Publications

4618

A novel small haemoplasma was detected following cytological examination of blood smears from a splenectomized dog with haemic neoplasia. The 16S rRNA and rnpB genes of the organism were partially sequenced and a phylogenetic tree constructed. The organism was most closely related to the small feline haemoplasma, ‘Candidatus Mycoplasma haemominutum’ (94 % 16S rRNA gene nucleotide sequence identity; 75 % rnpB) and was only distantly related to Mycoplasma haemocanis (78 % 16S rRNA gene nucleotide sequence identity; 65 % rnpB). As this organism has not been cultured in vitro, the candidate species name ‘Candidatus Mycoplasma haematoparvum’ is proposed.

Huanglongbing (ex-greening) disease is one of the most serious diseases of citrus. It is caused by the phloem-limited, gram-negative bacterium “Candidatus Liberibacter spp.”. This bacterium is not well characterized mainly because it is still uncultured. There are two known strains, Asian (“Candidatus Liberibacter asiaticus”) and African (“Candidatus Liberibacter africanus”) that cause severe damage to citrus plants including twig dieback, decline, and death. Symptoms first appear as leaf mottling and chlorosis occurring in one shoot or sector of trees. Later, leaf symptoms resemble nutritional deficiencies (Zn, Ca, and N) that vary depending on the strains, with more severe symptoms caused by “Ca. L. asiaticus”. The Asian strains are transmitted by the Asian citrus psyllid (AsCP), Diaphorina citri, which is present in Brazil. The bacterium has been detected in citrus plants in many geographic locations including China, Japan, Thailand, India, the Philippines, the Arabian Peninsula, and Africa. In 2004, plants showing Huanglongbing symptoms were observed in the Araraquara County, a central region of the State of Sao Paulo, the largest citrus-producing area in Brazil. To verify the presence of “Ca. L. spp.” in these plants, leaf samples of sweet orange cvs. Hamlin and Valencia were used for DNA extraction and polymerase chain reaction amplification using the specific OI1 and Oi2c primers (1). Amplification of the 16S rDNA was positive for 2 (cvs. Hamlin and Valencia) of 10 analyzed plants. The amplified fragments were cloned and sequenced. The amplicons obtained from both plants showed the same sequence, which differed from “Ca. L. africanus”, utilized as the positive control in the amplification experiment (27 divergent bases in 1,160). The sequences were used for BLAST searches, and the results showed identities ranging from 94.71 to 100% with “Ca. L. spp.” sequences available at the National Center for Biotechnology Information database (on-line publication). The highest scores were obtained with “Ca. L. asiaticus sequences. These analyses confirmed the presence of such agent in the State of Sao Paulo. To our knowledge, this is the first report of “Ca. L. asiaticus” in Brazil as well as elsewhere in the Americas. The significance of this report relates to the potential damage that this pathogen could cause to the citrus industry in the largest citrus-producing country in the world. It remains unclear how and when the pathogen entered Brazil. Reference: (1) S. Jagoueix et al. Mol. Cell Probes 10:43, 1996.

Summary The X‐bacteria which initiated organismic association with the D strain of Amoeba proteus in 1966 as parasites have changed to obligate endosymbionts on which the host depends for survival. Owing to the difficulty in cultivating the bacteria in vitro , the identity of X‐bacteria has not been determined. As the life cycle of X‐bacteria is similar to that of Legionella spp. in soil amoebae, we applied the polymerase chain reaction method with specific primers aimed at Legionella spp. for the detection and cloning of 16S rRNA gene. The identity and intracellular localization of the endosymbiont were confirmed by the application of a specific fluorescently labelled 16S rRNA‐targeted probe. In addition we cloned RNA polymerase β‐subunit gene ( rpoB ) of X‐bacteria by genomic library tagging. A phylogenetic analysis of the 16S rRNA gene placed the bacterium within a unique monophyletic group containing all other members of the genus Legionella . Phylogeny from rpoB and mip genes further confirmed the taxonomic context of X‐bacteria to be a Legionella sp. In all three phylogenic analyses, X‐bacterium was placed apart from Legionella ‐like amoebal pathogens present in soil amoebae. Thus, we propose the name ‘ Candidatus Legionella jeonii’ sp. nov. for the endosymbiotic X‐bacteria in Amoeba proteus .

While Helicobacter pylori is accepted as the major bacterial agent of gastric disease in humans, some patients and many animals are infected with a larger, tightly helical-shaped bacterium previously referred to as ‘Helicobacter heilmannii’ or ‘Gastrospirillum hominis’. Taxonomic classification of these bacteria has been hampered by the inability to cultivate them in vitro and by the inadequate discriminatory power of 16S rRNA gene sequence analysis. This study describes the detection and phylogenetic analysis of 26 different gastrospirillum isolates from humans and animals, which incorporates sequence data based on the 16S rRNA and urease genes. Fifteen gastrospirilla detected in humans, primates and pigs clustered with ‘Candidatus Helicobacter suis’, thus expanding the host range for this organism. By comparison, based on 16S rRNA data, the remaining 11 gastrospirilla could not be differentiated from Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis. However, urease gene sequence analysis allowed for the discrimination of this latter group into four discrete clusters, three of which contained the above recognized species. The fourth cluster contained isolates from human and feline hosts, and should provisionally be considered a unique bacterial species, for which the name ‘Candidatus Helicobacter heilmannii’ is proposed.

ABSTRACT To characterize intracellular gram-negative bacteria associated with epitheliocystis in farmed Atlantic salmon ( Salmo salar ), gills with proliferative lesions were collected for histopathology, conventional transmission and immunoelectron microscopy, in situ hybridization, and DNA extraction during epitheliocystis outbreaks in Ireland and Norway in 1999 and 2000, respectively, and compared by ultrastructure and immunoreactivity to nonproliferative gills from Ireland archived in 1995. Genomic DNA from proliferative gills was used to amplify 16S ribosomal DNA (rDNA) for molecular phylogenetic analyses. Epitheliocystis inclusions from proliferative gills possessed variably elongate reticulate bodies, examples of binary fission, and vacuolated and nonvacuolated intermediate bodies, whereas inclusions in nonproliferative gills had typical chlamydial developmental stages plus distinctive head-and-tail cells. Immunogold processing using anti-chlamydial lipopolysaccharide antibody labeled reticulate bodies from proliferative and nonproliferative gills. 16S rDNA amplified directly from Irish (1999) and Norwegian (2000) gill samples demonstrated 99% nucleotide identity, and riboprobes transcribed from cloned near-full-length 16S rDNA amplicons from Norwegian gills hybridized with inclusions in proliferative lesions from Irish (1999) and Norwegian (2000) sections. A 1,487-bp consensus 16S rRNA gene sequence representing the chlamydia-like bacterium (CLB) from proliferative gills had the highest percent nucleotide identity with endosymbionts of Acanthamoeba spp. (order Chlamydiales ). Molecular phylogenetic relationships inferred from 16S rRNA gene sequences using distance and parsimony indicated that the CLB from proliferative gills branched with members of the order Chlamydiales . “ Candidatus Piscichlamydia salmonis” is proposed for the CLB associated with epitheliocystis from proliferative gills of Atlantic salmon, which exhibits developmental stages different from those identified in nonproliferative gills.

Phylogenetic 16S rRNA gene analysis was used to assign the bacterial leaf-nodulating endosymbionts of two tropical African Psychotria species to the genus Burkholderia. The microsymbionts of the different Psychotria hosts were recognized as distinct and novel species of Burkholderia on the basis of relatively low intersequence similarities and sufficiently large evolutionary distances when compared with each other and their closest validly named neighbours. The obligate endosymbiotic nature of the bacteria prevented their in vitro cultivation and the deposition of type strains to culture collections. Therefore, the provisional status Candidatus is assigned to the bacterial partners of Psychotria calva and Psychotria nigropunctata, with the proposal of the names ‘Candidatus Burkholderia calva’ and ‘Candidatus Burkholderia nigropunctata’, respectively.

ABSTRACT “ Candidatus Glomeribacter gigasporarum” is an endocellular β-proteobacterium present in the arbuscular mycorrhizal (AM) fungus Gigaspora margarita . We established a protocol to isolate “ Ca . Glomeribacter gigasporarum” from its host which allowed us to carry out morphological, physiological, and genomic investigations on purified bacteria. They are rod shaped, with a cell wall typical of gram-negative bacteria and a cytoplasm rich in ribosomes, and they present no flagella or pili. Isolated bacteria could not be grown in any of the 19 culture media tested, but they could be kept alive for up to 4 weeks. PCR-based investigations of purified DNA from isolated bacteria did not confirm the presence of all genes previously assigned to “ Ca . Glomeribacter gigasporarum.” In particular, the presence of nif genes could not be detected. Pulsed-field gel electrophoresis analyses allowed us to estimate the genome size of “ Ca . Glomeribacter gigasporarum” to approximately 1.4 Mb with a ca. 750-kb chromosome and a 600- to 650-kb plasmid. This is the smallest genome known for a β-proteobacterium. Such small genome sizes are typically found in endocellular bacteria living permanently in their host. Altogether, our data suggest that “ Ca . Glomeribacter gigasporarum” is an ancient obligate endocellular bacterium of the AM fungus G. margarita .