DNA probes in detection of spiroplasmas and mycoplasma-like organisms in plants and insects

Abstract DNA probes were applied to detect spiroplasmas and uncultivable mycoplasma-like organisms (MLOs) in infected plants and insects. The probes consisted of pMC5, a plasmid carrying the RNA genes of Mycoplasma capricolum and pRA1, a plasmid recovered from Spiroplasma citri . Southern blot hybridization of pMC5 with digested DNAs of periwinkle plants infected with S. citri , or with various MLOs, yielded 2 heavy and several weaker bands. The heavy hybridization bands were shown to represent rRNA genes of the plant chloroplasts, indicating significant nucleotide sequence homology between the mycoplasmal rRNA genes and those of plant chloroplasts. Some of the weaker hybridization bands, not revealed in DNA of healthy plants, appeared to represent rRNA gene sequences of the infectious agent. Use of the spiroplasma plasmid as a probe enabled the detection of S. citri in infected plant material and in hemolymph of infected leafhoppers at a high sensitivity level.     

Endosymbiotic microbiota of the bamboo pseudococcid Antonina crawii (Insecta, Homoptera)

We characterized the intracellular symbiotic microbiota of the bamboo pseudococcid Antonina crawii by performing a molecular phylogenetic analysis in combination with in situ hybridization. Almost the entire length of the bacterial 16S rRNA gene was amplified and cloned from A. crawii whole DNA. Restriction fragment length polymorphism analysis revealed that the clones obtained included three distinct types of sequences. Nucleotide sequences of the three types were determined and subjected to a molecular phylogenetic analysis. The first sequence was a member of the gamma subdivision of the division Proteobacteria (gamma-Proteobacteria) to which no sequences in the database were closely related, although the sequences of endosymbionts of other homopterans, such as psyllids and aphids, were distantly related. The second sequence was a beta-Proteobacteria sequence and formed a monophyletic group with the sequences of endosymbionts from other pseudococcids. The third sequence exhibited a high level of similarity to sequences of Spiroplasma spp. from ladybird beetles and a tick. Localization of the endosymbionts was determined by using tissue sections of A. crawii and in situ hybridization with specific oligonucleotide probes. The gamma- and beta-Proteobacteria symbionts were packed in the cytoplasm of the same mycetocytes (or bacteriocytes) and formed a large mycetome (or bacteriome) in the abdomen. The spiroplasma symbionts were also present intracellularly in various tissues at a low density. We observed that the anterior poles of developing eggs in the ovaries were infected by the gamma- and beta-Proteobacteria symbionts in a systematic way, which ensured vertical transmission. Five representative pseudococcids were examined by performing diagnostic PCR experiments with specific primers; the beta-Proteobacteria symbiont was detected in all five pseudococcids, the gamma-Proteobacteria symbiont was found in three, and the spiroplasma symbiont was detected only in A. crawii.     

Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization

Direct cultivation-independent sequence retrieval of unidentified bacteria from histological tissue sections has been limited by the difficulty of selectively isolating specific bacteria from a complex environment. Here, a new DNA isolation approach is presented for prokaryotic cells. By this method, a potentially pathogenic strain of the genus Brachyspira from formalin-fixed human colonic biopsies were visualized by fluorescence in situ hybridization (FISH) with a 16S rRNA-targeting oligonucleotide probe, followed by laser capture microdissection (LCM) of the targeted cells. Direct 16S rRNA gene PCR was performed from the dissected microcolonies, and the subsequent DNA sequence analysis identified the dissected bacterial cells as belonging to the Brachyspira aalborgi cluster 1. The advantage of this technique is the ability to combine the histological recognition of the specific bacteria within the tissue with molecular analysis of 16S rRNA gene or other genes of interest. This method is widely applicable for the identification of noncultivable bacteria and their gene pool from formalin-fixed paraffin-embedded tissue samples.     

Detection of Streptobacillus spp. in feral rats by specific polymerase chain reaction

Streptobacillus moniliformis is an etiological agent of rat-bite fever and Haverhill fever in human infection. As the currently available methods for identifying the causative bacteria are not satisfactory, we attempted to establish them by PCR using newly designed primers for the 16S rRNA gene of S. moniliformis. We then determined the prevalence of Streptobacillus spp. in two species of feral rats that inhabit an urban region in Japan, because information on the prevalence of the bacteria in feral rats is obscure. The use of PCR with newly designed primers showed that an extremely high proportion of R. norvegicus harbored the bacteria (61/66, 92%), whereas the prevalence was only 58% in R. rattus (30/52). The nucleotide sequence analysis of the 16S rRNA gene of Streptobacillus spp. isolated from oral swabs of feral rats showed at least two different types of bacteria among isolates from R. norvegicus and R. rattus.     

Comparison of two PCR methods for detection of Leptospira interrogans in formalin-fixed and paraffin-embedded tissues

In this study we compared two polymerase chain reaction (PCR) methods using either 16S ribosomal RNA (rRNA) or 23S rRNA gene primers for the detection of different Leptospira interrogans serovars. The performance of these two methods was assessed using DNA extracted from bovine tissues previously inoculated with several bacterial suspensions. PCR was performed on the same tissues before and after the formalin-fixed, paraffin-embedding procedure (FFPE tissues). The 23S rDNA PCR detected all fresh and FFPE positive tissues while the 16S rDNA-based protocol detected primarily the positive fresh tissues. Both methods are specific for pathogenic L. interrogans. The 23S-based PCR method successfully detected Leptospira in four dubious cases of human leptospirosis from archival tissue specimens and one leptospirosis-positive canine specimen. A sensitive method for leptospirosis identification in FFPE tissues would be a useful tool to screen histological specimen archives and gain a better assessment of human leptospirosis prevalence, especially in tropical countries, where large outbreaks can occur following the rainy season.     

Detection of Helicobacter in the fecal material of the endangered Yangtze finless porpoise Neophocaena phocaenoides asiaeorientalis

In animals, infection by the Epsilonproteobacteria Helicobacter spp. and H. cetorum is widespread. It has been suggested that H. cetorum may cause gastritis in cetaceans. The aim of our study was to investigate the presence of Helicobacter spp. in the fecal material of the endangered Yangtze finless porpoise Neophocaena phocaenoides asiaeorientalis. The fecal material of 12 porpoises living in the wild in Poyang Lake and 1 porpoise living in captivity at the Wuhan Baiji Dolphinarium were examined by PCR for the presence of Helicobacter spp. The fecal material of 8 of 12 wild porpoises and the captive porpoise were positive for Helicobacter spp. as determined by PCR using Helicobacter-specific primers, which target the 16S rRNA gene. A 16S rRNA clone library was then prepared from 1 sample isolated from a female porpoise living in the wild. DNA sequence analysis from 3 of the clones showed 98 to 99% identity to the H. cetorum 16S rRNA gene. These results demonstrate the prevalence of Helicobacter spp. and H. cetorum in endangered freshwater finless porpoises.     

Analysis of DNA encoding 23S rRNA and 16S–23S rRNA intergenic spacer regions from Plesiomonas shigelloides

Amplification of the gene encoding 23S rRNA of Plesiomonas shigelloides by polymerase chain reaction (PCR), with primers complementary to conserved regions of 16S and the 3' end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria. The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in a further two clones. In one the sequence of a single tRNA(Glu) was found which was absent from the other two. This variation in sequence suggests that the different clones may be derived from different ribosomal RNA operons.     

Molecular detection methods developed for a systemic rickettsia-like bacterium (RLB) in Penaeus monodon (Decapoda: Crustacea)

Molecular detection methods were developed to aid in the diagnosis of a rickettsia-like bacterium (RLB) which caused severe mortalities of farm-raised Penaeus monodon in Madagascar. Using primers derived from the 16S rRNA gene of bacteria, a PCR assay was optimized to amplify this region of the genome of the RLB, using extracted DNA from infected P. monodon tissue as the template. The resulting amplified PCR product was sequenced and 2 novel primers were selected from the variable region of the gene. These primers amplified a 532 bp fragment of DNA originating from the rickettsia-infected samples. The PCR assay was optimized and tested on DNA extracted from specific pathogen-free (SPF) P. vannamei tissue and several other strains of bacteria. The PCR assay with the rickettsia-specific primers was specific for this RLB and did not amplify the other DNA samples tested. The 532 bp PCR-amplified fragment was labeled with digoxigenin (DIG) for in situ hybridization assays. This probe was tested on SPF, RLB and bacteria-infected shrimp specimens preserved in Davidson's fixative. The probe was specific for both natural and experimental rickettsial infections. Hybridization with this probe required a stringent temperature of 65 degrees C, otherwise cross-reactivity was observed with other types of bacteria.     

Selection and characterization of Spiroplasma citri mutants by random transposome mutagenesis

Phytopathogenic spiroplasmas can multiply in vascular plants and insects. A deeper understanding of this dual-host life could be furthered through the identification by random mutagenesis of spiroplasma genes required. The ability of the EZ::TN? Tnp transposome? system to create random insertional mutations in the genome of Spiroplasma citri was evaluated. The efficiency of electroporation-mediated transformation of S. citri BR3-3X averaged 28.8 CFUs/ng transposome for 10(9) spiroplasma cells. Many transformants appearing on the selection plates were growth impaired when transferred to broth. Altering broth composition in various ways did not improve their growth. However, placing colonies into a small broth volume resulted in robust growth and successful subsequent passages of a subset of transformants. PCR using primers for the dihydrofolate reductase gene confirmed the transposon's presence in the genomes of selected transformants. Southern blot hybridization and nucleotide sequencing suggested that insertion was random within the chromosome and usually at single sites. The insertions were stable. Growth rates of all transformants were lower than that of the wild-type S. citri, but none lost the ability to adhere to a Circulifer tenellus (CT-1) cell line. The EZ::TN? Tnp transposome? system represents an additional tool for genetic manipulation of the fastidious spiroplasmas.     

Discrimination of Bacillus cereus and Bacillus thuringiensis with 16S rRNA and gyrB gene based PCR primers and sequencing of their annealing sites

AIMS: To evaluate the possibility for discrimination of Bacillus cereus and B. thuringiensis using 16S rRNA and gyrB gene based PCR methods, and to obtain the sequences of the primer annealing sites so that the PCR results may be explained. METHODS AND RESULTS: Based on the sequence difference in the variable region (V1) of 16S rRNA and in the gyrB gene between B. cereus and B. thuringiensis, PCR primers specific to these Bacillus spp. were designed. When these primers were used to discriminate B. cereus and B. thuringiensis, six of 82 B. cereus strains were identified as B. thuringiensis while 67 of 73 B. thuringiensis strains were identified as B. cereus. Sequence analysis of the primer annealing sites showed that there is no clear-cut difference in the V1 region of 16S rRNA, and in the gyrB gene, between B. cereus and B. thuringiensis strains. CONCLUSIONS: Although 16S rDNA based probes and gyrB gene based PCR primers have been suggested for the discrimination of B. cereus and B. thuringiensis strains, when a large number of Bacillus strains was tested, results showed that discrimination between B. cereus and B. thuringiensis is difficult. Therefore, to distinguish B. thuringiensis from B. cereus, a single feature, such as the presence of a parasporal crystal protein or cry gene, may sometimes be reliable. SIGNIFICANCE AND IMPACT OF THE STUDY: Discrimination between B. cereus and B. thuringiensis is a challenging debate to which this paper makes a contribution.     

Identification of the fire blight pathogen, Erwinia amylovora, by PCR assays with chromosomal DNA.

Erwinia amylovora, the causative agent of fire blight, was identified independently from the common plasmid pEA29 by three different PCR assays with chromosomal DNA. PCR with two primers was performed with isolated DNA and with whole cells, which were directly added to the assay mixture. The oligonucleotide primers were derived from the ams region, and the PCR product comprised the amsB gene, which is involved in exopolysaccharide synthesis. The amplified fragment of 1.6 kb was analyzed, and the sequence was found to be identical for two E. amylovora strains. The identity of the PCR products was further confirmed by restriction analysis. The 1.6-kb signal was also used for detection of the fire blight pathogen in the presence of other plant-associated bacteria and in infected plant tissue. For further identification of isolated strains, the 16S rRNA gene of E. amylovora and other plant-associated bacteria was amplified and the products were digested with the restriction enzyme HaeIII. The pattern obtained for E. amylovora was different from that of other bacteria. The sequence of the 16S rRNA gene was determined from a cloned fragment and was found to be closely related to the sequences of Escherichia coli and other Erwinia species. Finally, arbitrarily primed PCR with a 17-mer oligonucleotide derived from the sequence of transposon Tn5 produced a unique banding pattern for all E. amylovora strains investigated. These methods expand identification methods for E. amylovora, which include DNA hybridization and a PCR technique based on plasmid pEA29.     

致病性维罗纳气单胞菌检测方法的建立

发掘维罗纳气单胞菌特异性更强的检测靶点和毒力相关基因靶点,建立能够检测致病性维罗纳气单胞菌的PCR检测方法.通过序列比对分析气单胞菌的16S rRNA基因序列,筛选对维罗纳气单胞菌特异的引物,用于检测种特异性,利用气单胞菌气溶素基因保守引物,检测菌株的致病性,并进行反应条件和反应体系的优化,灵敏度试验和特异性试验.发掘并设计的维罗纳气单胞菌16S rRNA特异性引物结合气单胞菌气溶素基因保守引物建立的检测方法,对12株气单胞菌和10株非气单胞菌的检测结果显示,所有致病性维罗纳气单胞菌都能扩增到大小分别为343 bp和232 bp的特异性条带,而非维罗纳气单胞菌的致病性气单胞菌只能扩增到232 bp的气溶素基因特异性条带,其它菌株都不能扩增到目的条带.灵敏度试验表明,该反应体系的检测灵敏度为1.35×10-3 mg/L.我们建立的致病性维罗纳气单胞菌检测方法能特异地检测致病性维罗纳气单胞菌,并具有高度灵敏性.     

Analysis of 23S rRNA genes in metagenomes - a case study from the Global Ocean Sampling Expedition

As an evolutionary marker, 23S ribosomal RNA (rRNA) offers more diagnostic sequence stretches and greater sequence variation than 16S rRNA. However, 23S rRNA is still not as widely used. Based on 80 metagenome samples from the Global Ocean Sampling (GOS) Expedition, the usefulness and taxonomic resolution of 23S rRNA were compared to those of 16S rRNA. Since 23S rRNA is approximately twice as large as 16S rRNA, twice as many 23S rRNA gene fragments were retrieved from the GOS reads than 16S rRNA gene fragments, with 23S rRNA gene fragments being generally about 100 bp longer. Datasets for 16S and 23S rRNA sequences revealed similar relative abundances for major marine bacterial and archaeal taxa. However, 16S rRNA sequences had a better taxonomic resolution due to their significantly larger reference database.Reevaluation of the specificity of previously published PCR amplification primers and group specific fluorescence in situ hybridization probes on this metagenomic set of non-amplified 23S rRNA sequences revealed that out of 16 primers investigated, only two had more than 90% target group coverage. Evaluations of two probes, BET42a and GAM42a, were in accordance with previous evaluations, with a discrepancy in the target group coverage of the GAM42a probe when evaluated against the GOS metagenomic dataset.     

Simultaneous detection and differentiation of pathogenic and nonpathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay

Leptospirosis, caused by pathogenic Leptospira, is one of the most important zoonoses in the world. Several molecular techniques have been developed for detection and differentiation between pathogenic and saprophytic Leptospira spp. The aim of this study was to develop a rapid and simple assay for specific detection and differentiation of pathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay using primers and probes targeting Leptospira genus specific 16S ribosomal RNA gene, the pathogen specific lig A/B genes and nonpathogen Leptospira biflexa specific 23S ribosomal RNA gene. Sixteen reference strains of Leptospira spp. including pathogenic and nonpathogenic and ten other negative control bacterial strains were used in the study. While the 16S primers amplified target from both pathogenic and non-pathogenic leptospires, the ligA/B and the 23S primers amplified target DNA from pathogenic and non-pathogenic leptospires, respectively. The multiplex real-time PCR (TaqMan) assay detection limit, that is, the sensitivity was found approximately 1 x 10(2) cells/ml for ligA/B gene and 23S ribosomal RNA gene, and 10 cells/ml 16S ribosomal RNA. The reaction efficiencies were 83-105% with decision coefficients of more than 0.99 in all multiplex assays. The multiplex real-time PCR (TaqMan) assay yielded negative results with the ten other control bacteria. In conclusion, the developed multiplex real-time PCR (TaqMan) assay is highly useful for early diagnosis and differentiation between pathogenic and non-pathogenic leptospires in a reaction tube as having high sensitivity and specificity.     

Characterization of the rRNA genes of Ehrlichia chaffeensis and Anaplasma phagocytophila

The rRNA genes of Ehrlichia chaffeensis and Anaplasma phagocytophila have been analyzed. The 16S rRNA genes were previously characterized for both of these agents. Southern hybridization was used to show that there are single copies of both the 16S and 23S rRNA genes in the genomes of each organism, and that the 16S rRNA genes were upstream from the 23S rRNA genes by at least 16 and 11 Kb for E. chaffeensis and A. phagocytophila, respectively. PCR amplification and gene walking was used to sequence the 23S and 5S rRNA genes, and show that these genes are contiguous and are likely expressed as a single operon. The level of homology between the E. chaffeensis and A. phagocytophila 23S and 5S rRNA genes, and 23S-5S spacers, was 91.8, 81.5, and 40%, respectively. To confirm the hybridization data, genome walking was used to sequence downstream of the 16S rRNA genes, and although no tRNA genes were identified, open reading frames encoding homologues of the Escherichia coli succinate dehydrogenase, subunit C, were found in both E. chaffeensis and A. phagocytophila. Phylogenetic analysis using the 23S rRNA gene suggests that reorganization of the phylum Proteobacteria by division of the class Alphaproteobacteria into two separate subclasses, may be appropriate.     

一株凡纳滨对虾源维氏气单胞菌的分离鉴定及药敏特性

【目的】凡纳滨对虾(Litopenaeus vannamei)是世界范围内最主要的对虾养殖品种之一,2017年5-6月上海某凡纳滨对虾养殖场出现不明原因的死亡病例,发病急,死亡率高。从患病凡纳滨对虾体内分离到一株优势菌AVZ01,旨在确定病因并筛选出敏感药物,为今后凡纳滨对虾维氏气单胞菌(Aeromonas veronii)的防治提供参考。【方法】从患病凡纳滨对虾肝胰腺和肠道中分离致病菌,通过理化特性及16S r RNA基因序列分析进行鉴定,通过人工感染试验确定病原,使用Bliss法计算出半数致死剂量(LD50),并通过纸片扩散法进行药敏试验。【结果】从患病凡纳滨对虾体内分离到一株优势菌AVZ01,进行人工回归感染试验后,对虾发病症状与自然发病症状相似,凡纳滨对虾的LD50为8.7×105 CFU/m L。根据该菌株的形态特征、理化特性、16S r RNA基因序列分析,综合判断该病原菌为维氏气单胞菌。药敏试验结果显示,该菌株对米诺环素、诺氟沙星、庆大霉素等16种抗生素高度敏感,对青霉素、苯唑西林、头孢氨苄等9种抗生素耐药。【结论】分离菌株AVZ01对凡纳滨对虾有较强的致病性,养殖过程中可选用庆大霉素及新霉素等药物进行防控。     

Identification of a haemomycoplasma species in anemic reindeer (Rangifer tarandus)

During an 18-mo period (May 2002-November 2003), 10 animals in a herd of 19 reindeer (Rangifer tarandus) at the National Animal Disease Center (NADC) experienced episodes of anemia. Affected animals had histories of weight loss, unthriftiness, occasionally edema of dependent parts and moderate anemia characterized by microcytosis or macrocytosis, hypochromasia, schistocytosis, keratocytosis, acanthocytosis, and dacryocytosis. Numerous basophilic punctate to ring-shaped bodies, measuring less than 1.0 microm, were found on the surface of red blood cells and were often observed encircling the outer margins of the cells. Based on cytologic findings, DNA preparations from selected affected animals in the NADC herd and one animal from a private herd experiencing similar episodes of anemia were assayed by polymerase chain reaction (PCR) for the presence of hemotropic bacteria using primers targeting the 16S rRNA genes of Mycoplasma (Eperythrozoon) suis, Mycoplasma (Haemobartonella) haemofelis, Anaplasma marginale, Anaplasma spp., and Ehrlichia spp. Amplification products were detected from four of the affected animals using primers specific for the 16S rRNA gene of M. haemofelis and Mycoplasma haemocanis. Product from one of the animals was sequenced and internal primers were designed from the resulting sequence to perform a nested PCR assay. Samples from 10 reindeer were positive using the nested PCR reaction and products from seven animals were sequenced; BLAST searches and phylogenetic analysis were performed on the resulting sequences. Sequence data from six animals revealed homology to an organism most closely related to Mycoplasma ovis, Mycoplasma wenyonii, and Mycoplasma haemolamae; sequence from a single animal was most closely related to M. haemofelis and M. haemocanis. This represents the first identification of a haemomycoplasma species in reindeer. Although several animals were also infected with abomasal nematodes, the presence of this newly described haemomycoplasma may have contributed to the anemic syndrome.     

分离自我国蜜蜂的一株新的致病螺原体及其基本特性

【目的】通过分析分离自我国患病蜜蜂体内的螺原体MF1006的基本生物学特征,初步确定其分类地位及致病性。【方法】应用暗视野显微镜和透射电子显微镜观察螺原体形态,运用常规螺原体分离和培养方法、分子生物学方法和血清学方法研究螺原体分离菌株可能的分类地位,并采用饲喂接种法研究其致病性。【结果】菌株MF1006具有典型的螺旋形和运动性,能通过0.22μm孔径的滤膜,与我国之前发现的引起蜜蜂螺原体病的参比菌株Spiroplasma melliferum CH-1的基本生物学特征差异较大。S.melliferum CH-1抗血清对其没有抑制作用。根据16S rDNA、ITS序列构建系统发育树显示,菌株MF1006与在法国发现的引起蜜蜂"五月病"的Spiroplasma apis的亲缘关系最近。此外,菌株MF1006对供试意蜂有较强的致病性。【结论】分离菌株MF1006是在我国蜜蜂体内发现的除S.melliferum以外的另一种致病螺原体。     

Genetic diversity of Datisca cannabina-compatible Frankia strains as determined by sequence analysis of the PCR-amplified 16S rRNA gene.

The presence of Frankia strains in soil samples collected from northern areas of Pakistan was detected by inoculating Coriaria nepalensis and Datisca cannabina plants. The abundance of compatible Frankia strains in some areas was indicated by profuse nodulation of the host plants, whereas soil samples from other localities failed to result in nodulation. An oligonucleotide probe (COR/DAT) directed against the 16S rRNA gene of the endophytes of Coriaria and Datisca spp. that did not cross-react with the RNA gene of Frankia strains isolated from other hosts was developed. Genetic diversity among Frankia strains nodulating D. cannabina was determined by sequence analysis of the partial 16S rRNA gene amplified from nodules induced by soil samples from different localities by PCR. Four types of Frankia sequences and one non-Frankia sequence were detected by hybridization with a Frankia genus probe and the COR/DAT probe as well as by sequence analysis of the cloned PCR products.