16S rRNA Gene Fragments Research Articles

16S rRNA gene for ribotyping Brucella: A monospecific genus

The aim of the study was to assess the usefulness of 16S rRNA gene in identification of Brucella spp. and phylogenetic study. For identification purposes, a 1477-bp fragment of 16S rRNA gene of 16 isolates and 3 control reference strains was amplified and 3 isolates with B. abortus S19 sequenced. The obtained sequences were submitted to GenBank for species identification. Sequence analysis of a 1477-bp 16S rRNA fragment allows the identification of Brucella spp. However, for discrimination of closely related species sequencing of the entire 16S rRNA gene, additional sequencing of the 23S rRNA gene or sequencing of the 16S-23S rRNA intergenic spacer is essential.

Inference about Monophyly of the Family Oedipodidae and the Classification of Subfamilies Based on 16S rDNA Sequences

Most of grasshoppers in the family Oedipodidae are the famous agriculture pests in China. However monophyly and the relationships among the subfamilies within this family are unclear up to now. Here the phylogeny of the Oedipodidae was reconstructed based on 16S rDNA sequence fragments by using Mekongiella kingdoni and Atractomorpha sinensis as outgroups under weighted MP, NJ and Bayesian criteria. The 408 bp fragments of mitochondrial 16S rRNA gene were sequenced for 15 species from 4 subfamilies of the family Oedipodidae, and the homologous sequences of other 15 species of grasshoppers were downloaded from the GenBank data library. The numbers of transitions and transversions among pairwise comparisons of the 16S fragments were respectively plotted against percentage sequence differences. Saturation of transitions was discovered, and transversions were not saturated with the increase of percentage sequence difference in the plots. All the individuals of the Oedipodidae excluding Trilophidia annulata were gathered together in the three trees. Our results are very different from the traditionary taxonomy of the Oedipodidae including 4 subfamilies. The Bryodemellinae is not supported as a subfamily, and neither Locustinae nor Oedipodinae are supported as a monophyletic group in this study.

전북지역 한우에서 분리한 기종저 균의 유전학적 특성 규명

Clostridium chauvoei is the etiologic agent of blackleg, a high mortality rated disease infection mainly cattle. In the present study, the partial sequences of 16S rRNA and flagellin gene of C. chauvoei isolated in Jeonbuk, Korea were determined and compared with those of reference strain. Oligonucleotide primers were designed to amplify a 811 bp fragment of 16S rRNA gene and 1229 bp fragment of flagellin gene. Sequencing analysis of 16S rRNA gene showed high homology to the reference strains ranging 82.3% to 100%, while flagellin gene were different from published foreign clostridia, showing 98.7% to 72.0% nucleotide sequence homology. Phylogenetic analysis based on 16S rRNA gene revealed the close phylogenetic relationship of C. chauvoei and C. septicum in cluster I, which includes C. carnis, C. tertium, C. quinii, C. celatum, C. perfringens, C. absonum, C. botulinum B. Phylogentic analysis also revealed that flagellin gene formed a single cluster with C. chauvoei, C. septicum, C. novyi A, C. novyi B, C. tyrobutylicum, C. acetobutylicum. The genetic informations obtained from this study could be useful for the molecular study of C. chauvoei.

Silver quartz crystal microbalance for differential diagnosis of Plasmodium falciparum and Plasmodium vivax in single and mixed infection

The most severe form of malaria is cerebral malaria caused by Plasmodium falciparum. Standard malaria diagnosis is Giemsa stained peripheral blood smear but false negative findings are always reported. Moreover, mixed infections are underestimated by routine microscopy. Many methods have been developed to overcome these disadvantages and the most specific and sensitive is molecular diagnosis. Specific malaria genes are amplified by polymerase chain reaction (PCR) and many post-PCR methods have been created. We developed a gold fabricated quartz crystal microbalance (QCM) as a post-PCR method of malaria diagnosis. In this work a cheaper silver fabricated QCM was developed to identify both single and mixed infection of P. falciparum and Plasmodium vivax. The biotinylated malaria probe was immobilized on silver surface via specific avidin–biotin interaction. The target DNA fragment of 18s rRNA gene was amplified and hybridized with a QCM immobilized probe. Mass changes due to DNA hybridization were indicated by changes of quartz resonance frequencies. Validation showed that malaria silver QCM had high diagnostic potency. Evaluation of suspected 67 febrile blood samples from malaria endemic area demonstrated that the malaria silver QCM could identify both false negative and misdiagnosis cases of routine microscopy. The analysis cost of malaria silver QCM was $1/sample and analysis time was 30min after blood collection. The malaria silver QCM is stable at tropical temperature for up to 6 months. Thus, it can be transported to be used in a remote endemic area. Thus, the malaria silver QCM is accurate, precise, rapid, cheap, and field applicable.

Experience in Genome Analysis Use in SAET Mobile Complex Facilities

Described is the first-ever experience in genome analysis use in SAET mobile complex facilities, obtained during the XXVII World-wide Summer Universiade in Kazan, 2013. Carried out was 16S rRNA sequencing of Salmonella enteric strain isolated from an infected. For that matter, employed was “MicroSEQ 500 16S rDNA Bacterial Identification Kits” (Applied Biosystems, USA), which allows for sequencing of DNA fragments (500 bp) of 16S rRNA gene in the bacteria under investigation.

Amblyomma hadanii n. sp. (Acari: Ixodidae), a tick from northwestern Argentina previously confused with Amblyomma coelebs Neumann, 1899.

All stages of Amblyomma hadanii n. sp. (Acari: Ixodidae) are described from northwestern Argentina. The diagnostic characters for males are a combination of the pattern of scutal ornamentation, basis capituli dorsally rectangular with cornua, coxa I with two subequal spurs (the internal wider, the external longer), coxae II-III with a single spur, coxa IV with a single spur not reaching level of anus, ventral plates irregular in shape (larger and sometimes with a small incision on festoons 4, 5 and 6) and hypostome spatulate with dental formula 3/3 in 7-8 rows. The diagnostic characters for the females are a combination of scutal ornamentation, postero-lateral margins of scutum slightly convex, coxa I with two subequal spurs (the internal wider, the external longer), basis capituli dorsally rectangular, porose areas rounded, genital aperture U-shaped, and hypostome spatulate with dental formula 3/3 in 7-8 rows. Diagnosis of nymphs can be performed by a combination of basis capituli rectangular, scutum with large punctations in the lateral fields and small punctations in the central field, and cervical groove short and ending as a small shallow depression at the eye level. Larvae are diagnosed by the shape of basis capituli, scutum with with posterior margin slightly convex, and legs with coxa I with 2 triangular spur (the external longer than the internal), and with coxae II and III each with 1 triangular spur. The hosts recorded for this new tick species are Tapirus terrestris (Linnaeus), horse, cattle, dog and humans. Analyses of a 410 bp fragment of the mitochondrial 16S rRNA gene and the complete sequence of the nuclear 18S rRNA gene supported the description of A. hadanii as a new species.

The first genotype determination of Acanthamoeba potential threat to human health, isolated from natural water reservoirs in Poland.

Different species of amoebae belonging to the genus Acanthamoeba are widely distributed in many parts of the world and known as free-living organisms. Some strains of the protozoans may exist as parasites and cause risk to human health as causative agents of serious human diseases. Currently, in Poland, there is no sufficient information about the distribution of Acanthamoeba strains and their genotypes in the environment. Therefore, 20 environmental surface water samples were collected from different sites located at five water reservoirs in Gdynia, Sopot, and Gdańsk (northern Poland). The material was cultured to obtain Acanthamoeba isolates that were then specifically analyzed with both PCR and real-time PCR assays. Of the 20 samples examined, Acanthamoeba DNA was found in 13 samples tested with the use of real-time PCR; in 10 of them, DNA of the amoeba was also detected using PCR technique. The comparison with sequences available in the GenBank confirmed that the PCR products are fragments of Acanthamoeba 18S rRNA gene and that isolates represent T4 genotype, known as the most common strains related to AK cases. This is the first investigation in Poland describing Acanthamoeba detection in environmental water samples with molecular techniques and genotyping. The results indicate that surface water in Poland may be a source of acanthamoebic strains potentially pathogenic for humans.

Rapid bovine and caprine species identification in ruminant feeds by duplex real-time PCR melting curve analysis using EvaGreen fluorescence dye.

A duplex real-time PCR assay with melting curve analysis, using the EvaGreen fluorescence dye, was developed for rapid and reliable identification of bovine and caprine in ruminant feeds. The method merges the use of bovine (Bos taurus) and caprine (Capra hircus) specific primers that amplify small fragments (bovine 96 bp and caprine 142 bp) of the mitochondrial 16S rRNA and 12S rRNA genes, respectively. DNA was isolated from heat-treated meats (133 °C/3 bar for 20 min) mixtures of bovine and caprine and was used to optimize the assay. Gene products of caprine and bovine produced two distinct melting peaks simultaneously at 82 and 86.8 °C, respectively. Duplex analysis of the reference samples showed that the detection limit of the assay was 0.003 % for bovine and 0.005 % for caprine species. The aim of this study was to develop a duplex real-time PCR assay followed by a melt curve step for sensitive, rapid, specific, and cost-effective detection of bovine and caprine species based on the amplicon melting peak in ruminant feeds to prevent Transmissible Spongiform Encephalopathies.

Identification of 'Candidatus Neoehrlichia mikurensis' and Anaplasma species in wildlife from Croatia

Vector borne diseases are classical emerging infectious diseases in human and animal populations. Ticks represent perfect vectors for number of bacteria, parasites and viruses. The genera Anaplasma, Ehrlichia and recently specified cluster ‘Candidatus Neoehrlichia’ comprise all bacteria from family Anaplasmataceae, transmitted by ixodid ticks to mammalian hosts causing infection in humans and animals. ‘Candidatus Neoehrlichia mikurensis’ is one of emrgening vector borne zoonosis recently recognized as human pathogen. First case of human infection was reported from Sweden in 2010 and up to now six human infections were described in Czech Republic, Germany and Switzerland. In 2011 first case of septicaemia in two dogs was reported indicating ability of Neoehrlichia to infect various mammalian species, while different rodent species may act as reservoir host. The aim of this study was to identify species from family Anaplasmataceae from different species of wild animals (851). The presence of selected pathogens from wild cervids, wild boars, small rodents, muflons, chamois, martens, bears, badgers, wolves, jackals and foxes were determined by performing PCR on spleen samples and subsequent sequencing of fragment of 16S rRNA gene. Sequence analysis revealed presence of N. mikurensis, two strains of Anaplasma phagocytophilum, Anaplasma centrale, A. bovis and Ehrlichia sp. Small rodents were infected mainly with N. mikurensis together with A. phagocytophilum. Neoehrlichia mikurensis was single species detected in wild boars, bears and badgers, which represent first finding in these mammalian species. In chamois all species have been found except A. bovis. Wild cervids were harbouring both strains of A. phagocytophilum, while muflons were infected with A. centrale, A. phagocytophilum, A. bovis and N. mikurensis. Martens and jackals were free from pathogens while sequencing failed in wolf samples. Foxes were infected with both strains of A. phagocytophilum and interestingly in single animal A. bovis was confirmed. Two isolates Ehrlichia sp. from roe deer and chamois were identical to Ehrlichia sp. from Sika deer from Japan. Data obtained from this study clearly present presence of two zoonotic pathogens: A. phagocytophilum, causative agent of human granulocytic anaplasmosis (HGA) and newly recognized pathogen N. mikurensis in various species. These results also imply importance of wildlife as potential reservoir for ticks and potential transmission to humans.

FEATURES OF CORROSION ACTIVITY OF MICROBIAL COMMUNITIES OF GAS-PIPELINE FERROSPHERE LAID IN SANDY SOIL

Aim. To study the sulfidogenic microbial communities road gas-pipeline ferrospheres laid in the sandy soil and the determination of its composition was the aim of this research work. Methods. The method of limiting dilution (plating cell suspension to appropriate liquid selection medium) was used for detection and isolation of the components of communities of bacteria and microscopic fungi complex. Due to quantitative and qualitative composition, sulfidogenic community of ferrospheres in corrosive dangerous. Pure cultures of sulfate-reducing bacteria were obtained by planting numerous individual colonies on solid and liquid medium Postgate «B». Results. The dominant strain of sulfate-reducing bacteria – М-4.1 described by morphological and cultural characteristics has been isolated and identified by using PCR, with the use of universal primers to the fragments of 16S rRNA genes. When conducting a comparative analysis of DNA sequencer which encodes 16S rRNA gene of tested bacteria with the strains of similar sequences from the GenBank database, sulfate reducing bacteria strain M-4.1 belonging to Desulfovibrio genus has been confirmed. Microfungi were isolated by planting soil suspension dilutions with agar Czapek-Dox in Petri dishes. Fungal identification was carried out on the basis of their morphological and cultural features using determinants. 78 microfungi cultures belonging to Anamorphic fungi group (12 species of 5 genera), have been isolated. Conclusions. Thus, this scientific study has shown that in the ferrospheres on the surface of damaged road gas-pipeline laid in the sandy soil the corrosion dangerous abundance and species composition of complex physiological bacteria group and micromycetes is formed.

Morphological and Molecular Analysis of the Andean SlugsColosius confusus, n. sp., A Newly Recognized Pest of Cultivated Flowers and Coffee from Colombia, Ecuador and Peru, andColosius pulcher(Colosi, 1921) (Gastropoda, Veronicellidae)

In this study, we propose C. confusus, new species, an Andean slug of the genus Colosius Thomé, 1975, and a newly recognized pest of coffee and cultivated flowers from Colombia, Ecuador and Peru. We compare it with C. pulcher (Colosi, 1921), a poorly known species with which it has been confused. Our study is based on morphological analysis of a large number of specimens, including interceptions on cut-flowers and live plants by federal agricultural inspectors of the United States Department of Agriculture (USDA) and the Department of Homeland Security (DHS), and material from eight museum collections. Genetic diversity within C. confusus, n. sp. and C. pulcher is also analysed based on fragments of cytochrome oxidase I (COI), and 16S rRNA. They are differentiated by reproductive characters and genes studied. In C. confusus, n. sp., the phallus has a deep longitudinal groove from the base, near the retractor muscle, to its distal region, close to the papilla. In C. pulcher, there is an oval to rectangular swelling on the basal region of the phallus. Some important differences between these species are also found in the digitiform gland and bursa copulatrix. We describe, illustrate and discuss the color variation, morphological similarities, diagnostic characters and its variation, habitat and distribution for each species. Genetic diversity within C. confusus, n. sp., and C. pulcher is low. In order to analyze their relationship with C. propinquus (Colosi, 1921) (currently a junior synonym of C. pulcher) and C. lugubris (Colosi, 1921) (type-species of Colosius), fragments of COI, 16S, and 28S rRNA genes are also analyzed in a sample of these species. C. confusus, n. sp., is a distinct lineage within the genus Colosius. It is not a sister species of C. pulcher, which has C. propinquus as a sister species, here recognized as valid. Colosius confusus , n. sp., is closer to the clade that includes C. pulcher and C. propinquus than it is to C. lugubris. Based on the phylogenetic reconstruction, C. lugubris is sister to all the other Colosius, although additional studies are required to formally test phylogenetic placements and monophyly of the genus. Associated imports and number of interceptions per year of C. confusus, n. sp., by agricultural inspectors are also presented.

Low Occurrence ofHelicobacterDNA in Tropical Wild Birds, Venezuela

Enteric Helicobacter species (Helicobacter pullorum, Helicobacter pametensis, Helicobacter canadensis, Helicobacter anseris, and Helicobacter brantae) have been found in birds from temperate latitudes. We evaluated the occurrence of Helicobacter spp. in terrestrial wild birds from Venezuela. A fragment of 16S rRNA gene was amplified by PCR with Helicobacter genus-specific primers. Helicobacter spp. were detected in four of 80 fecal and in three of 42 intestinal tissue samples. Analyses of 16S and 23S rRNA gene sequences confirm for the first time the presence of Helicobacter in tropical terrestrial wild birds. However, the occurrence of Helicobacter was low, suggesting these bacteria may be uncommon in the populations we studied.

Molecular, morphological and osteological differentiation of a new species of microhylid frog of the genus Stumpffia from northwestern Madagascar.

We describe a new small-sized microhylid frog of the genus Stumpffia and provide a preliminary survey of Stumpffia diversity in northern and northwestern Madagascar based on molecular and morphological data. Analysis of 68 previously published and 142 newly generated DNA sequences of a fragment of the mitochondrial 16S rRNA gene revealed a large genetic diversity within Stumpffia, with numerous strongly differentiated lineages occurring across Madagascar. The anal. ysis confirmed a clade formed exclusively by Stumpffia species occurring in north/northwestern Madagascar and contain. ing almost all species from this area. Based on DNA sequences of the RAG1 gene, we further assess that most of the deep mitochondrial lineages do not share haplotypes in this nuclear marker, even in the few cases of sympatry, which we interpret as confirmation of their status as independent evolutionary lineages. Micro-computed tomography revealed subtle differences in phalangeal formulae among some of these candidate species, providing additional taxonomic characters for future revisions of the genus. The newly described species Stumpffia analamaina sp. nov. occurs 27 km north-west of Antsohihy and is at present the southernmost confirmed Stumpffia of the north/northwestern clade. It is mainly characterized by a snout-vent length of 10-12 mm, advertisement calls consisting of a series of short melodious chirps, manus with four fingers and pes with five toes, with only slight length reduction of first finger and toe. It furthermore does not share haplotypes with any other Stumpffia species in 16S or RAG1, and has relatively longer hands and feet compared with other small-sized Stumpffia of the north/northwestern clade such as S. madagascariensis and S. pygmaea. The new species lives in an area of dry forest where it was found in leaf litter along a small stream bordered by degraded gallery vegetation. Due to the limited distributions of almost all species in the north/northwestern Stumpffia clade, the rareness of appropriate microhabitat around the type locality of S. analamaina sp. nov. and the rampant habitat destruction in this part of Madagascar, we propose a threat status of Critically Endangered for this new species.

Ecological Inferences from a deep screening of the Complex Bacterial Consortia associated with the coral, Porites astreoides

The functional role of the bacterial organisms in the reef ecosystem and their contribution to the coral well-being remain largely unclear. The first step in addressing this gap of knowledge relies on in-depth characterization of the coral microbial community and its changes in diversity across coral species, space and time. In this study, we focused on the exploration of microbial community assemblages associated with an ecologically important Caribbean scleractinian coral, Porites astreoides, using Illumina high-throughput sequencing of the V5 fragment of 16S rRNA gene. We collected data from a large set of biological replicates, allowing us to detect patterns of geographical structure and resolve co-occurrence patterns using network analyses. The taxonomic analysis of the resolved diversity showed consistent and dominant presence of two OTUs affiliated with the order Oceanospirillales, which corroborates a specific pattern of bacterial association emerging for this coral species and for many other corals within the genus Porites. We argue that this specific association might indicate a symbiotic association with the adult coral partner. Furthermore, we identified a highly diverse rare bacterial 'biosphere' (725 OTUs) also living along with the dominant bacterial symbionts, but the assemblage of this biosphere is significantly structured along the geographical scale. We further discuss that some of these rare bacterial members show significant association with other members of the community reflecting the complexity of the networked consortia within the coral holobiont.

Isolation and identification of Mycoplasma agalactiae by culture and polymerase chain reaction (PCR) from sheep of Qom province, Iran

Contagious agalactia (C.A) is an infectious syndrome of sheep that is characterized by mastitis and subsequent failure of milk production, arthritis, abortion and keratoconjunctivitis. Mycoplasma agalactiae (M. agalactiae) is the main cause of the disease in sheep. The aim of this study was isolation and identification of M. agalactiae with culture and polymerase chain reaction (PCR) assay from sheep of Qom province in Iran. A total of 102 samples were collected from milk secretion, eye, ear and joint exudates of sheep. All samples were cultured in PPLO broth supplemented for M. agalaciae isolation. The bacteria DNAs were extracted by phenol/chloroform method and the PCR assay was applied for detecting of Mycoplasma genus in 163bp fragment of 16S rRNA gene and M. agalactiae in 375bp fragment of lipoprotein gene from culture as same as in clinical samples. Out of the 102 samples, 19(18.63%) cultures were shown positive and typical Mycoplasma colonies in PPLO agar culture diagnostic method and 59(57.8%) were scored positive by Mycoplasma genus PCR, 19(18.62%) of the samples were scored positive by using M. agalactiae PCR as diagnostic method. Out of the 102 samples, 19 samples were shown both positive in the culture and PCR, 42 samples were shown both negative in the culture and PCR. 40 samples were negative in the culture and positive in PCR whereas only one sample was positive in culture and negative in PCR. The results showed that the more isolations of M. agalactiae were taken from milk and less in joint samples. M. agalactiae was one of the main factors of contagious agalactia that was detected for the first time from sheep in Qom province.

Sequence analysis of mitochondrial 16S rRNA gene to identify meat species

In the present study, mitochondrial 16S rRNA gene sequence analysis was used for identification of Cattle (Bos taurus), buffalo (Bubalus bubalis), sheep (Ovis aries), goat (Capra hircus) and pig (Sus scrofa) species in fresh and processed meat. The DNA was extracted from fresh and processed meat including autoclaved meat and meat emulsion followed by polymerase chain reaction (PCR) amplification of about 497 bp DNA fragments of mitochondrial 16S rRNA gene. Then the amplified PCR fragments were sequenced and analysed to differentiate the species. No adverse effect of ingredients and processing conditions was observed on PCR amplification of DNA extracted from heat-treated meat and meat emulsion. The closely related species such as cattle and buffalo, sheep and goat were differentiated from each other by sequence analysis. Thus, PCR amplification and sequence analysis of mitochondrial 16S rRNA gene can be used as a tool for authentic identification of meat species.

Ehrlichiosis and Zoonotic Anaplasmosis in Suburban Areas of Beijing, China

In 2006, an unusual nosocomial outbreak of anaplasmosis occurred in Anhui Province, China. To follow these emerging tickborne-rickettsioses, a larger survey of Ehrlichia chaffeensis and Anaplasma phagocytophilum seroprevalence among farm worker populations, and the divergence of the partial 16S rRNA gene sequences of A. phagocytophilum among domestic animals, were conducted in Yanqing, Miyun, and Tongzhou Counties in Beijing from March to April, 2009. Blood samples from 562 farmers, 90 goats, 73 cattle, and 2 dogs were collected. IgG antibodies against E. chaffeensis and A. phagocytophilum were assayed by micro-indirect immunofluorescence assay (IFA). Partial fragments of 16S rRNA genes of A. phagocytophilum were amplified from blood DNA from domestic animals and their sequences analyzed. The total E. chaffeensis and A. phagocytophilum seroprevalence among the farm worker population was 16.4% and 14.1%, respectively. For domestic animals, the seropositive rates of A. phagocytophilum for goats, cattle, and dogs, were 2.3%, 0%, and 0%, respectively. The PCR-positive rates for A. phagocytophilum in goats and cattle were 48.9% and 23.9%, respectively. Three dominant genetic groups of Chinese A. phagocytophilum isolates were determined for goats and cattle, and these isolate varieties were broadly identified in China, Japan, and Korea. The prevalence of E. chaffeensis and A. phagocytophilum among farmers and domestic animals in Beijing rural areas was also demonstrated. The diagnoses and differential diagnoses of these emerging infectious diseases should be emphasized in clinics, and further ecological investigation of E. chaffeensis and A. phagocytophilum vectors and hosts is needed.

Environmental bacteria and childhood asthma

We have previously found an inverse association of bacterial diversity with childhood asthma. It remains unclear whether certain bacteria account for the protective effect. The high variability of the bacterial 16S rRNA gene allows assessing diversity and specificity of bacterial communities by single-strand configuration polymorphism (SSCP). DNA was extracted from mattress dust samples of 489 school-age children from rural and suburban regions in Germany. A fragment of the bacteria-specific 16S rRNA gene was amplified by PCR, digested to single-strand DNA, and subjected to electrophoresis. The resulting band patterns reflect the underlying DNA sequences. The individual bands were tested for associations with asthma, hay fever, and atopy in quantitative and qualitative multivariable analyses. Significantly associated bands were isolated and sequenced. The sequences were compared to a database, and distinct bacteria were identified. Seven of 76 independent bands were found to be inversely associated with asthma, atopic sensitization, and hay fever with odds ratios ranging from 0.17 to 0.73. The bands contained the sequences of Acinetobacter sp., Lactobacillus spp., Neisseria spp., Staphylococcus sciuri, Jeotgalicoccus sp., Corynebacterium spp., and others. In a diverse microbial environment, certain bacteria may account for the protective effect on the development of asthma and atopy.

Molecular detection of tick-borne bacterial agents in Brazilian and exotic captive carnivores

The present study aims to detect and characterize by molecular techniques, the presence of tick-borne pathogens in wild captive carnivore blood samples from Brazil. Blood was collected from 76 Brazilian felids, 23 exotic felids, 3 European wolves (Canis lupus), and 97 Brazilian canids maintained in captivity in zoos located in São Paulo and Mato Grosso states, Brazil. DNA of each sample was used in PCR reactions for Ehrlichia, Anaplasma, and Rickettsia identification. The blood from 10/100 (10%) of canids (1 European wolf, 3 bush dogs, and 6 crab-eating foxes) and from 21/99 (21%) felids (4 pumas, 6 little spotted cats, 4 ocelots, 3 jaguarundis, 1 tiger, and 3 lions) contained fragments of 16S rRNA gene of Ehrlichia spp. Fragments of Anaplasma spp. groESL and 16S rRNA genes were detected in the blood of 1/100 (1%) canids (1 bush dog) and in 4/99 (3%) felids (4 little spotted cats), respectively. Rickettsia species infections were not identified. The present work showed that new strains of Ehrlichia and Anaplasma spp. circulate among wild carnivores in Brazil.

Detection and Identification of 16SrII Group Phytoplasmas Infecting Stylosanthes in China

AbstractIn July 2008, Stylosanthes guianensis with witches’‐broom symptoms and Stylosanthes capitata with yellows symptoms were observed in Hainan Province, China. From the symptomatic S. guianensis, the phytoplasma‐specific 16S rRNA gene fragment of 1.4 kb was amplified by a direct PCR, and a fragment of 16S rRNA gene of 1.2 kb was obtained from the symptomatic Stylosanthes capitata by a nested PCR. Sequencing results and NCBI BLASTn analysis showed that the two phytoplasmas belonged to group 16SrII. Polygenetic analysis and virtual RFLP classified the two phytoplasmas into subgroup 16SrII‐A.