Publications

4490

ABSTRACT Laser capture microdissection in combination with fluorescent in situ hybridization was used to identify an unknown species of spirochetes from the pig colonic mucosa. The 16S rRNA gene was PCR amplified, and the closest related type strain was Treponema bryantii T (90.1%). The spirochete, here named “ Candidatus Treponema suis, ” was associated with colitis, including invasion of the surface epithelium as well as superficial parts of the mucosa.

ABSTRACTTwo hemotropic mycoplasmas have been recognized in cats,Mycoplasma haemofelisand “CandidatusMycoplasma haemominutum.” We recently described a third feline hemoplasma species, designated “CandidatusMycoplasma turicensis,” in a Swiss cat with hemolytic anemia. This isolate induced anemia after experimental transmission to two specific-pathogen-free cats and analysis of the 16S rRNA gene revealed its close relationship to rodent hemotropic mycoplasmas. The agent was recently shown to be prevalent in Swiss pet cats. We sought to investigate the presence and clinical importance of “CandidatusMycoplasma turicensis” infection in pet cats outside of Switzerland and to perform the molecular characterization of isolates from different countries. A “CandidatusMycoplasma turicensis”-specific real-time PCR assay was applied to blood samples from 426 United Kingdom (UK), 147 Australian, and 69 South African pet cats. The 16S rRNA genes of isolates from different countries were sequenced and signalment and laboratory data for the cats were evaluated for associations with “CandidatusMycoplasma turicensis” infection. Infections were detected in samples from UK, Australian, and South African pet cats. Infection was associated with the male gender, and “CandidatusMycoplasma haemominutum” andM. haemofeliscoinfection. Coinfected cats exhibited significantly lower packed cell volume (PCV) values than uninfected cats. Phylogenetic analyses revealed that some Australian and South African “CandidatusMycoplasma turicensis” isolates branched away from the remaining isolates. In summary, “CandidatusMycoplasma turicensis” infection in pet cats exists over a wide geographical area and significantly decreased PCV values are observed in cats coinfected with other feline hemoplasmas.

ABSTRACTTo survive in host cells, intracellular pathogens or symbiotic bacteria require protective mechanisms to overcome the oxidative stress generated by phagocytic activities of the host. By genomic library tagging, we cloned adps(stands forDNA-bindingprotein fromstarved cells) gene of the symbiotic “CandidatusLegionella jeonii” organism (called the X bacterium) (dpsX) that grows inAmoeba proteus. The gene encodes a 17-kDa protein (pI 5.19) with 91% homology to Dps and DNA-binding ferritin-like proteins of other organisms. The cloned gene complemented thedpsmutant ofEscherichia coliand conferred resistance to hydrogen peroxide. DpsXproteins purified fromE. colitransformed with thedpsXgene were in oligomeric form, formed a complex with pBlueskript SKII DNA, and protected the DNA from DNase I digestion and H2O2-mediated damage. The expression of thedpsXgene in “CandidatusLegionella jeonii” was enhanced when the host amoeba was treated with 2 mM H2O2and by phagocytic activities of the host cell. These results suggested that the Dps protein has a function protective of the bacterial DNA and that its gene expression responds to oxidative stress generated by phagocytic activities of the host cell. With regard to the fact that invasion ofLegionellasp. into respiratory phagocytic cells causes pneumonia in mammals, further characterization ofdpsXexpression in theLegionellasp. that multiplies in a protozoan host in the natural environment may provide valuable information toward understanding the protective mechanisms of intracellular pathogens.

Two Gram-positive bacilli, designated as strains SMC 4352-1T and SMC 4352-2T, were isolated sequentially from the blood of a newborn child with sepsis. They could not be identified by using conventional clinical microbiological methods. 16S rRNA gene sequencing and phylogenetic analysis revealed that both strains belonged to the genus Bacillus but clearly diverged from known Bacillus species. Strain SMC 4352-1T and strain SMC 4352-2T were found to be closely related to Bacillus firmus NCIMB 9366T (98.2 % sequence similarity) and Bacillus cibi JG-30T (97.1 % sequence similarity), respectively. They also displayed low DNA–DNA reassociation values (less than 40 %) with respect to the most closely related Bacillus species. On the basis of their polyphasic characteristics, strain SMC 4352-1T and strain SMC 4352-2T represent two novel species of the genus Bacillus, for which the names Bacillus infantis sp. nov. (type strain SMC 4352-1T=KCCM 90025T=JCM 13438T) and Bacillus idriensis sp. nov. (type strain SMC 4352-2T=KCCM 90024T=JCM 13437T) are proposed.

An intracellular bacterium with the unique ability to enter mitochondria exists in the European vector of Lyme disease, the hard tick Ixodes ricinus. Previous phylogenetic analyses based on 16S rRNA gene sequences suggested that the bacterium formed a divergent lineage within the Rickettsiales (Alphaproteobacteria). Here, we present additional phylogenetic evidence, based on the gyrB gene sequence, that confirms the phylogenetic position of the bacterium. Based on these data, as well as electron microscopy (EM), in situ hybridization and other observations, we propose the name ‘Candidatus Midichloria mitochondrii’ for this bacterium. The symbiont appears to be ubiquitous in females of I. ricinus across the tick's distribution, while lower prevalence is observed in males (44 %). Based on EM and in situ hybridization studies, the presence of ‘Candidatus M. mitochondrii’ in females appears to be restricted to ovarian cells. The bacterium was found to be localized both in the cytoplasm and in the intermembrane space of the mitochondria of ovarian cells. ‘Candidatus M. mitochondrii’ is the first bacterium to be identified that resides within animal mitochondria.

Conventional PCR and two real‐time PCR (RTi‐PCR) methods were developed and compared using the primer pairs CQULA03F/CQULA03R and CQULA04F/CQULA04R, and TaqMan probe CQULAP1 designed from a species‐specific sequence of the rplJ/rplL ribosomal protein gene, for diagnosis of citrus huanglongbing (HLB) disease in southern China. The specificity and sensitivity of the three protocols for detecting ‘ Candidatus Liberibacter asiaticus’ in total DNA extracts of midribs collected from infected citrus leaves with symptoms in Guangxi municipality, Jiangxi Province and Zhejiang Province, were tested. Sensitivities using extracted total DNA (measured as copy number, CN per µ L of recombinant plasmid solution) were 439·0 (1·30 × 10 5 CN µ L −1 ), 4·39 (1·30 × 10 3 CN µ L −1 ) and 0·44 fg µ L −1 (1·30 × 10 2 CN µ L −1 ) for conventional PCR, TaqMan and SYBR Green I (SGI) RTi‐PCR, respectively. SGI RTi‐PCR was the most sensitive, but its specificity needed to be confirmed by running a melt‐curve assay. The TaqMan RTi‐PCR assay was rapid and had the greatest specificity. Concerning the correlation of PCR detection results with the various HLB symptoms, uneven mottling of leaves had the highest positive rate (96·50%), indicating that leaf mottling was the most reliable symptom for field surveys. Dynamic analysis results from the TaqMan assays showed that the titre (CN) g −1 citrus tissue of ‘ Ca. L. asiaticus’ was highest between October and December (threshold cycle ( C t ) average = 29·3, CN = 3·35 × 10 7 ) and lowest between March and May ( C t average = 32·0, CN = 5·10 × 10 6 ) in 2004 and 2005. The optimized molecular‐based assays should prove useful for presymptom diagnosis of HLB disease, monitoring and identification of ‘ Ca. L. asiaticus’, and field epidemic regulation.

Huanglongbing (greening) disease caused by a nonculturable, phloem-limited bacterium is a severe disease of citrus. On the basis of the influence of temperature on host symptoms and the causal agent, this disease can be categorized as Asian caused by “Candidatus Liberibacter asiaticus”, African caused by “Ca. L. africanus”, and American caused by “Ca. L. americanus”. Kumquat (Fortunella margarita (Lour.) Swingle), a member of the Rutaceae is an economically important crop for export and local consumption in Taiwan. Recently, a Huanglongbing-like disease was found on kumquat in Yilan County, the largest kumquat-producing area in northeastern Taiwan. Even though the disease has been reported on Citrus spp. from Taiwan, it has never been reported on kumquat. Symptoms of infected kumquat were mottling, yellowing, hardening, and curling of leaves followed by premature defoliation, twig dieback, decay of feeder rootlets and lateral roots, and ultimately the death of the entire plant. Typical sieve-tube-restricted bacteria were observed in kumquat cells by electron microscopy (1). In addition, psyllid-transmission tests demonstrated that the Asian psyllid (Diaphorina citri) could transmit this bacterium to healthy kumquats. Positive bud graft transmissions were obtained to F. margarita, F. japonica (Thunb.) Swingle, F. obovata Hort. ex Tanaka, Luchen sweet orange (Citrus sinensis (L.) Osb.), and Wentan pummelo (C. maxima f. sp. butan Hay.). These inoculated plants showed symptoms in 3 to 8 months, and bacteria could be detected by polymerase chain reaction (PCR) using a common primer pair that amplified a 226-bp specific DNA fragment (2). For further molecular identification, the bacterial DNA was extracted from the inoculated plants and PCR was performed by using two sets of primers selected from the 16S rRNA region (GenBank Accession No. L22532) and 16S/23S intergenic spacer region (GenBank Accession No. AB019793). The expected DNA fragments of 1,389 bp and 862 bp were, respectively, amplified from symptomatic plants but not from healthy plants. The PCR products were cloned and sequenced (GenBank Accession Nos. DQ302750 and DQ207841). The 16S rRNA has 98 to 99% identity and 16S/23S intergenic spacer region has 99% identity to the corresponding region of “Ca. L. asiaticus” in GenBank. These molecular analyses confirm the presence of “Ca. L. asiaticus” in kumquat. Since Huanglongbing has been rarely reported naturally on kumquat, further analysis of this bacterium as a special strain of “Ca. L. asiaticus” is needed. References: (1) M. Garnier et al. Ann. Microbiol. 135A:169, 1984. (2) T. H. Hung et al. J. Phytopathol. 147:599, 1999.