ompA Sequences Research Articles

Homology analysis and cross-immunogenicity of OmpA from pathogenic Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis.

The outer membrane protein A (OmpA) is one of the intra-species conserved proteins with immunogenicity widely found in the family of Enterobacteriaceae. Here we first confirmed OmpA is conserved in the three pathogenic Yersinia: Yersinia pestis, Yersinia pseudotuberculosis and pathogenic Yersinia enterocolitica, with high homology at the nucleotide level and at the amino acid sequence level. The identity of ompA sequences for 262 Y. pestis strains, 134 Y. pseudotuberculosis strains and 219 pathogenic Y. enterocolitica strains are 100%, 98.8% and 97.7% similar. The main pattern of OmpA of pathogenic Yersinia are 86.2% and 88.8% identical at the nucleotide and amino acid sequence levels, respectively. Immunological analysis showed the immunogenicity of each OmpA and cross-immunogenicity of OmpA for pathogenic Yersinia where OmpA may be a vaccine candidate for Y. pestis and other pathogenic Yersinia.

COMPARISON OF FOUR MOLECULAR TYPING METHODS FOR RIEMERELLA ANATIPESTIFER

The pathogen causing septicaemiae and infectious serositis in waterfowl, Riemerella anatipestifer has been categorized as a member of Flavobacteriaceae by 16S rDNA sequence analysis and at least 21 serotypes have been characterized through serum agglutination test. So far, the molecular determinants for R. anatipestifer serotyping are not clear; moreover, serotype analysis is time-consuming and labor-intensive. To find a better molecular typing method for R. anatipestifer, 58 field isolates collected from sick birds in central Taiwan from 2006 to 2008 were investigated by molecular methods to determine serotype-associated genetic variations. Genetic variations were determined by the methods of ITS nucleotide sequence, OmpA amino acid sequence, pulsed-field gel electrophoresis (PFGE), and repetitive-sequence PCR (Rep-PCR). The phylogenetic trees constructed by the ITS region showed nine clusters, while the amino acid sequence of OmpA clustered into seven clusters. OmpA sequence correlated much better with serotyping than ITS, PFGE, and Rep-PCR. Meanwhile, the biofilm formation activity was also tested for serotypes 2 and 8, which were the two largest groups in the study. Serotype 2 isolates showed a higher degree of biofilm formation than serotype 8 isolates. In conclusion, we demonstrated that phylogenetic analysis with OmpA amino acid sequence is an effective tool in epidemiology and may be a candidate method substituting serotyping for R. anatipestifer. Moreover, the biofilm forming activity may play an important role in field transmission of R. anatipestifer.

High detection rate of Rickettsia africae in Amblyomma variegatum but low prevalence of anti-rickettsial antibodies in healthy pregnant women in Madagascar

Tick-borne spotted fever group (SFG) rickettsioses are emerging infectious diseases in Sub-Saharan Africa. In Madagascar, the endemicity of tick-borne rickettsiae and their vectors has been incompletely studied. The first part of the present study was conducted in 2011 and 2012 to identify potential anthropophilic tick vectors for SFG rickettsiae on cattle from seven Malagasy regions, and to detect and characterize rickettsiae in these ticks. Amblyomma variegatum was the only anthropophilic tick species found on 262 cattle. Using a novel ompB-specific qPCR, screening for rickettsial DNA was performed on 111 A. variegatum ticks. Rickettsial DNA was detected in 96 of 111 ticks studied (86.5%). Rickettsia africae was identified as the only infecting rickettsia using phylogenetic analysis of ompA and ompB gene sequences and three variable intergenic spacers from 11 ticks. The second part of the study was a cross-sectional survey for antibodies against SFG rickettsiae in plasma samples taken from healthy, pregnant women at six locations in Madagascar, two at sea level and four between 450 and 1300m altitude. An indirect fluorescent antibody test with Rickettsia conorii as surrogate SFG rickettsial antigen was used. We found R. conorii-seropositives at all altitudes with prevalences between 0.5% and 3.1%. Our results suggest that A. variegatum ticks highly infected with R. africae are the most prevalent cattle-associated tick vectors for SFG rickettsiosis in Madagascar. Transmission of SFG rickettsiosis to humans occurs at different altitudes in Madagascar and should be considered as a relevant cause of febrile diseases.

Genotyping and Source Tracking of Cronobacter sakazakii and C. malonaticus Isolates from Powdered Infant Formula and an Infant Formula Production Factory in China.

Cronobacter spp. (formerly defined as Enterobacter sakazakii) are opportunistic bacterial pathogens of both infants and adults. In this study, we analyzed 70 Cronobacter isolates from powdered infant formula (PIF) and an infant formula production facility in China to determine possible contamination routes. The strains were profiled by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), PCR-based O-antigen serotyping, and ompA and rpoB sequence analyses. The isolates were primarily Cronobacter sakazakii (66/70) or Cronobacter malonaticus (4/70). The strains were divided into 38 pulsotypes (PTs) using PFGE and 19 sequence types (STs) by MLST. In contrast, rpoB and ompA sequence analyses divided the strains into 10 overlapping clusters each. PCR serotyping of the 66 C. sakazakii and 4 C. malonaticus strains resulted in the identification of four C. sakazakii serotypes (O1, O2, O4, and O7) and a single C. malonaticus serotype, O2. The dominant C. sakazakii sequence types from PIF and an infant formula production factory in China were C. sakazakii clonal complex 4 (CC4) (n = 19), ST1 (n = 14), and ST64 (n = 11). C. sakazakii CC4 is a clonal lineage strongly associated with neonatal meningitis. In the process of manufacturing PIF, the spray-drying, fluidized-bed-drying, and packing areas were the main areas with Cronobacter contamination. C. sakazakii strains with the same pulsotypes (PT3 and PT2) and sequence types (ST1 and ST64) were isolated both from processing equipment and from the PIF finished product.

Chlamydia trachomatis genotypes in school adolescents, Italy.

Chlamydia trachomatis genogroups using ompA and multilocus sequence typing (MLST) were determined in consecutive isolates from school students aged 18 or older in the district of Brescia, Italy, 2012-2013. Among 40 samples, 4 ompA genovars and 18 STs were identified. Genovar E predominated (70 %) including five STs derived from ST59 (29 % of all isolates). This study, combining ompA and MLST typing of C. trachomatis school teenagers, suggests limited mixing and sexual interchange in this population.

Distribution of the ompA-types among ruminant and swine pneumonic strains of Pasteurella multocida exhibiting various cap-locus and toxA patterns

Pasteurella multocida is an important pathogen in food-producing animals and numerous virulence genes have been identified in an attempt to elucidate the pathogenesis of pasteurellosis. Currently, some of these genes including the capsule biosynthesis genes, the toxA and the OMPs-encoding genes have been suggested as epidemiological markers. However, the number of studies concerning ruminant isolates is limited, while, no attempt has ever been made to investigate the existence of ompA sequence diversity among P. multocida isolates. The aim of the present study was the comparative analysis of 144 P. multocida pneumonic isolates obtained from sheep, goats, cattle and pigs by determining the distribution of the ompA-types in conjunction with the cap-locus and toxA patterns. The ompA genotypes of the isolates were determined using both a PCR-RFLP method and DNA sequence analysis. The most prevalent capsule biosynthesis gene among the isolates was capA (86.1%); a noticeable, however, rate of capD-positive isolates (38.6%) was found among the ovine isolates that had been associated primarily with the capsule type A in the past. Moreover, an unexpectedly high percentage of toxA-positive pneumonic isolates was noticed among small ruminants (93.2% and 85.7% in sheep and goats, respectively), indicating an important epidemiological role of toxigenic P. multocida for these species. Despite their great heterogeneity, certain ompA-genotypes were associated with specific host species, showing evidence of a host preference. The OmpA-based PCR-RFLP method developed proved to be a valuable tool in typing P. multocida strains.

Chlamydia trachomatis genovars causing urogenital infections in Santiago, Chile

Background: Chlamydia trachomatis is a common sexually transmitted infection in Chile, but little is known about the genovar distribution in genital infections. Thus, the objective of this study was to determine the distribution of C. trachomatis genovars in such cases. Methods: A total of 522 urogenital specimens, 403 from women and 119 from men, were analyzed for C. trachomatis by nested polymerase chain reaction (PCR) targeting of the ompA gene. Positive specimens were genotyped by DNA sequencing of the amplicons. Results: Sixty-two (11.9%) specimens were positive. Of these, 43 (69.4%) were collected from men and 19 (30.6%) from women (p < 0.0001). Eight genovars were identified in men and seven in women. Genovar E was the most common in both men and women, followed by genovar Da in men, and F in women. Together these three genovars accounted for 84% of infections. Genovar D was the third most common genovar (n = 4). Genovar G was detected in two samples, and sequences of genovars Ba, H, and Ja were each found in single samples. One sample (1.6%) contained mixed sequences. No association was found between gender and specific genovars. Fifty-six (92%) sequences were identical to those reported for the respective reference genovars and the other two have been described in several regions. Conclusions: Our findings add to the results of most studies, which indicate that genovars E, F, and D/Da are the most frequent. No association was found between gender and specific genovars. Despite the heterogeneous population of genovars, most ompA sequences were conserved.

Epidemiology of Chlamydia psittaci Infection in Racing Pigeons and Pigeon Fanciers in Beijing, China.

Over 3 million racing pigeons (Columba livia) are registered in Beijing City Center for gambling purposes. During 2008-2010, we evaluated the occurrence and prevalence of Chlamydia psittaci in racing pigeons as well as the possible zoonotic transmission to pigeon fanciers in six districts of Beijing where pigeon races are particularly popular. C. psittaci-specific serum antibody titres were obtained from 370 pigeons and 79 fanciers using enzyme-linked immunosorbent assay. In addition, 206 and 67 throat swabs were, respectively, collected from pigeons and fanciers and tested for the presence of chlamydial antigen using immunofluorescence. C. psittaci-specific serum antibody was detected in 37 of 370 pigeons and 19 of 79 fanciers. Of 206 pigeon clinical specimens, 55 were positive for C. psittaci antigen, while 16 of 67 swabs from the pigeon fanciers were positive. Based on ompA sequence analysis, the genotype of several avian and human isolates was genotype B. Thus, both high-titre C. psittaci-specific antibody and C. psittaci-specific antigen were found with relatively high frequency in the pigeon flocks as well as in the pigeon fanciers. Our study suggests that C. psittaci infection is prevalent among the racing pigeon population in Beijing. Moreover, detection of serum antibodies and antigen in pigeon fanciers suggests that exposure and possible zoonotic transmission of C. psittaci from racing pigeons to humans does occur. In view of the life-threatening respiratory illness C. psittaci may cause in humans, regulatory public health measures, to prevent further spread of the pathogen in avian populations and possible transmission to exposed humans, are urgently needed.

Detection of an undescribed Rickettsia sp. in Ixodes boliviensis from Costa Rica

Ixodes boliviensis is a tick of carnivores that is common on domestic dogs. The only Rickettsia that has been detected previously in this species is ‘Candidatus Rickettsia andeanae’. We report the detection of an undescribed Rickettsia sp., named strain IbR/CRC, in I. boliviensis collected from dogs in Costa Rica. Analyses of gltA, ompA, and htrA partial sequences place Rickettsia sp. strain IbR/CRC in the group of R. monacensis, also close to an endosymbiont of Ixodes scapularis and other undescribed rickettsiae. It was not possible to isolate Rickettsia sp. strain IbR/CRC in Vero E6 or C6/36 cell lines. Isolation and further characterization of Rickettsia sp. strain IbR/CRC and the other undescribed rickettsiae are required to determine their taxonomic status and pathogenic potential.

Seroprevalence and genotype of Chlamydia in pet parrots in China

Parrots are one of the most popular pet birds in China, and can harbour Chlamydia which has significance for human and animal health. We investigated, by indirect haemagglutination assay, the seroprevalence of Chlamydia infection in four species of parrots, namely budgerigars (Melopsittacus undulatus), lovebirds (Agapornis sp.), cockatiels (Nymphicus hollandicus) and Alexandrine parakeets (Psittacula eupatria) that were collected from Weifang and Beijing cities, North China and explored the association between potential risk factors and chlamydial seropositivity. We further determined the genotype of Chlamydia in 21 fresh faecal samples based on the ompA sequence by reconstruction of phylogenetic relationships. Of the 311 parrots examined, 35·37% (95% confidence interval 30·06-40·68) were seropositive, and species, gender, age, season and geographical location were identified as risk factors. Two PCR-positive samples represented Chlamydia psittaci genotype A. The occurrence of C. psittaci genotype A in the droppings of two pet parrots in China suggests potential environmental contamination with Chlamydiaceae and may raise a public health concern.

Detecção e classificação molecular de Chlamydophila psittaci em amostras fecais de aves assintomáticas

Chlamydophila psittaci é uma bactéria que causa doença respiratória ou sistêmica em aves e em seres humanos. Em vista do risco de transmissão para humanos, o objetivo deste estudo foi detectar a presença de Chlamydophila spp. em amostras de fezes ou suabes cloacais de aves assintomáticas. Foram colhidas 403 amostras fecais ou suabes cloacais, provenientes de aves domésticas, selvagens ou exóticas. As amostras foram submetidas à PCR em tempo real para C. psittaci, para amplificação de fragmento parcial do gene da subunidade 16S do rRNA, utilizando o SsoFastTM EvaGreen® Supermix (Bio-Rad) e análise da curva de dissociação. Para determinação do genótipo de C. psittaci, foi usada a hemi-nested PCR visando à amplificação de fragmento parcial do gene OMP-A, realizada nas amostras positivas pela PCR em tempo real, seguida de sequenciamento dos fragmentos amplificados. A PCR em tempo real revelou positividade em 17 (4,21%) amostras. A hemi-nested foi positiva em 2 amostras positivas pela PCR em tempo real. O genótipo A de C. psittaci foi identificado pelo sequenciamento de uma amostra amplificada pela hemi-nested PCR. Os resultados deste experimento demonstram que a PCR em tempo real, visando à amplificação de fragmento parcial da subunidade 16S do rRNA, seguida da análise da curva de dissociação, pode ser utilizada para detecção de DNA de Chlamydophila sp. em amostras fecais de aves assintomáticas. A classificação da espécie de Chlamydophila e do genótipo de C. psittaci deve ser realizada por meio de PCR tendo como alvo o gene ompA e sequenciamento dos fragmentos amplificados.

Culture-independent sequence analysis of Chlamydia trachomatis in urogenital specimens identifies regions of recombination and in-patient sequence mutations

A culture-independent genome sequencing approach was developed and used to examine genomic variability in Chlamydia trachomatis-positive specimens that were collected from patients in the Seattle, WA, USA, area. The procedure is based on an immunomagnetic separation approach with chlamydial LPS-specific mAbs, followed by DNA purification and total DNA amplification, and subsequent Illumina-based sequence analysis. Quality of genome sequencing was independent of the total number of inclusion-forming units determined for the sample and the amount of non-chlamydial DNA in the Illumina libraries. A geographically and temporally linked clade of isolates was identified with evidence of several different regions of recombination and variable ompA sequence types, suggesting that recombination is common within outbreaks. Culture-independent sequence analysis revealed a linkage pattern at two nucleotide positions that was unique to the genomes of isolates from patients, but not in C. trachomatis recombinants generated in vitro. These data demonstrated that culture-independent sequence analysis can be used to rapidly and inexpensively collect genome data from patients infected by C. trachomatis, and that this approach can be used to examine genomic variation within this species.

Spotted Fever Group Rickettsiae in Questing Ticks, Central Spain

Spotted Fever Group Rickettsiae in Questing Ticks, Central Spain

Detection of a new Non-Classified Chlamydia Species in Hens in Poland

Abstract The outbreak of chlamydiosis in one of the western provinces of Poland, was diagnosed accidentally as a concurrent infection in a commercial laying hen flock during an outbreak of fowl pox. For histological examination, skin and subcutaneous tissue samples from lesions on heads of the birds were collected. Swabs from throat and trachea have been examined by nested PCR, real-time PCR, and partial ompA sequencing. Detailed electron microscopy analysis revealed fowl pox intracytoplasmic inclusions, called Bollinger bodies, and the presence of other intracytoplasmic inclusions; specific for Chlamydia sp. Results of nested PCR confirmed the presence of Chlamydiaceae sp. in two tested samples. Surprisingly, one of the two Chlamydiaceae-positive cases turned out to be infected with a non-classified strain. Results of real-time PCR and sequencing confirmed the presence of a new Chlamydia species that has not been found in Poland to date. Partial sequencing and BLAST analysis of ompA gene sequence confirmed the highest homology to non-classified poultry strains of Chlamydia sp. that were previously detected in Germany and France. The zoonotic potential and the exact taxonomic status of this atypical strain have yet to be defined.

Molecular evidence for the presence of Rickettsia Felis in the feces of wild-living African apes.

Background Rickettsia felis is a common emerging pathogen detected in mosquitoes in sub-Saharan Africa. We hypothesized that, as with malaria, great apes may be exposed to the infectious bite of infected mosquitoes and release R. felis DNA in their feces.MethodsWe conducted a study of 17 forest sites in Central Africa, testing 1,028 fecal samples from 313 chimpanzees, 430 gorillas and 285 bonobos. The presence of rickettsial DNA was investigated by specific quantitative real-time PCR. Positive results were confirmed by a second PCR using primers and a probe targeting a specific gene for R. felis. All positive samples were sequenced.ResultsOverall, 113 samples (11%) were positive for the Rickettsia-specific gltA gene, including 25 (22%) that were positive for R. felis. The citrate synthase (gltA) sequence and outer membrane protein A (ompA) sequence analysis indicated 99% identity at the nucleotide level to R. felis. The 88 other samples (78%) were negative using R. felis-specific qPCR and were compatible with R. felis-like organisms.ConclusionFor the first time, we detected R. felis in wild-living ape feces. This non invasive detection of human pathogens in endangered species opens up new possibilities in the molecular epidemiology and evolutionary analysis of infectious diseases, beside HIV and malaria.

Multilocus Sequence Typing of Genital Chlamydia trachomatis in Norway Reveals Multiple New Sequence Types and a Large Genetic Diversity

BackgroundThe Chlamydia trachomatis incidence rate in Finnmark, the most northern and sparsely populated county in Norway, has been twice the national average. This population based cross-sectional study among Finnmark high school students had the following aims: i) to examine distribution of multilocus sequence types (STs) of C. trachomatis in a previously unmapped area, ii) to compare chlamydia genetic diversity in Finnmark with that of two urban regions, and iii) to compare discriminatory capacity of multilocus sequence typing (MLST) with conventional ompA sequencing in a large number of chlamydia specimens.Methodology ompA sequencing and a high-resolution MLST system based on PCR amplification and DNA sequencing of five highly variable genetic regions were used. Eighty chlamydia specimens from adolescents aged 15–20 years in Finnmark were collected in five high schools (n = 60) and from routine clinical samples in the laboratory (n = 20). These were compared to routine clinical samples from adolescents in Tromsø (n = 80) and Trondheim (n = 88), capitals of North and Central Norway, respectively.Principal Findings ompA sequencing detected 11 genotypes in 248 specimens from all three areas. MLST displayed 50 STs providing a five-fold higher resolution. Two-thirds of all STs were novel. The common ompA E/Bour genotype comprised 46% and resolved into 24 different STs. MLST identified the Swedish new variant of C. trachomatis not discriminated by ompA sequencing. Simpson's discriminatory index (D) was 0.93 for MLST, while a corrected Dc was 0.97. There were no statistically significant differences in ST genetic diversity between geographic areas. Finnmark had an atypical genovar distribution with G being predominant. This was mainly due to expansion of specific STs of which the novel ST161 was unique for Finnmark.Conclusions/SignificanceMLST revealed multiple new STs and a larger genetic diversity in comparison to ompA sequencing and proved to be a useful tool in molecular epidemiology of chlamydia infections.

Molecular detection and analysis of spotted fever group Rickettsia in patients with fever and rash at a tertiary care centre in Tamil Nadu, India

Background: Detection of specific targets by PCR is used to confirm a diagnosis of spotted fever, but serological tests are still widely used. In this prospective study, nested PCR was performed on skin biopsy specimens to confirm the diagnosis of spotted fever.Methods: In 58 clinically suspected cases of spotted fever, nested PCR, to detect gltA, 17 kDa lipoprotein antigen gene (17 kDa), ompA and ompB, from skin biopsy of the rash was performed. Sequencing was carried on amplicons representing the four targets to confirm specificity of amplification. This was followed by phylogenetic analysis using MEGA version 4.0 software.Results: The gltA, 17 kDa, ompA, and ompB genes were detected from skin biopsy specimens in 38, 23, 27, and 22 individuals. Sequence analysis revealed that the gltA, 17 kDa, ompA, and ompB sequences belonged to spotted fever group (SFG) rickettsia. Of the six partial ompA gene sequences, only one was dissimilar to the previously reported ‘Candidatus Rickettsia kellyi’.Conclusion: Further evidence indicates that SFG rickettsiae resembling ‘Candidatus Rickettsia kellyi’ cause fever and rash in southern India. More detailed phylogenetic analysis following isolation of rickettsia in culture is required for providing irrefutable proof for the occurrence of novel spotted fever rickettsiae in this region.

First report on the occurrence of Rickettsia slovaca and Rickettsia raoultii in Dermacentor silvarum in China

BackgroundRickettsioses are among both the longest known and most recently recognized infectious diseases. Although new spotted fever group rickettsiae have been isolated in many parts of the world including China, Little is known about the epidemiology of Rickettsia pathogens in ticks from Xinjiang Autonomous Region of China.MethodsIn an attempt to assess the potential risk of rickettsial infection after exposure to ticks in Xinjiang Uygur Autonomous Region of China, a total of 200 Dermacentor silvarum ticks collected in Xinyuan district were screened by polymerase chain reaction based on the outer membrane protein A gene.Results22 of the 200 specimens (11%) were found to be positive by PCR. Phylogenetic analysis of OmpA sequences identified two rickettsial species, Rickettsia raoultii (4.5%) and Rickettsia slovaca (6.5%).ConclusionsThis study has reported the occurrence of Rickettsia raoultii and Rickettsia slovaca in Xinjiang Autonomous Region of China and suggests that Dermacentor silvarum could be involved in the transmission of rickettsial agents in China. Further studies on the characterization and culture of rickettsial species found in Dermacentor silvarum should be performed to further clarify this. Additionally, the screening of human specimens for rickettsial disease in this region will define the incidence of infection.

More than classical Chlamydia psittaci in urban pigeons

In the literature, studies of Chlamydia infection in birds have usually been confined to the search for Chlamydia (C., formerly Chlamydophila) psittaci, so that little is known about the presence of other chlamydial agents. In the present study, cloacal swabs and faeces samples of urban pigeons have been examined by real-time PCR, DNA microarray assays and partial ompA sequencing. Whilst C. psittaci was the predominant chlamydial agent in this pigeon population (75.8% of all Chlamydiaceae positives), the combined use of highly specific and sensitive molecular assays facilitated the detection of atypical serovars of C. psittaci, as well as other species of Chlamydia, such as C. abortus. Detection of C. pecorum and C. trachomatis from an avian host is reported here for the first time. Rather unexpectedly, 19.5% of all Chlamydiaceae-positive cases turned out to be infected with non-classified organisms. The considerable prevalence of these novel agents raises the question of their epidemiological importance and possible role as pathogens. Future surveys in domestic and wild birds will have to take the extended variety of chlamydial organisms into account.

Impact of urban environment and host phenotype on the epidemiology ofChlamydiaceaein feral pigeons (Columba livia)

Chlamydiaceae are obligate intracellular Gram-negative bacteria found all over the world and known to cause various forms of disease in animals and humans. Urban pigeons are known to be an important reservoir of Chlamydia psittaci, the agent of human psittacosis. In this study, we examined the influence of pigeon houses used to regulate pigeon populations and of melanin-based coloration on several epidemiological parameters of Chlamydiaceae in 708 urban pigeons in Paris. We also identified species and genotypes of Chlamydiaceae present in Parisian populations. First, our results revealed that pigeons roosting and breeding in pigeon houses were equally infected by Chlamydiaceae as those that did not. Second, we found that dark melanic pigeons excreted more Chlamydiaceae than pale melanic ones. Finally, species and strain diversities were very low: all samples were of C. psittaci genotype B. Nevertheless, two atypical Chlamydiaceae were identified based on 16S rRNA and ompA sequences. Our study thus highlights the importance of considering environmental and host phenotype when investigating the epidemiology of infectious diseases.