Flagellin Gene Sequence Research Articles

Molecular Detection of Novel Borrelia Species, Candidatus Borrelia javanense, in Amblyomma javanense Ticks from Pangolins.

A novel Borrelia species, Candidatus Borrelia javanense, was found in ectoparasite ticks, Amblyomma javanense, from Manis javanica pangolins seized in anti-smuggling operations in southern China. Overall, 12 tick samples in 227 (overall prevalence 5.3%) were positive for Candidatus B. javanense, 9 (5.1%) in 176 males, and 3 (5.9%) in 51 females. The phylogenetic analysis, based on the 16S rRNA gene and the flagellin gene sequences of the Borrelia sp., exhibited strong evidence that Candidatus B. javanense did not belong to the Lyme disease Borrelia group and the relapsing fever Borrelia group but another lineage of Borrelia. The discovery of the novel Borrelia species suggests that A. javanense may be the transmit vector, and the M. javanica pangolins should be considered a possible origin reservoir in the natural circulation of these new pathogens. To our knowledge, this is the first identification of a novel Borrelia species agent in A. javanense from pangolins. Whether the novel agent is pathogenic to humans is unknown and needs further research.

Selection and characterization of two Bacillus thuringiensis strains showing nematicidal activity against Caenorhabditis elegans and Meloidogyne incognita.

Bacillus thuringiensis has been widely used as a biological control agent against insect pests. Additionally, nematicidal strains have been under investigation. In this report, 310 native strains of B. thuringiensis against Caenorhabditis elegans were tested. Only the LBIT-596 and LBIT-107 strains showed significant mortality. LC50s of spore-crystal complexes were estimated at 37.18 and 31.89μg/mL for LBIT-596 and LBIT-107 strains, respectively, while LC50s of partially purified crystals was estimated at 23.76 and 20.25μg/mL for LBIT-596 and LBIT-107, respectively. The flagellin gene sequence and plasmid patterns indicated that LBIT-596 and LBIT-107 are not related to each other. Sequences from internal regions of a cry5B and a cyt1A genes were found in the LBIT-596 strain, while a cry21A, a cry14A and a cyt1A genes were found in the LBIT-107 strain. Genome sequence of the LBIT-107 strain showed new cry genes, along with other virulence factors, hence, total nematicidal activity of the LBIT-107 strain may be the result of a multifactorial effect. The highlight of this contribution is that translocation of spore-crystal suspensions of LBIT-107 into tomato plants inoculated at their rhizosphere decreased up to 90% the number of galls of Meloidogyne incognita, perhaps the most important nematode pest in the world.

Retrospective Survey of Borrelia spp. From Rodents and Ticks in Thailand.

Borrelia is a genus of spirochetal bacteria with several species known to cause disease in humans. The distribution of Borrelia has rarely been studied in Thailand. In this study, a retrospective survey of Borrelia was conducted in ticks and wild rodents to better characterize the prevalence, diversity, and distribution of Borrelia across Thailand. Several pools of DNA from tick samples were positive for Borrelia spp. (36/258, 13.9%). Borrelia theileri/B. lonestari was found in 17 tick samples (16 pools of Haemaphysalis bandicota and 1 pool of Rhipicephalus sp.), and Borrelia yangtzensis was found in 8 tick samples (2 pools of H. bandicota and 6 pools of Ixodes granulatus). Borrelia spp. were detected at low prevalence levels in rodent tissue samples (24/2001, 1.2%), with 19 identified as B. theileri or B. lonestari and 5 identified as B. miyamotoi. Several geographic and species-specific infection trends were apparent, with Ixodes ticks infected with B. yangtzensis and Haemaphysalis and Rhipicephalus ticks infected with both B. yangtzensis and B. theileri/B. lonestari. Notably, B. yangtzensis showed a similar geographic distribution to B. miyamotoi, which was identified in new areas of Thailand in this study. The flagellin gene sequence from B. miyamotoi was more similar to European (99.3-99.9%) than Japanese (96.9-97.6%) genotypes. This study greatly expands the knowledge of Borrelia in Thailand and identified several Borrelia species for the first time. It also found several ticks and rodents infected with the pathogen that were not previously known to carry Borrelia.

Purification and Characterization of a Novel Antifungal Flagellin Protein from Endophyte Bacillus methylotrophicus NJ13 against Ilyonectria robusta

Endophyte Bacillus methylotrophicus NJ13 was isolated from Panax ginseng. Its sterile fermentation liquid showed a significant inhibitory effect against Ilyonectria robusta, causing the rusty root rot of P. ginseng and P. quinquefolius. The antifungal protein was obtained after precipitation by 20% saturated ammonium sulfate, desalted by Sephadex G-25, weak anion exchange chromatography, and gel filtration chromatography. SDS-PAGE showed that the purified protein was approximately 29 KDa. The antifungal protein after desalting was not resistant to temperatures higher than 100 °C, resistant to acid conditions, and did not tolerate organic solvents and protease K. The amino acid sequence of purified antifungal protein had an identity of 76% to flagellin from Bacillus velezensis. The isoelectric point of the protein was 4.97 and its molecular mass was 27 KDa. Therefore, a specific primer G1 was designed based on the flagellin gene sequence, and a 770 bp gene sequence was cloned in NJ13 genomic DNA, which shared the same size of flagellin. There were ten base differences between the gene sequences of flagellin and the cloned gene, however, the amino acid sequence encoded by the cloned gene was identical to the flagellin. In conclusion, the antifungal protein produced by biocontrol agent NJ13 contained a flagellin protein.

Characterization of a highly toxic strain of Bacillus thuringiensis serovar kurstaki very similar to the HD-73 strain.

The LBIT-1200 strain of Bacillus thuringiensis was recently isolated from soil, and showed a 6.4 and 9.5 increase in toxicity, against Manduca sexta and Trichoplusia ni, respectively, compared to HD-73. However, LBIT-1200 was still highly similar to HD-73, including the production of bipyramidal crystals containing only one protein of ∼130000 kDa, its flagellin gene sequence related to the kurstaki serotype, plasmid and RepPCR patterns similar to HD-73, no production of β-exotoxin and no presence of VIP genes. Sequencing of its cry gene showed the presence of a cry1Ac-type gene with four amino acid differences, including two amino acid replacements in domain III, compared to Cry1Ac1, which may explain its higher toxicity. In conclusion, the LBIT-1200 strain is a variant of the HD-73 strain but shows a much higher toxicity, which makes this new strain an important candidate to be developed as a bioinsecticide, once it passes other tests, throughout its biotechnological development.

Geographical and genospecies distribution of Borrelia burgdorferi sensu lato DNA detected in humans in the USA

The present study investigated the cause of illness in human patients primarily in the southern USA with suspected Lyme disease based on erythema migrans-like skin lesions and/or symptoms consistent with early localized or late disseminated Lyme borreliosis. The study also included some patients from other states throughout the USA. Several PCR assays specific for either members of the genus Borrelia or only for Lyme group Borrelia spp. (Borrelia burgdorferi sensu lato), and DNA sequence analysis, were used to identify Borrelia spp. DNA in blood and skin biopsy samples from human patients. B. burgdorferi sensu lato DNA was found in both blood and skin biopsy samples from patients residing in the southern states and elsewhere in the USA, but no evidence of DNA from other Borrelia spp. was detected. Based on phylogenetic analysis of partial flagellin (flaB) gene sequences, strains that clustered separately with B. burgdorferi sensu stricto, Borrelia americana or Borrelia andersonii were associated with Lyme disease-like signs and symptoms in patients from the southern states, as well as from some other areas of the country. Strains most similar to B. burgdorferi sensu stricto and B. americana were found most commonly and appeared to be widely distributed among patients residing throughout the USA. The study findings suggest that human cases of Lyme disease in the southern USA may be more common than previously recognized and may also be caused by more than one species of B. burgdorferi sensu lato. This study provides further evidence that B. burgdorferi sensu stricto is not the only species associated with signs and/or symptoms consistent with Lyme borreliosis in the USA.

Study on Salmonella Typhi occurrence in gallbladder of patients suffering from chronic cholelithiasis—a predisposing factor for carcinoma of gallbladder

Cholelithiasis is frequently associated with carcinoma of gallbladder, and the presence of Salmonella Typhi in gallbladder of patients suffering from cholelithiasis is implicated as a predisposing factor for carcinogenesis. This study was conducted on patients suffering from chronic cholelithiasis from a region in North India–endemic area for enteric fever with high incidence of gallstones and gallbladder cancer. Since culture studies rarely reveal the chronic Salmonella Typhi persistence, we use PCR assay to specifically amplify the H1-d flagellin gene sequence homologous with Salmonella Typhi. Seven cases (17.5%), none of which were positive for culture, showed positive PCR results for Salmonella Typhi, 4 (10%) of which were tissue, 2 bile (5%), and 1 gallstone (2.5%). The chronic existence of Salmonella Typhi in gallbladder disease was confirmed. Thus, the study would indicate the importance of vaccination so as to prevent chronic infection and need for early diagnostic tools to prevent any further complications.

Borrelia burgdorferi Bacteria in Domestic Animals China

Background: Borreliosis are among both the longest known and most recently recognized infectious diseases. Although Borrelia species have been isolated in many parts of the world including China, Little is known about the epidemiology of Borrelia burgdorferi pathogen in domestic animals from Xinjiang Autonomous Region of China (XUAR). Methods: In an attempt to assess the potential risk of B. burgdorferi infection in Xinjiang Uygur Autonomous Region of China, a total of 99 blood samples collected from domestic animals in the north of XUAR were screened by polymerase chain reaction based on the 5S-23S rRNA intergenic spacer and flagellin gene. Results: 14 of the 99 specimens (14%) were found to be positive by PCR. Phylogenetic analysis of 5S-23S rRNA intergenic spacer and flagellin gene sequences identified a B. burgdorferi- like bacteria, Ili district (10/72, 13.8%) and Altay district (4/27, 14.8%). Conclusions: This study has reported the occurrence of B. burgdorferi- like bacteria in Xinjiang Autonomous Region of China and suggested that B. burgdorferi bacteria encompassed a large area in China. Further studies on the characterization and culture of B. burgdorferi- like bacteria found in domestic animals should be performed to further clarify this. Additionally, the screening of human specimens for borreliosis in this region will define the incidence of infection.

Isolation of Salmonella typhi from apparently healthy liver

It is suggested that Salmonella typhi resides mostly in hepatobiliary system especially in gallbladder in chronic typhoid carriers. It is not very clear whether in gallbladder lumen or on its wall or in liver. However, we had been successful in detecting S. typhi in liver by PCR targeting flagellin gene sequences. Therefore, in the present study, we tried to isolate the bacterium from liver tissue collected from dead bodies brought for post mortem examination. We could isolate S. typhi in 2 of 20 such liver tissues examined by using conventional isolation techniques. The isolates were identified by routine phenotypic characters and were confirmed by amplification and sequencing of two conserved genes i.e. 16S rDNA and flagellin (fliC) gene followed by blasting on www.ncbi.nlm.nih.gov.

The presence of Borrelia valaisiana-related genospecies in ticks and a rodent in Taiwan

A field survey was conducted to investigate the presence of Borrelia burgdorferi sensu lato (s.l.) in six counties of Taiwan. Spirochetes were successfully isolated from one rodent ear sample out of 485 rodent ears and 53 live, fed tick (Ixodes granulatus) samples. The spirochetes were confirmed to be B. burgdorferi s.l. by real-time PCR. In addition, 23 of 113 tick samples were tested positive for Borrelia DNA according to real-time PCR. The Borrelia isolate from the rodent and the 23 Borrelia DNA samples from the ticks were identified as B. valaisiana-related genospecies by phylogenetic analysis based on flagellin gene sequences. These findings suggest that the Borrelia valaisiana-related strains are maintained in a zoonotic cycle between tick vectors and reservoir hosts in Taiwan.

Flagellin gene structure of flaA and flaB and adjacent gene loci in urease‐positive thermophilic Campylobacter (UPTC)

Two flagellin gene (flaA and flaB) sequences and the adjacent gene loci of urease-positive thermophilic Campylobacter (UPTC)(1) were examined. The flagellin gene sequences (1.7 kb) and adjacent gene loci of the two UPTC isolates (89049 and A3), obtained from animal hosts, were very similar to those of C. lari RM2100, C. jejuni, and C. coli. However, the structure and loci of the two flagellin genes (1.46-1.47 kb) and the adjacent gene loci of a UPTC strain obtained from the natural environment (NCTC12892) clearly differed from those of C. lari RM2100 and UPTC obtained from animal hosts. The two flagellin genes of UPTC 89049 and A3 were located between topA /CLA0518 and CLA0521, whereas those of NCTC12892 were located between topA and CLA0521. The sequences involved in regulation of flagellin expression, like sigma(28), sigma(54) and transcription termination signals, were conserved in all isolates. The characteristic direct repeat sequences containing a complete repeat unit of 5'-TCTTTAAAACAAC-3' were located in the intergenic regions between flaA and flaB in UPTC89049 and A3, but not in NCTC12892. The deduced amino acid sequence alignment revealed that the two flagellin genes in NCTC12892 had a deletion of the variable region of flagellin, which was reported previously to be modified by pseudaminic acid in C. jejuni and C. coli.Consequently, these results may possibly suggest that the length of the flagellin is related to pathogenicity and colonization of Campylobacter.

Detection of Borrelia garinii, Borrelia tanukii and Borrelia sp. closely related to Borrelia valaisiana in Ixodes ticks removed from dogs and cats in Japan

Ticks removed from 1136 dogs and 134 cats all over Japan were examined for Borrelia infection by PCR and sequencing. The 5S-23S rDNA intergenic spacer of Borrelia was detected from two Ixodes persulcatus ticks from two dogs and two unidentified Ixodes spp. from another two dogs in Hokkaido, and two Ixodes granulatus ticks from two cats in Okinawa. Additional 2 I. granulatus from the same cats also showed positive. Sequence analysis of the PCR products revealed that the one from Hokkaido was similar to B. garinii, the three from Hokkaido to B. tanukii, and the four from Okinawa to a novel Borrelia sp. closely related to B. valaisiana. The data was confirmed by analysis of the flagellin gene sequence. Infected ticks carried by companion animals can be introduced into the human environment.

Characterization of a spirochaete isolated from a case of bovine digital dermatitis

The aim of the study was to characterize a spirochaete isolated from the lesions of a cow with digital dermatitis (DD). The characterization was on the basis of its light and electron microscopic appearance, enzymic profile and DNA sequence analysis of its flagellin and 16S rRNA genes. The spirochaete was 6-8-microm long and 0.2-0.3 microm in diameter, and possessed seven to eight periplasmic flagella, with three to five helical turns. The enzymic profile of the bacterium resembles, but is not identical to that of Treponema brennaborense. Its flagellin gene sequence was identical to that of Treponema phagedenis but distinct from that of an ovine spirochaete. Analysis of a 1477-bp region of the 16S rRNA genes indicated that this is a Treponema species and that it is indistinguishable from some isolates made from cases of bovine DD in the United States. Finally, electron microscopy revealed the presence of myovirus-like bacteriophage particles in all cultures of the treponeme examined. The spirochaete isolate was identified as a Treponema species closely related to some isolates from the United States (by 16S rDNA) and to T. phagedenis (by flagellin gene sequence) and is associated with bacteriophage particles. The fact that the isolates with the same or very similar 16S rDNA sequences have been obtained from cases of bovine DD in cattle in different countries at different times, lends further support to the hypothesis that treponemes play a role in the pathogenesis of this disease.

Diagnostic Utility of Borrelia burgdorferi Cerebrospinal Fluid Polymerase Chain Reaction in Children with Lyme Meningitis

Cerebrospinal fluid (CSF) laboratory tests are frequently collected to help differentiate Lyme meningitis from other causes of aseptic meningitis. Previous studies using Lyme CSF polymerase chain reaction (PCR) have yielded varied results (sensitivity between 10 and 90%). No studies have specifically examined the diagnostic utility of Lyme CSF-PCR in North American children with Lyme meningitis. Retrospective chart review of children presenting to a children's hospital in a Lyme-endemic region between October 1999 and September 2004. Patients were included if they had both Lyme serology and Lyme CSF-PCR performed during the same hospital encounter and had documented meningitis. Patients were considered to have Lyme meningitis if they had meningitis and met CDC criteria for Lyme disease. The Lyme CSF-PCR assay amplified a Borrelia burgdorferi DNA flagellin gene sequence. Of 108 patients with meningitis who qualified for the study, 20 patients met criteria for Lyme meningitis and 88 were classified as aseptic meningitis. Positive Lyme CSF-PCR was found in 1 patient (1 of 20, 5%) with Lyme meningitis and one patient classified as aseptic meningitis (1 of 88, 1%). Lyme CSF-PCR had a sensitivity of 5% and a specificity of 99%. The only Lyme meningitis patient with positive Lyme CSF-PCR had the highest CSF white blood cell count and CSF protein values compared with the other Lyme meningitis patients. This is the first study to evaluate Lyme CSF-PCR exclusively in North American children. This commercially available laboratory test is not generally helpful for identifying Lyme meningitis because of its low sensitivity.

Characterization of Borrelia burgdorferi sensu lato isolated in Moscow province – a sympatric region for Ixodes ricinus and Ixodes persulcatus

The relationships between Borrelia species and the vector ticks, Ixodes persulcatus and Ixodes ricinus were examined in a molecular epidemiological study. We conducted a survey in the Moscow region which is a sympatric region for both species of tick. We examined 630 unfed I. ricinus and I. persulcatus, ticks collected from four different regions around Moscow within an area of 250 km 2. Eighty-four ticks were culture positive (13.3%) and the prevalence rate varied in each region from 5.7% to 42.3%. No difference was found between the total prevalence rate for both species. Eight Borrelia afzelii-like variant isolates from I. ricinus and Clethrionomys glareolus were identified as B. afzelii by flagellin gene and 16S rDNA sequence analyses. Most isolates from I. ricinus were identified as Borrelia garinii type 20047 and B. afzelii. Two isolates were identified as Borrelia burgdorferi sensu stricto (s.s.) and Borrelia valaisiana, respectively, but no B. garinii type NT29 was found. In contrast, isolates from I. persulcatus were identified as both types 20047 and NT29 of B. garinii, and B. afzelii. No B. burgdorferi s.s. isolate was found among isolates from I. persulcatus.

New World relapsing fever Borrelia found in Ornithodoros porcinus ticks in central Tanzania.

Ticks were collected from 8 houses in Mvumi Mission village, near Dodoma, Tanzania. All ticks were examined for Borrelia infestation by flagellin gene-based nested polymerase chain reaction. All houses were highly infested with ticks, and all ticks collected were of the Ornithodoros porcinus species. Fifty-one out of 120 ticks were infected with spirochetes, and a flagellin gene sequence comparison showed that most of the spirochetes belonged to Borrelia duttonii, which is the causative agent of tick-borne relapsing fever in East Africa. The rest of the spirochetes were quite different from B. duttonii and instead resembled the New World tick-borne relapsing fever borreliae. Phylogenetic analysis using 16S ribosomal RNA gene sequences also supported the interpretation that the spirochete was a Borrelia species distinct from previously described members of the genus.

Detection and characterization of Borrelia bissettii in rodents from the central California coast.

This is the first report of Borrelia burgdorferi sensu lato in rodents from San Luis Obispo county, with most isolates obtained from a previously unreported host, Neotoma lepida Thomas. B. burgdorferi sensu lato was identified in seven rodent species, including the California vole, Microtus californicus Peale; dusky-footed woodrat, Neotoma fuscipes Baird; desert woodrat, Neotoma lepida Thomas; brush mouse, Peromyscus boylii Baird; California mouse, Peromyscus californicus Gambel; deer mouse, Peromyscus maniculatus Wagner; and western harvest mouse, Reithrodontomys megalotis Baird. Ear punch biopsies were cultured in BSK-H medium from 179 rodents trapped at six different study sites. Overall, prevalence of rodent infection was 44/179 (24.6%), with 34 of these isolates from N. lepida. Spirochete isolates were obtained from rodents at all study sites, indicating widespread prevalence of B. burgdorferi sensu lato across rodent species and habitats. Nucleotide sequences for 14 of these isolates have been submitted to GenBank. Isolates from three N. lepida and one P. boylii had identical flagellin gene sequences, and phylogenetic analysis placed these spirochetes in B. burgdorferi sensu lato group DN127, now known as B. bissettii Postic, Marti Ras, Lane, Hendson & Baranton. Additional sequencing of the intergenic spacer regions between the 5S and 23S ribosomal genes was performed on three of these isolates. Phylogenetic analysis separated these isolates into two clusters that grouped with Colorado or California isolates. The role of B. bissettii and related species other than B. burgdorferi sensu stricto Johnson, Schmid, Hyde, Steigerwalt & Brenner as human pathogens in the United States warrants further investigation.

New genomospecies related to Borrelia valaisiana, isolated from mammals in Okinawa archipelago, Japan.

In 2000 and 2001, a survey was conducted of Borrelia isolated from various mammals in the southernmost islands of Japan, including Okinawa (main island), Izena, Iriomote and Ishigaki. Isolates obtained from the ear tissues of Suncus murinus (10 isolates), Mus calori (four isolates), Rattus norvegicus (one isolate) and Crocidura watasei (one isolate), were characterized by RFLP of the 5S-23S rDNA intergenic spacer and sequence analysis of the intergenic spacer, 16S rDNA and flagellin gene. While these isolates showed identical RFLPs to Borrelia valaisiana found in Korea, Taiwan and the southern and central parts of China, their RFLP patterns differed from those of B. valaisiana found in European countries, and strain Am501 isolated from Ixodes columnae in Japan. It was found that these isolates clustered with each other on a phylogenetic tree based on flagellin gene and 16S rDNA sequences, but were relatively divergent from the European B. valaisiana and strain Am501. These findings suggest that these isolates found in East Asia should be classified as a new genomospecies of Borrelia burgdorferi sensu lato.

Lateral flagella are required for increased cell adherence, invasion and biofilm formation by Aeromonas spp.

Two types of flagella are responsible for motility in mesophilic Aeromonas strains. A polar unsheathed flagellum is expressed constitutively that allows the bacterium to swim in liquid environments and, in media where the polar flagellum is unable to propel the cell, Aeromonas express peritrichous lateral flagella. Recently, Southern blot analysis using a DNA probe based on the Aeromonas caviae Sch3N lateral flagellin gene sequence showed a good correlation between strains positive for the DNA probe, swarming motility and the presence of lateral flagella by microscopy. Here, we conclude that the easiest method for the detection of the lateral flagellin gene(s) is by PCR (polymerase chain reaction); this showed good correlation with swarming motility and the presence of lateral flagella. This was despite the high degree of DNA heterogeneity found in Aeromonas gene sequences. Furthermore, by reintroducing the laf (lateral flagella) genes into several mesophilic lateral-flagella-negative Aeromonas wild-type strains, we demonstrate that this surface structure enhances the adhesion to and invasion of HEp-2 cells and the capacity for biofilm formation in vitro. These results, together with previous data obtained using Laf − mutants, demonstrate that lateral flagella production is a pathogenic feature due to its enhancement of the interaction with eukaryotic cell surfaces.

Rapid diagnosis of typhoid fever by PCR assay using one pair of primers from flagellin gene of Salmonella typhi

Using the polymerase chain reaction (PCR) assay, we developed a rapid diagnosis method for Salmonella typhi infection in blood specimens from patients with typhoid fever. Primers were designed from the flagellin gene sequence, which would give an amplification product of 367 base pairs. In this study, the specificity of the assay, with no amplification, was seen for the other Salmonella strains with the flagellin gene, and not for non- Salmonella bacteria. For the sensitivity test, the protocol described allowed the detection of two to three copies of the Salmonella typhi genome, as determined by serial dilution of genomic DNA from Salmonella typhi. With the PCR technique, genomic DNA of Salmonella typhi was detected in 46 of 73 blood samples collected from patients with clinically suspected typhoid fever who had fever within 3 days of admission to the General Hospital, Makassar, South Sulawesi, Indonesia, and who had had no prior antibiotic treatment. The PCR results (63% positive cases) were compared with those of blood culture (13.7% positive cases) and the Widal test (35.6% positive cases), using the same samples from each of the 73 patients admitted to the General Hospital in Makassar. The time taken for PCR analysis of each sample was less than 12 h, compared with 3 to 5 days for blood or clot culture. The PCR with one pair of primers can be used as a novel, rapid diagnostic method for typhoid fever, particularly when results of standard culture assays are negative.