Spotted Fever Group <Emphasis Type="Italic">Rickettsiae</Emphasis> in Ticks in Cyprus

In two surveys conducted from March 1999 to March 2001 and from January 2004 to December 2006, a total of 3,950 ticks (belonging to ten different species) were collected from seven domestic and wild animals (goat, sheep, cattle, dog, fox, hare, and mouflon) from different localities throughout Cyprus. In order to establish their infection rate with Spotted Fever Rickettsiae (SFG), ticks were pooled and tested by polymerase chain reaction targeting gltA and ompA genes, followed by sequencing analysis. When tick pools tested positive, individual ticks were then tested one by one, and of the 3,950 ticks screened, rickettsial DNA was identified in 315 ticks (infection rate, 8%). Five SFG Rickettsiae were identified: Rickettsia aeschlimannii in Hyalomma marginatum marginatum, Rickettsia massiliae in Rhipicephalus turanicus and Rhipicephalus sanguineus, Rickettsia sibirica mongolotimonae in Hyalomma anatolicum excavatum, and a Rickettsia endosymbiont of Haemaphysalis sulcata (later described as Rickettsia hoogstraalii) in Haemaphysalis punctata. Two additional genes, 17 kDa and ompB, were targeted to characterize a new genotype of “Candidatus Rickettsia barbariae” genotype in R. turanicus, designated here as “Candidatus Rickettsia barbariae” Cretocypriensis. These results confirm the presence of a spectrum of SFG Rickettsiae on the island. Further studies are necessary to gain better knowledge on the epidemiology of SFG Rickettsiae in Cyprus.     

Associations Between Coinfection Prevalence of <Emphasis Type="Italic">Borrelia lusitaniae</Emphasis>, <Emphasis Type="Italic">Anaplasma</Emphasis> sp., and <Emphasis Type="Italic">Rickettsia</Emphasis> sp. in Hard Ticks Feeding on Reptile Hosts

An increasing number of studies reveal that ticks and their hosts are infected with multiple pathogens, suggesting that coinfection might be frequent for both vectors and wild reservoir hosts. Whereas the examination of associations between coinfecting pathogen agents in natural host–vector–pathogen systems is a prerequisite for a better understanding of disease maintenance and transmission, the associations between pathogens within vectors or hosts are seldom explicitly examined. We examined the prevalence of pathogen agents and the patterns of associations between them under natural conditions, using a previously unexamined host–vector–pathogen system—green lizards Lacerta viridis, hard ticks Ixodes ricinus, and Borrelia, Anaplasma, and Rickettsia pathogens. We found that immature ticks infesting a temperate lizard species in Central Europe were infected with multiple pathogens. Considering I. ricinus nymphs and larvae, the prevalence of Anaplasma, Borrelia, and Rickettsia was 13.1% and 8.7%, 12.8% and 1.3%, and 4.5% and 2.7%, respectively. The patterns of pathogen prevalence and observed coinfection rates suggest that the risk of tick infection with one pathogen is not independent of other pathogens. Our results indicate that Anaplasma can play a role in suppressing the transmission of Borrelia to tick vectors. Overall, however, positive effects of Borrelia on Anaplasma seem to prevail as judged by higher-than-expected Borrelia–Anaplasma coinfection rates.     

Discrimination between <Emphasis Type="Italic">Haemaphysalis longicornis</Emphasis> and <Emphasis Type="Italic">H. qinghaiensis</Emphasis> based on the partial 16S rDNA and the second internal transcribed spacer (ITS-2)

In the present study, two hard tick species, Haemaphysalis longicornis and H. qinghaiensis from North-western China were characterized genetically by the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA and partial 16S rDNA. Based on a fragment within the hypervariable region of 16S rDNA with the length of approximately 453 bp, the phylogenetic trees were constructed by Neighbor-Joining and Maximum-parsimony methods. The results indicated that the phylogenetic status of H. qinghaiensis was distant from that of H. longicornis and closer to H. flava. Furthermore, the ITS-2 rDNA was amplified by PCR and sequenced from individual ticks. The length of ITS-2 is 1,606 bp for H. longicornis and 1,162 bp for H. qinghaiensis. Although sequence variation between the immature stages of H. longicornis was 0.1–0.4%, nucleotide differences between the tested species ranged 2.1–23.2%, indicating that ITS-2 rDNA sequences are genetic markers for the differentiation of the two hard ticks in China. Hence, a PCR-linked restriction fragment length polymorphism (RFLP) approach was developed for their unequivocal differentiation based on ITS-2 rDNA, which provides the foundation for further studies on ticks in China and has implications for studying the population genetic structure of the ticks and for identification and differentiation of closely related ticks.     

Molecular analysis of <Emphasis Type="Italic">Ixodes granulatus</Emphasis>, a possible vector tick for <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato in Taiwan

The genetic identity of Ixodes granulatus ticks was determined for the first time in Taiwan. The phylogenetic relationships were analyzed by comparing the sequences of mitochondrial 16S ribosomal DNA gene obtained from 19 strains of ticks representing seven species of Ixodes and two outgroup species (Rhipicephalus sanguineus and Haemaphysalis inermis). Four major clades could be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. All these I. granulatus ticks of Taiwan were genetically affiliated to a monophyletic group with highly homogeneous sequences (92.2–99.3% similarity), and can be discriminated from other Ixodes species and other genera of ticks with a sequence divergence ranging from 11.7 to 30.8%. Moreover, intraspecific analysis revealed that two distinct lineages are evident between the same species of I. granulatus ticks collected from Taiwan and Malaysia. Our results demonstrate that all these I. granulatus ticks of Taiwan represent a unique lineage distinct from the common vector ticks (I. ricinus complex) for Borrelia burgdorferi spirochetes.     

Molecular detection of <Emphasis Type="Italic">Rickettsia</Emphasis>, <Emphasis Type="Italic">Coxiella</Emphasis> and <Emphasis Type="Italic">Rickettsiella</Emphasis> DNA in three native Australian tick species

Three Australian native animal species yielded 60 samples composed of three indigenous ticks. Hosts included twelve koalas, two echidnas and one wombat from Victoria, and ticks were of the species Ixodes tasmani (n = 42), Bothriocroton concolor (n = 8) and B. auruginans (n = 10), respectively. PCR screening and sequencing detected a species of Coxiella, sharing closest sequence identity to C. burnetii (>98%), in all B. auruginans, as well as a species of Rickettsia, matching closest to R. massiliae, in 70% of the same samples. A genotype sharing closest similarity to Rickettsia bellii (>99%) was identified in three female B. concolor collected from one of the echidnas. Three samples of I. tasmani, taken from three koalas, yielded different genotypes of Rickettsiella. These results represent the first detection of the three genera in each tick species and identify a high level of previously undetected bacterial diversity in Australian ticks.     

Molecular detection of <Emphasis Type="Italic">Ehrlichia ruminantium</Emphasis> infection in <Emphasis Type="Italic">Amblyomma variegatum</Emphasis> ticks in The Gambia

In West Africa, losses due to heartwater disease are not known because the incidence/prevalence has not been well studied or documented. To develop a diagnostic tool for molecular epidemiology, three PCR-based diagnostic assays, a nested pCS20 PCR, a nested map1 PCR and a nested reverse line blot (RLB) hybridization assay, were evaluated to determine their ability to detect infection in vector ticks, by applying them simultaneously to A. variegatum field ticks to detect Ehrlichia ruminantium, the causative agent of heartwater. The nested pCS20 PCR assay which amplified the pCS20 gene fragment showed the highest detection performance with a detection rate of 16.6%; the nested map1 PCR, which amplified the gene encoding the major antigenic protein1 (map1 gene) showed a detection rate of 11% and the RLB, based on the 16S rDNA sequence of anaplasma and ehrlichial species, detected 6.2%. The RLB, in addition, demonstrated molecular evidence of Ehrlichia ovina, Anaplasma marginale and Anaplasma ovis infections in The Gambia. Subsequently, the pCS20 assay was applied to study the prevalence and distribution of E. ruminantium tick infection rates at different sites in five divisions of The Gambia. The rates of infection in the country ranged from 1.6% to 15.1% with higher prevalences detected at sites in the westerly divisions (Western, Lower River and North Bank; range 8.3–15.1%) than in the easterly divisions (Central River and Upper River; range 1.6–7.5%). This study demonstrated a gradient in the distribution of heartwater disease risk for susceptible livestock in The Gambia which factor must be considered in the overall design of future upgrading programmes.     

Mitochondrial <Emphasis Type="Italic">cox</Emphasis>1 and plastid <Emphasis Type="Italic">rbc</Emphasis>L genes of <Emphasis Type="Italic">Gracilaria vermiculophylla</Emphasis> (Gracilariaceae,Rhodophyta)

The mitochondrial cytochrome c oxidase subunit I gene sequence was recently developed for DNA barcoding of red algal species. We determined the 1245 base pairs of the gene from 27 taxa of an agar-producing species, Gracilaria vermiculophylla, and putative relatives and compared the results with rbcL data from the same species. A total of 392 positions (31.5%) were variable, 282 positions (22.6%) were parsimoniously informative, and average sequence divergence was 13% in an ingroup. Within G. vermiculophylla, pairwise divergence of the gene was variable up to 11 bp (0.9%). Seven recognized haplotypes of cox1 tended to be geographically related. In the aligned 1386 bp of rbcL, three haplotypes were recognized. These results suggest that cox1 is a valuable molecular marker within species and will be very useful in haplotype analyses.     

Purification and properties of aminoaldehyde dehydrogenase from <Emphasis Type="Italic">Avena</Emphasis><Emphasis Type="Italic">sativa</Emphasis>

NAD-dependent aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.-) from Avena shoots was purified by DEAE Sephacel, hydroxyapatite, 5′-AMP Sepharose 4B, Mono Q, and TSK-GEL column chromatographies to homogeneity by the criterion of native PAGE. SDS–PAGE yielded a single band at a molecular mass of 55 kDa. IEF studies showed a band with a pI value of 5.3. In contrast to AMADHs from other species, the TSK-GEL chromatography showed that Avena AMADH exists as a monomer in the native state. The purified enzyme catalyzed the oxidations of 3-aminopropionaldehyde (APAL), 4-aminobutyraldehyde (ABAL) N-(3-aminopropyl)-4-aminobutyraldehyde (APBAL), and 4-guanidinobutyraldehyde (GBAL), but not of betaine aldehyde or indoleacetaldehyde. The K _m values for APAL, ABAL, and GBAL were 1.5×10^–6, 2.2×10^–6, and 1.3×10^–5 M, respectively. Although N-terminal amino acid sequence of Avena AMADH could not be determined due to a modification of the amino residue, the sequence of the fragment of AMADH cleaved by V8 protease showed greater similarity to the barley BADH than to the pea AMADH. Electronic Publication     

Invasion of the Lyme Disease Vector <Emphasis Type="Italic">Ixodes scapularis</Emphasis>: Implications for <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> Endemicity

Lyme disease risk is increasing in the United States due in part to the spread of blacklegged ticks Ixodes scapularis, the principal vector of the spirochetal pathogen Borrelia burgdorferi. A 5-year study was undertaken to investigate hypothesized coinvasion of I. scapularis and B. burgdorferi in Lower Michigan. We tracked the spatial and temporal dynamics of the tick and spirochete using mammal, bird, and vegetation drag sampling at eight field sites along coastal and inland transects originating in a zone of recent I. scapularis establishment. We document northward invasion of these ticks along Michigan’s west coast during the study period; this pattern was most evident in ticks removed from rodents. B. burgdorferi infection prevalences in I. scapularis sampled from vegetation in the invasion zone were 9.3% and 36.6% in nymphs and adults, respectively, with the majority of infection (95.1%) found at the most endemic site. There was no evidence of I. scapularis invasion along the inland transect; however, low-prevalence B. burgdorferi infection was detected in other tick species and in wildlife at inland sites, and at northern coastal sites in years before the arrival of I. scapularis. These infections suggest that cryptic B. burgdorferi transmission by other vector-competent tick species is occurring in the absence of I. scapularis. Other Borrelia spirochetes, including those that group with B. miyamotoi and B. andersonii, were present at a low prevalence within invading ticks and local wildlife. Reports of Lyme disease have increased significantly in the invasion zone in recent years. This rapid blacklegged tick invasion—measurable within 5 years—in combination with cryptic pathogen maintenance suggests a complex ecology of Lyme disease emergence in which wildlife sentinels can provide an early warning of disease emergence.     

Multiple paternity in <Emphasis Type="Italic">Rhipicephalus</Emphasis> (<Emphasis Type="Italic">Boophilus</Emphasis>) <Emphasis Type="Italic">microplus</Emphasis> confirmed by microsatellite analysis

The aim of this study was to determine if individual ticks among the progeny of a single female Rhipicephalus (Boophilus) microplus tick removed from cattle under natural conditions are the result of mating with one or several males. To this end, simulations were run using an existing dataset of genotypes from 8 microsatellite loci to predict the number of samples required and the best locus. Subsequently, 14–22 progeny from each of 15 engorged female ticks removed from three cows, and the engorged females themselves, were genotyped for the BmM1 locus and the minimum number of potential male parents was determined for each progeny group. Of the 15 progeny groups, 10 must have been sired by more than one male, as indicated by the presence of five unique alleles among the progeny or three unique alleles that could not have been contributed by the female. This finding demonstrates multiple paternity in R. microplus.     

Bio-resolution of glycidyl (<Emphasis Type="Italic">o</Emphasis>, <Emphasis Type="Italic">m</Emphasis>, <Emphasis Type="Italic">p</Emphasis>)-methylphenyl ethers by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

A newly isolated Bacillus megaterium with epoxide hydrolase activity resolved racemic glycidyl (o, m, p)-methylphenyl ethers to give enantiopure epoxides in 84–99% enantiomeric excess and with 21–73 enantiomeric ratios. The (S)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ether while the (R)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained at the level by enzyme-substrate docking studies.     

Prevalence of tick-borne pathogens in ixodid ticks (Acari: Ixodidae) collected from European wild boar (<Emphasis Type="Italic">Sus scrofa</Emphasis>) and Iberian red deer (<Emphasis Type="Italic">Cervus elaphus hispanicus</Emphasis>) in central Spain

Emerging tick-borne diseases of humans and animals have occurred frequently during the past 30 years. These disease outbreaks appear to result from changes in the distribution of tick and vertebrate hosts, and the introduction of humans and domestic animals into tick–pathogen–wildlife cycles. Use of molecular technologies now available for identification of pathogens in ticks can provide valuable information that allows for risk analysis of emerging tick-borne diseases. In this study, the prevalence of selected pathogens in ticks collected in six locations in central Spain from the major wild ungulate species, European wild boar (Sus scrofa) and Iberian red deer (Cervus elaphus hispanicus), was determined by PCR. Tick species collected included Ixodes ricinus, Dermacentor marginatus, Rhipicephalus bursa and Hyalomma m. marginatum. Pathogens identified in ticks included piroplasmids, Anaplasma spp., Ehrlichia spp. and Rickettsia spp. Piroplasmids were identified in all tick species except I. ricinus. Ehrlichia spp. were detected in all tick species and collection locations, while Rickettsia spp., which proved to be R. slovaca and a recently identified Rickettsia sp. DnS28, were identified only in D. marginatus. A. marginale and A. phagocytophilum were detected in D. marginatus, R. bursa and Hy. m. marginatum. Concurrent infections of these pathogens were frequently observed in ticks. Notably, A. phagocytophilum, which is infective for a broad host range that includes humans and domestic and wild animals, was identified in ticks from all collection locations. The variety of ticks and tick-borne pathogens demonstrated in this study suggests a risk in central Spain for the emergence of tick-borne diseases in humans and domestic animals.     

Acaricidal effects of <Emphasis Type="Italic">Corymbia citriodora</Emphasis> oil containing <Emphasis Type="Italic">para</Emphasis>-menthane-3,8-diol against nymphs of <Emphasis Type="Italic">Ixodes ricinus</Emphasis> (Acari: Ixodidae)

The toxicity of para-menthane-3,8-diol (PMD), the main arthropod-repellent compound in the oil of the lemon eucalyptus, Corymbia citriodora, was evaluated against nymphs of Ixodes ricinus using five methods (A–E) of a contact toxicity bioassay. Mortality rates were estimated by recording numbers of dead nymphs at 30 min intervals during the first 5 h after the start of exposure and at longer intervals thereafter. The mortality rate increased with increasing concentration of PMD and duration of exposure with a distinct effect after 3.5 h. From the results obtained by methods A, C and E, the LC₅₀ range was 0.035–0.037 mg PMD/cm² and the LC₉₅ range was 0.095–0.097 mg PMD/cm² at 4 h of exposure; the LT₅₀ range was 2.1–2.8 h and the LT₉₅ range was 3.9–4.2 h at 0.1 mg PMD/cm². To determine the duration of toxic activity of PMD, different concentrations (0.002, 0.01, 0.1 mg PMD/cm²) were tested and mortality was recorded at each concentration after 1 h; thereafter new ticks were tested. This test revealed that the lethal activity of PMD remained for 24 h but appeared absent after 48 h. The overall results show that PMD is toxic to nymphs of I. ricinus and may be useful for tick control.     

Incongruent effects of two isolates of <Emphasis Type="Italic">Rickettsia conorii</Emphasis> on the survival of <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> ticks

Rickettsia conorii, the etiologic agent of Mediterranean spotted fever is widely distributed in Southern Europe, the Middle East, Africa, India and the Caspian region. In the Mediterranean region, the brown dog tick, Rhipicephalus sanguineus, is the recognized vector of R. conorii. To study tick-pathogen relationships and pathogenesis of infection caused in model animals by the bite of an infected tick, we attempted to establish a laboratory colony of Rh. sanguineus persistently infected with R. conorii. Rhipicephalus sanguineus ticks of North American and Mediterranean origin were exposed to R. conorii isolates of African (R. conorii conorii strain Malish) and Mediterranean (R. conorii israelensis strain ISTT) origin. Feeding of ticks upon infected mice and dogs, intra-hemocoel inoculation, and submersion in suspensions of purified rickettsiae were used to introduce the pathogen into uninfected ticks. Feeding success, molting success and the longevity of molted ticks were measured to assess the effects of R. conorii on the survival of Rh. sanguineus. In concordance with previously published results, Rh. sanguineus larvae and nymphs from both North American and Mediterranean colonies exposed to R. conorii conorii Malish experienced high mortality during feeding and molting or immediately after. The prevalence of infection in surviving ticks did not exceed 5%. On the other hand, exposure to ISTT strain had lesser effect on tick survival and resulted in 35–66% prevalence of infection. Rh. sanguineus of Mediterranean origin were more susceptible to infection with either strain of R. conorii than those from North America. Previous experimental studies had demonstrated transovarial and transstadial transmission of R. conorii in Rh. sanguineus; however, our data suggest that different strains of R. conorii may employ different means of maintenance in nature. The vertebrate host may be a more important reservoir than previously thought, or co-feeding transmission between different generations of ticks may obviate or lessen the requirement for transovarial maintenance of R. conorii.     

Cattle ticks of the genera <Emphasis Type="Italic">Rhipicephalus</Emphasis> and <Emphasis Type="Italic">Amblyomma</Emphasis> of economic importance in Tanzania: distribution assessed with GIS based on an extensive field survey

In order to implement a robust integrated tick and tick-borne disease control programme in Tanzania, based on ecological and epidemiological knowledge of ticks and their associated diseases, a national tick and sero-surveillance study was carried out in all 21 regions of the mainland, as well as on Mafia Island, between 1998 and 2001. The current distributions of Rhipicephalus appendiculatus, R. pravus, Amblyomma variegatum, A. gemma, and A. lepidum are illustrated and discussed. Tick distribution maps were assessed using the Weights-of-Evidence method (WofE), and employing temperature, humidity, NDVI, rainfall, and land-cover predictive data. Ground-truthing was done to check correspondence both of the data employed in prediction with land-cover characteristics discerned in the field as well as of the surveyed and predicted tick distributions. Statistical methods were used to analyse associations of the tick species with their environment, cattle density, and other ticks. Except for R. appendiculatus, no appreciable changes were demonstrated in the predicted and observed tick distributions compared to the existing maps that originated in the 1950–1960s. Cattle density influenced the distribution of A. variegatum and, to a certain extent, of A. lepidum, but had no appreciable influence on the distribution of any of the other ticks discussed in this paper, neither did livestock movement. Distinct differences for environmental requirements where observed between different tick species within the same genus. The predictive maps of R. appendiculatus and R. pravus suggest their mutually exclusive distribution in Tanzania, and simultaneous statistical analysis showed R. pravus as a greater specialist. Of the three Amblyomma species, A. variegatum is the most catholic tick species in Tanzania, while both A. gemma and A. lepidum belong to the more specialized species. Despite dissimilar habitat preferences, all three Amblyomma spp. co-exist in central Tanzania, where very heterogeneous habitats may simultaneously satisfy the environmental requirements of all three species. The current study, conducted about 4 decades after the last major survey activities, has shown that changing livestock policies, unrestricted livestock movement and a continuous change in climatic/environmental conditions in Tanzania have brought about only limited changes in the distribution patterns of R. appendiculatus, R. pravus and the three Amblyomma species investigated. Whether this observation indicates a relative indifference of these ticks to environmental and/or climate changes allows room for speculation.     

Genetic variation in populations of <Emphasis Type="Italic">Allothrombium pulvinum</Emphasis> (Acari: Trombidiidae) from Northern Iran revealed by mitochondrial <Emphasis Type="Italic">cox</Emphasis>I and nuclear rDNA <Emphasis Type="Italic">ITS</Emphasis>2 sequences

Allothrombium pulvinum Ewing is a common natural enemy of aphids and some other arthropods. So far, there are no studies that have addressed genetic variation of this predatory mite. We investigated genetic variation of A. pulvinum across its whole known range in Iran. A 410 bp portion of the mitochondrial cytochrome c oxidase subunit I gene (coxI) and 797–802 bp portion of the internal transcribed spacer 2 of rDNA (ITS2) were sequenced for 55 individuals from 11 populations, resulting in 12 and 26 haplotypes, respectively. In the coxI region, haplotype and nucleotide diversities varied among populations from 0.00 to 0.90 and from 0.0000 to 0.0110, respectively. In the ITS2 region they varied from 0.20 to 0.91 and from 0.0006 to 0.0023, respectively. For both gene regions the highest haplotype and nucleotide diversities were detected in population Mahmoud Abad from northern Iran. Statistically significant population differentiation (F _ST) was detected in most pair-wise population comparisons. The results of population differentiation for both gene regions were generally congruent indicating that A. pulvinum from Iran consists of genetically different populations. This suggests that A. pulvinum comprises at least two geographically distinct populations or even more than one species. This study is an initial step towards understanding genetic variation of A. pulvinum, a taxon for which little molecular information is available. More intensive sampling and analysis of additional DNA regions are necessary for more detailed classification of this taxon.     

Molecular analysis of the genus <Emphasis Type="Italic">Anoxybacillus</Emphasis> based on sequence similarity of the genes <Emphasis Type="Italic">recN</Emphasis>, <Emphasis Type="Italic">flaA</Emphasis>, and <Emphasis Type="Italic">ftsY</Emphasis>

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies. We analyzed three genes, recN, flaA, and ftsY, for determining if these genes are useful tools for systematic analyses in the genus Anoxybacillus. The genes encoding a DNA repair and genetic recombination protein (recN), the flagellin protein (flaA), and GTPase signal docking protein (ftsY) were sequenced for ten Anoxybacillus species. The sequence comparisons revealed that recN sequence similarities range between 61% and 99% in the genus Anoxybacillus. Comparisons to other bacterial recN genes indicated that levels of similarity did not differ from the levels within genus Anoxybacillus. These data showed that recN is not a useful marker for the genus Anoxybacillus. A 550–600-bp region of the flagellin gene was amplified for all Anoxybacillus strains except for Anoxybacillus contaminans. The sequence similarity of flaA gene varies between 61% and 76%. Comparisons to other bacterial flagellin genes obtained from GenBank (Bacillus, Pectinatus, Proteus, and Vibrio) indicated that the levels of similarity were lower (3–42%). Based on these data, we concluded that the variability in this single gene makes it a particularly useful marker. Another housekeeping gene ftsY suggested to reflect the G+C (mol/mol) content of whole genome was analyzed for Anoxybacillus strains. A mean difference of 1.4% was observed between the G+C content of the gene ftsY and the G+C content of the whole genome. These results showed that the gene ftsY can be used to represent whole G+C content of the Anoxybacillus species.     

<Emphasis Type="Italic">Humulus japonicus</Emphasis> Accelerates the Decomposition of <Emphasis Type="Italic">Miscanthus sacchariflorus</Emphasis> and <Emphasis Type="Italic">Phragmites australis</Emphasis> in a Floodplain

Humulus japonicus in communities of Miscanthus sacchariflorus and Phragmites australis can grow large enough to overtop other species in the Amsa-dong floodplain. Because of strong winds and the weight of Humulus, plants of M. sacchariflorus and P. australis fell in mid-August and were subject to decomposition under its dense shading. To assess the effects of H. japonicus on nutrient cycling in these communities, we collected fresh samples of M. sacchariflorus and P. australis in litterbags and decomposed them under H. japonicus for 9 months, beginning in August. Biomass and organic contents from M. sacchariflorus during this incubation period were 49–51% and 44–48%, whereas those of P. australis were 49–61% and 32–52%, respectively. Their annual k values were 1.61–1.74 and 1.46–3.54, respectively. Initial N concentrations in M. sacchariflorus and P. australis were 13 and 20 mg g⁻¹, while C:N ratios were 31 and 21, respectively. These results indicate that H. japonicus is responsible for the collapse of M. sacchariflorus and P. australis in August and also accelerates their nutrient cycling through rapid decomposition, thereby increasing nutrient circulation in floodplains.     

<Emphasis Type="Italic">Shewanella upenei</Emphasis> sp. nov., a lipolytic bacterium isolated from bensasi goatfish <Emphasis Type="Italic">Upeneus bensasi</Emphasis>

A Gram-staining-negative, motile, non-spore-forming and rod-shaped bacterial strain, 20-23R^T, was isolated from intestine of bensasi goatfish, Upeneus bensasi, and its taxonomic position was investigated by using a polyphasic study. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain 20-23R^T belonged to the genus Shewanella. Strain 20-23R^T exhibited 16S rRNA gene sequence similarity values of 99.5, 99.2, and 97.5% to Shewanella algae ATCC 51192^T, Shewanella haliotis DW01^T, and Shewanella chilikensis JC5^T, respectively. Strain 20-23R^T exhibited 93.1–96.0% 16S rRNA gene sequence similarity to the other Shewanella species. It also exhibited 98.3–98.4% gyrB sequence similarity to the type strains of S. algae and S. haliotis. Strain 20-23R^T contained simultaneously both menaquinones and ubiquinones; the predominant menaquinone was MK-7 and the predominant ubiquinones were Q-8 and Q-7. The fatty acid profiles of strain 20–23R^T, S. algae KCTC 22552^T and S. haliotis KCTC 12896^T were similar; major components were iso-C_15:0, C_16:0, C_16:1 ω7c and/or iso-C_15:0 2-OH and C_17:1 ω8c. The DNA G+C content of strain 20-23R^T was 53.9 mol%. Differential phenotypic properties and genetic distinctiveness of strain 20–23R^T, together with the phylogenetic distinctiveness, revealed that this strain is distinguishable from recognized Shewanella species. On the basis of the data presented, strain 20-23R^T represents a novel species of the genus Shewanella, for which the name Shewanella upenei sp. nov. is proposed. The type strain is 20–23R^T (=KCTC 22806^T =CCUG 58400^T).     

Detection of <Emphasis Type="Italic">Rickettsia</Emphasis> and a Novel <Emphasis Type="Italic">Haemaphysalis shimoga</Emphasis> Symbiont Bacterium in Ticks in Thailand

In this study, we identified two Haemaphysalis species present at the Khao Yai National Park in Thailand and investigated the presence of rickettsia in these ticks. A total of 166 Haemaphysalis specimens were collected randomly under leaves along visitor paths at five locations in the park. Male and female adults of two different Haemaphysalis species, H. shimoga and H. lagrangei, were identified. Polymerase chain reaction (PCR) analysis revealed Rickettsia bacteria in these two Haemaphysalis species; this study represents the first time such presence has been reported in Thailand. The infection rates of Rickettsia were in both H. shimoga (7.41%) and H. lagrangei (10.17%) at these locations in addition to two pools of Haemahysalis nymphs (28.57%). Furthermore, 25.93% of H. shimoga showed positive results that matched Haemaphysalis longicornis symbionts (92% sequence identity) and the Coxeilla burnetti 16S ribosomal RNA gene (90% sequence identity). We propose that this is a novel H. shimoga symbiont bacterium in Thailand and might be a novel Coxeilla-like agent or Coxeilla sp. found in H. shimoga. In contrast, we did not observe any Wolbachia bacteria, which also belong to the order Rickettsiales, in the same group of Haemaphysalis ticks. Furthermore, PCR was used to detect three other genera of bacteria, Anaplasma, Ehrlichia and Borrelia, none of which were identified in the Haemaphysalis ticks studied.