Multi Locus Sequence Typing of Chlamydia Reveals an Association between Chlamydia psittaci Genotypes and Host Species

Chlamydia comprises a group of obligate intracellular bacterial parasites responsible for a variety of diseases in humans and animals, including several zoonoses. Chlamydia trachomatis causes diseases such as trachoma, urogenital infection and lymphogranuloma venereum with severe morbidity. Chlamydia pneumoniae is a common cause of community-acquired respiratory tract infections. Chlamydia psittaci, causing zoonotic pneumonia in humans, is usually hosted by birds, while Chlamydia abortus, causing abortion and fetal death in mammals, including humans, is mainly hosted by goats and sheep. We used multi-locus sequence typing to asses the population structure of Chlamydia. In total, 132 Chlamydia isolates were analyzed, including 60 C. trachomatis, 18 C. pneumoniae, 16 C. abortus, 34 C. psittaci and one of each of C. pecorum, C. caviae, C. muridarum and C. felis. Cluster analyses utilizing the Neighbour-Joining algorithm with the maximum composite likelihood model of concatenated sequences of 7 housekeeping fragments showed that C. psittaci 84/2334 isolated from a parrot grouped together with the C. abortus isolates from goats and sheep. Cluster analyses of the individual alleles showed that in all instances C. psittaci 84/2334 formed one group with C. abortus. Moving 84/2334 from the C. psittaci group to the C. abortus group resulted in a significant increase in the number of fixed differences and elimination of the number of shared mutations between C. psittaci and C. abortus. C. psittaci M56 from a muskrat branched separately from the main group of C. psittaci isolates. C. psittaci genotypes appeared to be associated with host species. The phylogentic tree of C. psittaci did not follow that of its host bird species, suggesting host species jumps. In conclusion, we report for the first time an association between C. psittaci genotypes with host species.     

Genome sequencing and comparative analysis of three Chlamydia pecorum strains associated with different pathogenic outcomes

Background

Chlamydia pecorum is the causative agent of a number of acute diseases, but most often causes persistent, subclinical infection in ruminants, swine and birds. In this study, the genome sequences of three C. pecorum strains isolated from the faeces of a sheep with inapparent enteric infection (strain W73), from the synovial fluid of a sheep with polyarthritis (strain P787) and from a cervical swab taken from a cow with metritis (strain PV3056/3) were determined using Illumina/Solexa and Roche 454 genome sequencing.

Results

Gene order and synteny was almost identical between C. pecorum strains and C. psittaci. Differences between C. pecorum and other chlamydiae occurred at a number of loci, including the plasticity zone, which contained a MAC/perforin domain protein, two copies of a >3400 amino acid putative cytotoxin gene and four (PV3056/3) or five (P787 and W73) genes encoding phospholipase D. Chlamydia pecorum contains an almost intact tryptophan biosynthesis operon encoding trpABCDFR and has the ability to sequester kynurenine from its host, however it lacks the genes folA, folKP and folB required for folate metabolism found in other chlamydiae. A total of 15 polymorphic membrane proteins were identified, belonging to six pmp families. Strains possess an intact type III secretion system composed of 18 structural genes and accessory proteins, however a number of putative inc effector proteins widely distributed in chlamydiae are absent from C. pecorum. Two genes encoding the hypothetical protein ORF663 and IncA contain variable numbers of repeat sequences that could be associated with persistence of infection.

Conclusions

Genome sequencing of three C. pecorum strains, originating from animals with different disease manifestations, has identified differences in ORF663 and pseudogene content between strains and has identified genes and metabolic traits that may influence intracellular survival, pathogenicity and evasion of the host immune system.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-23) contains supplementary material, which is available to authorized users.     

Comparative genomics of koala,cattle and sheep strains of Chlamydia pecorum

Background

Chlamydia pecorum is an important pathogen of domesticated livestock including sheep, cattle and pigs. This pathogen is also a key factor in the decline of the koala in Australia. We sequenced the genomes of three koala C. pecorum strains, isolated from the urogenital tracts and conjunctiva of diseased koalas. The genome of the C. pecorum VR629 (IPA) strain, isolated from a sheep with polyarthritis, was also sequenced.

Results

Comparisons of the draft C. pecorum genomes against the complete genomes of livestock C. pecorum isolates revealed that these strains have a conserved gene content and order, sharing a nucleotide sequence similarity > 98%. Single nucleotide polymorphisms (SNPs) appear to be key factors in understanding the adaptive process. Two regions of the chromosome were found to be accumulating a large number of SNPs within the koala strains. These regions include the Chlamydia plasticity zone, which contains two cytotoxin genes (toxA and toxB), and a 77 kbp region that codes for putative type III effector proteins. In one koala strain (MC/MarsBar), the toxB gene was truncated by a premature stop codon but is full-length in IPTaLE and DBDeUG. Another five pseudogenes were also identified, two unique to the urogenital strains C. pecorum MC/MarsBar and C. pecorum DBDeUG, respectively, while three were unique to the koala C. pecorum conjunctival isolate IPTaLE. An examination of the distribution of these pseudogenes in C. pecorum strains from a variety of koala populations, alongside a number of sheep and cattle C. pecorum positive samples from Australian livestock, confirmed the presence of four predicted pseudogenes in koala C. pecorum clinical samples. Consistent with our genomics analyses, none of these pseudogenes were observed in the livestock C. pecorum samples examined. Interestingly, three SNPs resulting in pseudogenes identified in the IPTaLE isolate were not found in any other C. pecorum strain analysed, raising questions over the origin of these point mutations.

Conclusions

The genomic data revealed that variation between C. pecorum strains were mainly due to the accumulation of SNPs, some of which cause gene inactivation. The identification of these genetic differences will provide the basis for further studies to understand the biology and evolution of this important animal pathogen.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-667) contains supplementary material, which is available to authorized users.     

Chlamydia buteonis,a new Chlamydia species isolated from a red-shouldered hawk

Chlamydiaceae are obligate intracellular bacterial pathogens for humans and animals. A recent study highlighted that a Chlamydiaceae intermediary between C. psittaci and C. abortus can infect hawks. Here, an isolate was obtained upon passage of cloacal and conjunctival sac material collected from a female hatch-year red-shouldered hawk (Buteo lineatus) in cultured cells. The diseased bird, one of 12 birds housed in a rehabilitation center, developed conjunctivitis and later died. Swabs from both sites tested positive for Chlamydia using the QuickVue Chlamydia test. The isolate, named RSHA, tested negative in qPCR assays specific for C. psittaci and C. abortus, respectively. Analysis of the 16S rRNA, 23S rRNA and whole genome sequences as well as MLST, ANIb and TETRA values reveal that C. psittaci and C. abortus are the closest relatives of RSHA. However, the overall results strongly suggest a phylogenetic intermediate position between these two species. Therefore, we propose the introduction of a new species designated Chlamydia buteonis with RSHA^T as the type strain.     

Molecular genetic characterization of avian <Emphasis Type="Italic">Chlamydophila psittaci</Emphasis> isolates

RFLP analysis of the omp1 gene was used to characterize 51 avian Chlamydophila psittaci isolates. The analysis confirmed the predominance of genotype A in parrot C. psittaci isolates and revealed new omp1 genotypes in corvid C. psittaci isolates. The corvid isolates proved to lack an extrachromosomal plasmid. The omp1 and rRNA IGS sequences were determined for the new isolates. Phylogenetic analysis showed that isolate 1V, obtained from a crow, is intermediate in several characters between C. abortus and C. psittaci. The results were compared with data on the phylogenetic relationships of earlier chlamydium isolates.     

A New Primer Pair for Detection of Chlamydia pneumoniae by Polymerase Chain Reaction

In order to improve the detection and identification of Chlamydia pneumoniae, new primers for polymerase chain reaction (PCR) were designed based on the DNA base sequence within the 53-kDa protein gene, which is specific for C. pneumoniae. The primers permitted the identification of 24 C. pneumoniae strains collected from different geographical locations, but no reaction was observed with C. trachomatis, C. psittaci nor C. pecorum. The primers were unable to amplify the DNA of bacteria commonly related to respiratory tract infections. The positive amplification was achieved with only 9 EBs/assay. Therefore, the new primers seem to be useful in the diagnosis of C. pneumoniae infections.     

Full Genome Sequences of All Nine Chlamydia psittaci Genotype Reference Strains

Chlamydia psittaci primarily infects birds, but zoonotic transmission occurs in people in close contact with infected birds. The clinical outcome ranges from inapparent disease to pneumonia. Here we report the genome sequences of all 9 Chlamydia psittaci genotype reference strains.     

Detection of a novel Chlamydia species in captive spur-thighed tortoises (Testudo graeca) in southeastern Spain and proposal of Candidatus Chlamydia testudinis

The spur-thighed tortoise (Testudo graeca) is an endangered Mediterranean tortoise that lives in North Africa, Southern Europe and Southwest Asia. In the wake of recent legislation making their keeping as domestic animals illegal, many of these animals have been returned to wildlife recovery centers in Spain. In the present study, a population of such tortoises showing signs of ocular disease and nasal discharge was examined for the presence of Chlamydia spp. Cloacal, conjunctival and/or choanal swabs were collected from 58 animals. Using a real-time PCR specific for the family Chlamydiaceae, 57/58 animals tested positive in at least one sample. While only a few samples proved positive for C. pecorum, sequencing of the 16S rRNA gene revealed a sequence identical to previously published sequences from specimens of German and Polish tortoises. Whole-genome sequences obtained from two conjunctival swab samples, as well as ANIb, TETRA values and a scheme based on 9 taxonomic marker genes revealed that the strain present in the Spanish tortoises represented a new yet non-classified species, with C. pecorum being its closest relative. We propose to designate the new species Candidatus Chlamydia testudinis.     

Absence of intervening sequences and point mutations in the V domain within 23S rRNA in Campylobacter lari isolates

Although the absence of intervening sequences (IVSs) within the 23S rRNA genes in Campylobacter lari isolates has been described, there are apparently no reports regarding correlations between the nucleotide sequences of 23S rRNA genes and erythromycin (Ery) susceptibility in C. lari isolates. Here, we determined the minimum inhibitory concentrations of 35 C. lari isolates [n?=?19 for urease-positive thermophilic Campylobacter (UPTC); n?=?16 urease-negative (UN) C. lari] obtained from Asia, Europe, and North America. We found that the 18 isolates were resistant to the Ery (defined as ≧8 μg/mL), and three isolates, UPTC A1, UPTC 92251, and UPTC 504, showed increased resistance (16 μg/mL). No correlations between the IVSs in the helix 45 region within the 23S rRNA gene sequences and Ery resistance were identified in the C. lari isolates examined. In addition, no point mutations occurred at any expected or putative position within the V domain in the isolates. In conclusion, antibiotic resistance against the macrolide erythromycin is mediated through an alternative pathway to that described above.     

Evaluation of the Sensitivity and Specificity of Polymerase Chain Reaction Test Kit,AMPLICOR Chlamydia trachomatis

The sensitivity and specificity of the polymerase chain reaction (PCR) test kit, AMPLICOR Chlamydia trachomatis, were examined by the use of purified elementary bodies (EBs), cells having inclusions containing reticulate bodies alone and 20 clinical isolates. The numbers of EB and inclusion of C. trachomatis at the detection limit were determined to be approximately 2 to 4 EBs and one inclusion per assay, respectively. No reaction occurred for C. psittaci and C. pneumoniae. All clinical isolates were positively reacted in the PCR assay.     

The Chlamydia psittaci genome: a comparative analysis of intracellular pathogens

Background

Chlamydiaceae are a family of obligate intracellular pathogens causing a wide range of diseases in animals and humans, and facing unique evolutionary constraints not encountered by free-living prokaryotes. To investigate genomic aspects of infection, virulence and host preference we have sequenced Chlamydia psittaci, the pathogenic agent of ornithosis.

Results

A comparison of the genome of the avian Chlamydia psittaci isolate 6BC with the genomes of other chlamydial species, C. trachomatis, C. muridarum, C. pneumoniae, C. abortus, C. felis and C. caviae, revealed a high level of sequence conservation and synteny across taxa, with the major exception of the human pathogen C. trachomatis. Important differences manifest in the polymorphic membrane protein family specific for the Chlamydiae and in the highly variable chlamydial plasticity zone. We identified a number of psittaci-specific polymorphic membrane proteins of the G family that may be related to differences in host-range and/or virulence as compared to closely related Chlamydiaceae. We calculated non-synonymous to synonymous substitution rate ratios for pairs of orthologous genes to identify putative targets of adaptive evolution and predicted type III secreted effector proteins.

Conclusions

This study is the first detailed analysis of the Chlamydia psittaci genome sequence. It provides insights in the genome architecture of C. psittaci and proposes a number of novel candidate genes mostly of yet unknown function that may be important for pathogen-host interactions.     

Plaque Formation in Chick Embryo Fibroblast Cells by Chlamydia Isolated From Avian and Mammalian Sources

Fifteen strains of chlamydial agents, 2 Chlamydia trachomatis and 13 Chlamydia psittaci, were tested for plaque formation on chick embryo fibroblast cells. C. trachomatis strains did not form plaques; 12 strains of C. psittaci did form plaques and 1 strain did not. The distribution of plaque sizes with C. psittaci strains was studied and it was found that the 12 strains could be tentatively grouped into three plaquing types: small (<0.50 mm), intermediate (0.50 to 0.90 mm), and large (>0.90 mm). Small plaques were predominantly associated with strains M56, ET, CP3, CT4, EBA, CS1, and CT2. Intermediate size plaques were predominantly associated with strains NJ1, VT1, and H81. Large plaques were predominantly associated with strains WC and OG. Plaque size appears to be related to virulence of Chlamydia for laboratory animals.     

Indirect Hemagglutination Test for Chlamydial Antibodies

An indirect hemagglutination (IHA) test is described for chlamydial antibodies in psittacosis diagnostic sera; for this test tanned sheep erythrocytes sensitized with a deoxycholate extract of Chlamydia psittaci grown in Vero cell monolayers were used. Adaptation of the IHA test to the Microtiter system decreased sensitivity; nevertheless, the Microtiter-IHA test was more sensitive than the complement fixation test. Lymphogranuloma venereum antibodies also were detected by using antigen extracted from C. psittaci.     

Determination of internal transcribed spacer regions (ITS) in Trichomonas vaginalis isolates and differentiation among Trichomonas species

The nucleotide sequence of the 5.8S rRNA gene and the flanked internal transcribed spacer (ITS) regions of six Trichomonas vaginalis isolates with different metronidazole sensitivity and geographic origin were genotyped. A multiple sequence alignment was performed with different sequences of other isolates available at the GenBank/EMBL/DDBJ databases, which revealed 5 different sequence patterns. Although a stable mutation in position 66 of the ITS1 (C66T) was observed in 26% (9/34) of the T. vaginalis sequences analyzed, there was 99.7% ITS nucleotide sequence identity among isolates for this sequence. The nucleotide sequence variation among other species of the genus Trichomonas ranged from 3.4% to 9.1%. Surprisingly, the % identity between T. vaginalis and Pentatrichomonas hominis was ~ 83%. There was > 40% divergence in the ITS sequence between T. vaginalis and Tritrichomonas spp., including Tritrichomonas augusta, Tritrichomonas muris, and Tritrichomonas nonconforma and with Tetratrichomonas prowazeki. Dendrograms grouped the trichomonadid sequences in robust clades according to their genera. The absence of nucleotide divergence in the hypervariable ITS regions between T. vaginalis isolates suggests the early divergence of the parasite. Importantly, these data show this ITS1-5.8S rRNA-ITS2 region suitable for inter-species differentiation.     

Genetic Analysis of Type E Botulinum Toxin-Producing Clostridium butyricum Strains

Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area.     

A Real-Time PCR Assay for the Detection of Atypical Strains of Chlamydiaceae from Pigeons

Recent evidence of the occurrence of atypical Chlamydiaceae strains in pigeons, different from the established Chlamydiaceae, requires the development of a specific and rapid detection tool to investigate their prevalence and significance. Here is described a new real-time PCR assay that allows specific detection of atypical Chlamydiaceae from pigeons. The assay has been used to assess the dissemination of these strains in field samples collected from Parisian pigeon populations in 2009. The results suggest a limited dissemination compared to the usually higher prevalence of Chlamydia psittaci that is the main species associated with avian chlamydiosis.     

Genetic Diversity of Clostridium sporogenes PA 3679 Isolates Obtained from Different Sources as Resolved by Pulsed-Field Gel Electrophoresis and High-Throughput Sequencing

Clostridium sporogenes PA 3679 is a nonpathogenic, nontoxic model organism for proteolytic Clostridium botulinum used in the validation of conventional thermal food processes due to its ability to produce highly heat-resistant endospores. Because of its public safety importance, the uncertain taxonomic classification and genetic diversity of PA 3679 are concerns. Therefore, isolates of C. sporogenes PA 3679 were obtained from various sources and characterized using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. The phylogenetic relatedness and genetic variability were assessed based on 16S rRNA gene sequencing and whole-genome single nucleotide polymorphism (SNP) analysis. All C. sporogenes PA 3679 isolates were categorized into two clades (clade I containing ATCC 7955 NCA3679 isolates 1961-2, 1990, and 2007 and clade II containing PA 3679 isolates NFL, UW, FDA, and Campbell and ATCC 7955 NCA3679 isolate 1961-4). The 16S maximum likelihood (ML) tree clustered both clades within proteolytic C. botulinum strains, with clade I forming a distinct cluster with other C. sporogenes non-PA 3679 strains. SNP analysis revealed that clade I isolates were more similar to the genomic reference PA 3679 (NCTC8594) genome (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AGAH00000000.1","term_id":"364889226","term_text":"AGAH00000000.1"}}AGAH00000000.1) than clade II isolates were. The genomic reference C. sporogenes PA 3679 (NCTC8594) genome and clade I C. sporogenes isolates were genetically distinct from those obtained from other sources (University of Wisconsin, National Food Laboratory, U.S. Food and Drug Administration, and Campbell''s Soup Company). Thermal destruction studies revealed that clade I isolates were more sensitive to high temperature than clade II isolates were. Considering the widespread use of C. sporogenes PA 3679 and its genetic information in numerous studies, the accurate identification and genetic characterization of C. sporogenes PA 3679 are of critical importance.     

广西不同时期IBV分离株S1基因高变区Ⅰ的遗传变异分析

对广西1985-2007年间分离到的22株传染性支气管炎病毒（IBV）的S1基因高变区I（HVRI）进行序列测定,并与发表的其他IBV参考株及鸽子分离的冠状病毒株的基因序列进行比较和分析。系统进化关系显示毒株可分为5个基因群,其中有16个广西分离株属第1群,它们与鸽子冠状病毒分离株的氨基酸序列同源性较高,与Massachusetts（Mass）型疫苗株的同源性较低。有15个分离株在33-34位和34～35之间分别有4个和3个氨基酸残基的插入,GX-NN6在33～34位和34～35位之间则均有4个氨基酸残基的插入;GX-YL1、GX-NN2与常用的Mass型疫苗株的亲缘关系最近,同属于第Ⅱ群;GX-G、GX-XD与日本同一时期分离的毒株JP Miyazaki 89亲缘关系最近,属于第Ⅲ群;GX-YL6、GX-NN7与欧洲毒株4／91亲缘关系较近,属于第V群。结果表明广西存在着多种类型IBV毒株的流行,毒株S1基因HVRI碱基的突变或插入比较普遍,可导致其氨基酸序列的变化,绝大部分毒株与目前常用的Mass型疫苗株的亲缘关系较低。同一时期的分离株同源性较高,但无明显的地域性差异。     

Phenotypic and molecular identification of Sporothrix isolates of clinical origin in Northeast China

Sporotrichosis is the most common deep mycosis in Northeast China which is an area of high epidemicity due to contact with reeds or cornstalks. In this study, we have characterized a total of 74 clinical isolates from fixed cutaneous, lymphocutaneous and disseminated clinical forms and from Heilongjiang, Jilin, and Liaoning provinces, respectively. All isolates (previously as Sporothrix schenckii) were identified as Sporothrix globosa according to their phenotypic characteristics and calmodulin gene sequences analysis. They were subdivided into two sub-clades (S. globosa I and S. globosa II). Most of our isolates (71/74) presented restricted growth at 37 °C, which differed from a previous report. Up to now, S. globosa is the only pathogenic species in Northeast China, no matter what kind of clinical form and which region it is isolated from. Most of our clinical isolates (68/74) were clustered with three Chinese environmental isolates reported in the literature. The new findings of S. globosa isolates on division and thermotolerance at 37 °C described in this study will help us gain a better understanding of S. globosa.