Publications

4366

ABSTRACT “ Candidatus Chlorothrix halophila” is a recently described halophilic, filamentous, anoxygenic photoautotroph (J. A. Klappenbach and B. K. Pierson, Arch. Microbiol. 181: 17-25, 2004) that was enriched from the hypersaline microbial mats at Guerrero Negro, Mexico. Analysis of the photosynthetic apparatus by negative staining, spectroscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the photosynthetic apparatus in this organism has similarities to the photosynthetic apparatus in both the Chloroflexi and Chlorobi phyla of green photosynthetic bacteria. The chlorosomes were found to be ellipsoidal and of various sizes, characteristics that are comparable to characteristics of chlorosomes in other species of green photosynthetic bacteria. The absorption spectrum of whole cells was dominated by the chlorosome bacteriochlorophyll c (BChl c ) peak at 759 nm, with fluorescence emission at 760 nm. A second fluorescence emission band was observed at 870 nm and was tentatively attributed to a membrane-bound antenna complex. Fluorescence emission spectra obtained at 77 K revealed another complex that fluoresced at 820 nm, which probably resulted from the chlorosome baseplate complex. All of these results suggest that BChl c is present in the chlorosomes of “ Ca . Chlorothrix halophila,” that BChl a is present in the baseplate, and that there is a membrane-bound antenna complex. Analysis of the proteins in the chlorosomes revealed an ∼6-kDa band, which was found to be related to the BChl c binding protein CsmA found in other green bacteria. Overall, the absorbance and fluorescence spectra of “ Ca . Chlorothrix halophila” revealed an interesting mixture of photosynthetic characteristics that seemed to have properties similar to properties of both phyla of green bacteria when they were compared to the photosynthetic characteristics of Chlorobium tepidum and Chloroflexus aurantiacus .

Although Mycoplasma haemofelis and "Candidatus Mycoplasma haemominutum" infections have been reported in wild cats from United States, their presence among native and captive wild cats in Brazil is still unknown. A 12 year old healthy male lion (Panthera leo) from the Zoological Garden of Curitiba, Brazil was anesthetized for transportation and dental evaluation. A blood sample was obtained for a complete blood cell count (CBC) and PCR analysis. DNA was extracted and fragments of Mycoplasma haemofelis and "Candidatus Mycoplasma haemominutum" 16S ribosomal RNA gene were amplified in PCR assays. CBC results were within reference intervals. A weak band of 192 pb for "Candidatus Mycoplasma haemominutum" was observed, and no band was amplified from Mycoplasma haemofelis reaction. A weak PCR band associated with normal CBC results and without visible parasitemia or clinical signs may suggest a chronic subclinical infection with "Candidatus Mycoplasma haemominutum". The lack of clinical signs may also represent the low pathogenicity of this organism; however, it is noteworthy that immune suppression caused by management and/or corticoids treatment may induce parasitemia and anemia in this animal. This detection suggests further studies in captive wild cats in Brazilian Zoological Gardens.

Huanglongbing (HLB), also known as greening, is one of the most important diseases of citrus worldwide. The causal agent is a gram-negative bacterium known to inhabit the phloem of infected plants. Three different candidate species infect citrus: ‘Candidatus Liberibacter africanus’ found in the African continent; ‘Ca. L. asiaticus’ found in Asia, Brazil, and the United States; and ‘Ca. L. americanus’ found in Brazil. (1). Tobacco is an easily transformable plant species that can be used as an experimental host system to quickly screen for candidate genes useful to control plant pathogens. However, no evidence exists on the ability of this plant species to sustain populations of ‘Ca. L. americanus’. With the purpose of transmitting ‘Ca. L. americanus’ from citrus to tobacco, fragments of healthy stems of Cuscuta spp. (dodder) were used to connect an HLB-infected sweet orange plant to each of 10 healthy plants of Nicotiana tabacum L. cv. Xanthi and allowed to remain connected for 30, 45, and 50 days. Three different HLB-infected orange plants and 30 tobacco plants were used in three independent experiments. Most HLB-exposed Xanthi plants exhibited chlorotic leaves after 50 days of exposure probably because of the parasitic effect of dodder; however, an average of 6, 1, and 3 Xanthi plants exhibited a unique blotchy mottle symptom after 30, 45, and 50 days of exposure, respectively. Symptomatic and asymptomatic leaves were collected and analyzed by PCR. The results consistently confirmed the presence of ‘Ca. L. americanus’ only in symptomatic leaves. Sequencing of the PCR product and comparison to the NCBI database also confirmed the identity of the pathogen as ‘Ca. L. americanus’. Electron microscopy analysis of four symptomatic leaves indicated the presence of bacterium-like bodies with round to elongated bacilliform shapes and surrounded by two membranes. These bodies resembled those already described in HLB-infected citrus in Brazil (1). The evidence presented above confirms the successful transmission of ‘Ca. L. americanus’ from citrus to Xanthi using the parasitic plant Cuscuta spp. Reference: (1) F. A. O. Tanaka et al. Fitopatol. Bras. 31:99, 2006.

Filamentous bacteria frequently occurring in the pelagic zone of natural freshwater lakes and ponds were previously identified as being related to Haliscomenobacter hydrossis based upon their 16S rRNA gene sequences. These bacteria exhibit a specific morphology characterized by the formation of straight, stick-like filaments of variable length (5 to >100 μm) and quite stable, but narrow, width (0.25 to 0.35 μm). Bacteria with these morphological characteristics form a monophyletic but broad phylogenetic group with a maximal divergence of 16S rRNA gene sequences of 12.0 %. This monophyletic group consists of at least three monophyletic subclusters. H. hydrossis is affiliated to one of these subclusters and represents the sole recognized species affiliated to the broad monophyletic group. ‘Candidatus Haliscomenobacter calcifugiens' and ‘Candidatus Aquirestis calciphila’ are uncultured representatives of the other two subclusters and have 16S rRNA gene sequence dissimilarities of 5.4 % and 8.2 %, respectively, with the type strain of H. hydrossis. ‘Candidatus H. calcifugiens' and ‘Candidatus A. calciphila’ have a 16S rRNA gene sequence dissimilarity of 8.5 %. These large ribosomal divergences justify the classification of these environmentally important bacteria as a novel species and a new genus, respectively. Intensive attempts to cultivate these filamentous bacteria have resulted in the establishment of mixed cultures, however, attempts to establish pure cultures have failed.

Six strains (CB7T, CB18, CB23, CB26, CB28 and CB35T) were isolated from human faeces. Based on phylogenetic analysis, phenotypic characteristics, cellular fatty acid profiles and menaquinone profiles, these strains could be included within the genusPrevotellaand made up two clusters. 16S rRNA gene sequence analysis indicated that five strains were most closely related toPrevotella veroralis, sharing about 92 % sequence similarity; the remaining strain was most closely related toPrevotella shahii, sharing about 90 % sequence similarity. All six strains were obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative rods. The cellular fatty acid compositions of the six strains differed significantly from those of otherPrevotellaspecies. Five strains (CB7T, CB18, CB23, CB26 and CB28) contained dimethyl acetals and the major menaquinones of these strains were MK-11, MK-12 and MK-13. The major menaquinones of CB35Twere MK-12 and MK-13. Based on phenotypic and phylogenetic findings, two novel species,Prevotella coprisp. nov. andPrevotella stercoreasp. nov., are proposed, representing the two different strain clusters. The DNA G+C contents of strains CB7Tand CB35Twere 45.3 and 48.2 mol%, respectively. The type strains ofP. copriandP. stercoreaare CB7T(=JCM 13464T=DSM 18205T) and CB35T(=JCM 13469T=DSM 18206T), respectively.

The objective of this study was to evaluate the use of real-time TaqMan PCR assays for detection of coinfections with “ Candidatus Mycoplasma haemominutum” (Mhm), and Mycoplasma haemofelis (Mhf), in vitro and over time in experimentally infected cats. First, the ability of each real-time PCR assay to detect and quantify mixed infections was determined in vitro by testing mixtures of plasmids containing Mhm and Mhf 16S rDNA with each assay. Subsequently, 4 specific pathogen-free (SPF) cats, 2 of which were splenectomized, were inoculated with blood from a cat infected with both Mhm and Mhf. Sixteen blood samples were then collected from each cat over a 55-day period. Each of the 64 postinoculation samples was tested using both conventional polymerase chain reaction (cPCR) and real-time PCR for the 16S rRNA gene of each organism. When applied to mixtures of plasmid DNA from each species, the results of quantitation with each of the real-time PCR assays approximately reflected the number of plasmid copies present. Forty-nine of 64 post-inoculation samples (77%) were positive using both cPCR and real-time PCR, 4 (6%) were positive using cPCR only, and 3 (5%) were positive using real-time PCR only. Both organisms were detected in 23 samples using real-time PCR. Mixed infections were not detected using cPCR. The size of the corresponding cPCR products suggested infection with Mhm in 4 and Mhf in 18 of these samples. The use of multiple separate real-time PCR assays rather than cPCR alone should thus be considered for epidemiologic studies of hemoplasmosis in cats.