays indicated that either gene sequence can be used to distinguish P. pneumotropica from other members of the Pasteurellaceae family. However, alignment of rpoB sequences from the Pasteurella pneumotropica Heyl (15 sequences) and Jawetz (16 sequences) biotypes with other Pasteurellaceae sequences from GenBank indicated that although rpoB DNA sequencing could be used for diagnosis, development of diagnostic primers and probes would be difficult, because the sequence variability between Heyl and Jawetz biotypes is not clustered in any particular region of the rpoB sequence. In contrast, alignment of 16S rRNA sequences revealed a region with unique and stable nucleotide motifs sufficient to permit development of a specific fluorogenic real-time PCR assay to confirm P. pneumotropica isolated by culture and to differentiate Heyl and Jawetz biotypes.     

Accuracy of the slide agglutination method evaluated with PCR in typing Haemophilus influenzae isolates

In 1998, the Colombian government initiated an immunization program for children under one year of age with a Haemophilus influenzae capsular type b conjugate vaccine. After two years, the surveillance program of the Colombian Instituto Nacional de Salud Microbiology Group reported a 40% decrease in meningitis cases caused by H. influenzae. This effect was attributed to the vaccination. The surveillance program uses the standardized slide agglutination technique to serotype H. influenzae. The current study validated the accuracy of the slide agglutination method by means of the PCR technique. From children under five years of age, 146 isolates were obtained. These were collected between 1999 and 2002, and were characterized by biochemical tests and serotyped by the INS as part of the surveillance program. PCR confirmed 93% of the H. influenzae serotype b and 92% of the other serotypes. When the slide agglutination technique is conducted under a strict quality control program, it remains a sensitive and specific tool for serotyping H. influenzae.     

嗜肺巴氏杆菌蛋白及抗原图谱初步分析

对不同来源的 1 1株嗜肺巴氏杆菌进行了全菌可溶性蛋白及抗原图谱分析。使用SDS -聚丙烯酰胺凝胶电泳 (SDS -PAGE) ,梯度凝胶电泳结果显示 ,嗜肺巴氏杆菌蛋白主要分布于相对分子质量 ( 1 4 4～ 97 4)× 1 0 3,且带型基本一致。 6株菌与参考菌株有完全相同的蛋白带 ,另外 5株则缺乏 40× 1 0 3带。分别用嗜肺巴氏杆菌免疫血清和自然感染嗜肺巴氏杆菌小鼠血清对 1 1株菌进行了免疫印迹 (Westernblots)试验。与免疫小鼠血清的反应显示 ,1 1株受试菌主要有相对分子质量大约 ( 1 7、31 )× 1 0 3两条反应带 ;在与自然感染血清的反应中主要的反应带在 1 7× 1 0 3处 ,缺乏 40× 1 0 3蛋白的 5株菌有 31× 1 0 3反应带 ,其余 7株均有大约 ( 2 0、2 8)× 1 0 3的反应带 ,故可以认为该菌主要抗原大约为 ( 1 7、31 )× 1 0 3;1 0个流行株根据 40× 1 0 3蛋白和 ( 2 0、2 8)× 1 0 3抗原的有无可被分为两型。本研究为精制血清学方法的诊断抗原 ,以及将SDS -PAGE和Westernblot法用于嗜肺巴氏杆菌检测奠定了基础。     

Phenotypic characterization and description of two major O-serotypes in Tenacibaculum maritimum strains from marine fishes

Tenacibaculum maritimum is the etiological agent of marine flexibacteriosis disease, with the potential to cause severe mortalities in various cultured marine fishes. The development of effective preventive measures (i.e. vaccination) requires biochemical, serological and genetic knowledge of the pathogen. With this aim, the biochemical and antigenic characteristics of T. maritimum strains isolated from sole, turbot and gilthead sea bream were analysed. Rabbit antisera were prepared against sole and turbot strains to examine the antigenic relationships between the 29 isolates and 3 reference strains. The results of the slide agglutination test, dot-blot assay and immunoblotting of lipopolysaccharides (LPS) and membrane proteins were evaluated. All bacteria studied were biochemically identical to the T. maritimum reference strains. The slide agglutination assays using O-antigens revealed cross-reaction for all strains regardless of the host species and serum employed. However, when the dot-blot assays were performed, the existence of antigenic heterogeneity was demonstrated. This heterogeneity was supported by immunoblot analysis of the LPS, which clearly revealed 2 major serological groups that were distinguishable without the use of absorbed antiserum: Serotypes O1 and O2. These 2 serotypes seem to be host-specfic. In addition, 2 sole isolates and the Japanese reference strains displayed cross-reaction with both sera in all serological assays, and are considered to constitute a minor serotype, O1/O2. Analysis of total and outer membrane proteins revealed that all strains share a considerable number of common bands that are antigenically related.     

Evaluation of the antiserum agar method for the serogroup identification of Neisseria meningitidis.

The antiserum agar method (ASA), which is based on the formation of immunoprecipitates around bacterial growth on agar containing meningococcal hyperimmune horse serum, was evaluated for serogroup identification of Neisseria meningitidis. Four hundred meningococcal stains were serogrouped by ASA employing horse antisera to serogroups A, B, C, Y, W135, Z, and 29E and compared to serogroup identification by bacterial slide agglutination (BA) employing rabbit antisera. Overall, there was 95% agreement between the two methods. The ASA proved to be more accurate than BA since 15 strains which cross-reacted with Y and W135 rabbit antisera by BA were specifically serogrouped as either Y or W135 by ASA. In addition, 5 out of 75 strains which were ungroupable by BA were serogrouped as either B or 29E by ASA. Repeat serogroup identification of 100 meningococcal strains by ASA provided identical results thus showing the reproducibility of the method. The ASA is advantageous to BA since it is more reliable, utilizes standard antisera which do not have to be absorbed to remove cross-reactions, does not require the preparation of standardized bacterial antigen, and is simple to perform.     

Development of a rapid identification method for Aeromonas species by multiplex-PCR

Existing biochemical methods cannot distinguish among some species of Aeromonads, while genetic methods are labor intensive. In this study, primers were developed to three genes of Aeromonas: lipase, elastase, and DNA gyraseB. In addition, six previously described primer sets, five corresponding to species-specific signature regions of the 16S rRNA gene from A. veronii, A. popoffii, A. caviae, A. jandaei, and A. schubertii, respectively, and one corresponding to A. hydrophila specific lipase (hydrolipase), were chosen. The primer sets were combined in a series of multiplex-PCR (mPCR) assays against 38 previously characterized strains. Following PCR, each species was distinguished by the production of a unique combination of amplicons. When the assays were tested using 63 drinking water isolates, there was complete agreement in the species identification (ID) for 59 isolates, with ID established by biochemical assays. Sequencing the gyrB and the 16S rRNA gene from the remaining four strains established that the ID obtained by mPCR was correct for three strains. For only one strain, no consensus ID could be obtained. A rapid and reliable method for identification of different Aeromonas species is proposed that does not require restriction enzyme digestions, thus simplifying and speeding up the process.     

A PCR-based strategy for simple and rapid identification of rough presumptive Salmonella isolates

The purpose of the present study was to investigate the application of ready-to-go Salmonella PCR tests, based on dry chemistry, for final identification of rough presumptive Salmonella isolates. The results were compared with two different biotyping methods performed at two different laboratories. The sensitivity of the BAX Salmonella PCR test was assessed by testing a total of 80 Salmonella isolates, covering most serogroups, which correctly identified all the Salmonella strains by resulting in one 800-bp band in the sample tubes. The specificity of the PCR was assessed using 20 non-Salmonella strains, which did not result in any DNA band. A total of 32 out of the 36 rough presumptive isolates were positive in the PCR. All but one isolate were also identified as Salmonella by the two biochemical methods. All 80 Salmonella strains were also tested in the two multiplex serogroup tests based on PCR beads. All strains belonging to the serogroups B, C1, C2-C3, and D were grouped correctly. Among the 32 rough presumptive isolates identified, 19 isolates resulted in a band of 882 bp (serogroup B), 11 isolates resulted in a band of 471 bp (serogroup C1), and two isolates showed a band of 720 bp (serogroup D). In conclusion, rough presumptive Salmonella isolates can be conveniently confirmed to the serogroup-level, using the pre-mixed PCR tests. The system can be easily implemented in accredited laboratories with limited experience in molecular biology.     

Elimination of Pasteurella pneumotropica from a contaminated mouse colony by oral administration of Enrofloxacin.

Enrofloxacin, a fluoroquinolone bactericidal antibiotic, was administered in an attempt to eradicate Pasteurella pneumotropica (P. pneumotropica) from a contaminated mouse colony. Contaminated mice, maintained within 4 animal rooms, were administered Enrofloxacin in drinking water at a daily dosage of 25.5 mg/kg for 2 weeks. Following one week of Enrofloxacin treatment, mice were selected randomly from each room and examined for P. pneumotropica. This procedure was repeated two or three times until all mice examined tested negative for the Pasteurella strain. With the exception of one room, treated mice consistently tested negative for P. pneumotropica for up to 45 weeks following completion of Enrofloxacin treatment. Thus, oral administration of Enrofloxacin significantly eliminated P. pneumotropica from a contaminated mouse colony.     

Serogrouping of Rhodococcus equi

The serological relationships among 27 isolates of Rhodococcus equi selected from a total of 1,195 isolates were investigated by cross-agglutination and absorption tests. The presence of capsular material was demonstrated in all the 27 isolates by electron microscopic observation. Antisera were prepared by employing formalized antigen of each isolate. In the cross-agglutination test with formalized antigen, 13 antisera reacted with homologous antigens alone, but the remaining 14 antisera reacted not only with homologous antigens but also with one to four heterologous antigens. When these 14 antisera possessing heterologous agglutinins were absorbed with each of the cross-reacting antigens, 14 specific antisera were obtained. The cross-agglutination test with these 27 antisera proved the 27 strains examined to be serologically distinct from one another. These strains were designated serogroups 1 to 27. Thus the same number of grouping antisera were prepared. The distribution of each serogroup among the 1,195 isolates and 15 reference strains was investigated by the slide agglutination test. All the strains were found to be groupable. Most of them belonged to serogroups 1 to 4, 7 to 9, 11, 14, and 15. Of the serogroups designated, 4, 16, 2, 12, 21, 1, and 9 were identical with Prescott's serovars 1, 2, 3, 4, 5, 6, and 7, respectively.     

A proposed serotyping system for Flavobacterium psychrophilum

AIMS: To develop a common serological system for rapid and routine identification of the fish pathogen Flavobacterium psychrophilum. METHODS: Thirty-four isolates of Fl. psychrophilum from different fish species and different geographical areas were typed using a slide agglutination test and an enzyme-linked immunosorbent assay (ELISA). RESULTS: Seven host-dependent serovars (1: salmon; 2: trout; 3: trout; 4: eel; 5: carp; 6: tench; 7: ayu) were found. Serovar 2 was divided into two antigenic subgroups (2a and 2b). The results achieved by both slide agglutination and ELISA methods were totally consistent with each other. Although both techniques proved to be simple to carry out and useful, only the ELISA allowed identification of Fl. psychrophilum serovars using unabsorbed antiserum and whole-cells as antigens. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper proposes a harmonized scheme for serological identification of Fl. psychrophilum to be used for diagnostic and seroepidemiological studies of the diseases it causes.     

酯酶试验与聚合酶链反应鉴别中间普氏菌的比较研究

目的 研究脂酶试验和常规生化方法与聚合酶链反应比较在鉴别中间普氏菌Pi中的敏感度和特异度。方法 对207株牙周临床分离的产黑色素G厌氧杆菌分别进行脂酶试验、常规生化法鉴定和16srRNA特异引物PCR鉴定。结果 207株实验菌中PCR鉴定出Pi97株,脂酶试验检测出Pi126株,其中有85株PCR阳性,其敏感度为87．6％,特异度为63．7％。常规生化方法和脂酶试验共同鉴定出Pi80株,其中有55株PCR为阳性,其敏感度为67％,特异度为86．4％。结论 脂酶与常规生化方法对Pi的鉴别能力低于PCR（ P＜0．05）,尚不能作为Pi菌种的可靠鉴定方法。     

APPLICATION OF A COLONY PCR TECHNIQUE WITH FUNG'S DOUBLE TUBE METHOD FOR RAPID DETECTION AND CONFIRMATION OF CLOSTRIDIUM PERFRINGENS

A colony polymerase chain reaction (PCR) technique was applied with an established Fung's double tube (FDT) method for rapid detection and confirmation of Clostridium perfringens. Published sequences of PCR primers for C. perfringens alpha toxin gene were used and PCR conditions were optimized. From the detection of C. perfringens by FDT tube to the confirmation by a colony PCR assay took as short as 16–18 h. The method was applied to 147 isolates of anaerobic sulfite reducing bacteria isolated from foods, sewages and animal clinical specimens. The results were compared with standard methods for the confirmation of C. perfringens. Of those 147 suspected isolates, 97 and 99 were confirmed as C. perfringens by standard methods and the colony PCR technique, respectively. We found the developed method simple, rapid, cost-effective, and most importantly, very reliable for the detection and confirmation of C. perfringens.

PRACTICAL APPLICATIONS

With Fung's double tube method and a colony polymerase chain reaction technique, a completed determination of Clostridium perfringens contamination can be accomplished within 16–18 h. This saves at least 2–3 days when compared with standard methods. Moreover, it minimizes the cost and labor needed since an anaerobic chamber as well as steps of Gram staining and biochemical testing can be avoided. The developed method is a powerful tool and an alternative for the enumeration of C. perfringens . It can be applied to samples from various sources and highly reliable results are expected. Most microbiological laboratories have a thermocycler and reagents as parts of their basic instruments. Therefore, the developed method can be easily applied without massive investment.     

Anomalies in species identification of enterococci from veterinary sources using a commercial biochemical identification system

AIMS: A commercial biochemical panel ID kit was used to identify presumptive enterococci isolates of veterinary or agricultural origin obtained during different steps of culture. METHODS AND RESULTS: Fifty isolates identified as enterococci using a genus PCR assay were tested for genus and species identification using the BBL Crystal Identification Gram-Positive ID kit (Becton Dickinson, Sparks, MD, USA). Following sub-culture of the isolates three times, 59% agreement with the original panel ID was obtained. After four and six sub-cultures, percentage agreement increased to 61 and 64%, respectively. Nineteen of the 50 cultures were identified as both Enterococcus faecalis and E. faecium. CONCLUSIONS: As a result of the variability between speciation of isolates following re-culture, additional methods for speciation are warranted. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the identification of the genus and species of non-human enterococcal isolates can vary greatly during successive passages when using this kit.     

New Method of Grouping Beta-Hemolytic Streptococci Directly on Sheep Blood Agar Plates by Coagglutination of Specifically Sensitized Protein A-Containing Staphylococci

A technique is described that allows the grouping of beta-hemolytic streptococci directly upon the primary colony. This was accomplished by applying a small drop of specifically sensitized protein A-containing Staphylococcus aureus over a colony of streptococci, rocking the plate to allow mixing of the particles with the soluble group-specific polysaccharide, which in the case of beta-hemolytic streptocicci was produced in abundance during colony formation, and observing for agglutination of the sensitized particles. Such a simple test for group A beta-hemolytic streptococci should allow accurate identification of group A streptococci in small laboratories, such as in clinics or physicians' offices, as well as in the larger public health and private laboratories.     

Comparison of two commercial methods with PCR restriction fragment length polymorphism of the tuf gene in the identification of coagulase-negative staphylococci

AIMS: Two commercial methods for the identification of coagulase-negative staphylococci (CNS) were compared with the restriction fragment length polymorphism (RFLP) of the amplified tuf gene, which served as the reference method. METHODS AND RESULTS: One hundred and forty-five CNS were evaluated using the API 32 Staph ID and the Crystal GP/ID BBL systems. The PCR-RFLP of the tuf gene served as the reference method. The APIStaph and the GP/ID BBL had an overall rate of agreement with the molecular method of 58.6% and 46.2% respectively, with the inability of the GP/ID BBL to characterize 11.7% of the isolates. The APIStaph showed higher sensitivity and better agreement than the GP/ID BBL with the PCR-RFLP, except for Staphylococcus hominis and Staphylococcus capitis. CONCLUSIONS: Neither of the commercial systems was as reliable as the PCR-RFLP method for identifying isolates of CNS. Overall the APIStaph had better agreement with the PCR-RFLP than the GP/ID system. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that the PCR-RFLP method is more reliable than the two commercial systems tested, suggesting that it is more reliable for routinely identifying CNS.