groESL Heat Shock Operon Research Articles

A novel Ehrlichia strain (Rickettsiales: Anaplasmataceae) detected in Amblyomma triste (Acari: Ixodidae), a tick species of public health importance in the Southern Cone of America

ABSTRACT The aim of this work was to report the detection of a putative novel Ehrlichia strain associated with the tick Amblyomma triste. Free-living adult ticks determined as A. triste were collected by drag-sampling in Argentina and Uruguay. Molecular detection of Ehrlichia agents was performed targeting three different loci: 16S rRNA gene, dsb gene and a fragment of groESL heat shock operon. In total, 164 adults of A. triste (38 from INTA E.E.A Delta del Paraná in Argentina and 126 from Toledo Chico in Uruguay) were analyzed. One tick (0.6%) collected in INTA E.E.A. Delta del Paraná (Argentina) was positive. The phylogenetic analyses show that the Ehrlichia strain found in this study (named Ehrlichia sp. strain Delta) represents an independent lineage within the genus Ehrlichia, close to E. chaffeensis and E. muris. This is also the first report of an Ehrlichia agent infecting the tick A. triste. The medical and veterinary significance of Ehrlichia sp. strain Delta remains to be demonstrated. However, it is important to mention that adults of A. triste are aggressive to humans and domestic mammals. Therefore, the potential role of A. triste in the transmission of Ehrlichia agents to humans or domestic animals across its distributional range should be highlighted, even more considering that Ehrlichia sp. strain Delta is phylogenetically related to the zoonotic E. chaffeensis, which is recognized as pathogenic to both humans and animals.

Two novel Ehrlichia strains detected in Amblyomma tigrinum ticks associated to dogs in peri-urban areas of Argentina.

The aim of this work was to describe two novel strains of Ehrlichia associated to Amblyomma tigrinum from Argentina. Molecular detection of agents belonging to the family Anaplasmataceae was performed targeting three different loci: 16S rRNA gene, dsb gene and a fragment of groESL heat shock operon. The results have shown that two different strains of Ehrlichia sp. associated to A. tigrinum are circulating in peri-urban areas of Argentina. The Ehrlichia strain detected in ticks from San Luis Province, named as Ehrlichia sp. strain San Luis, is closely related to the Ehrlichia chaffeensis. The novel Ehrlichia strain detected in Córdoba Province, named as Ehrlichia sp. strain Córdoba, is phylogenetically related to three Ehrlichia strains from Brazil, two of them isolated from wild carnivorous and the third one isolated from horse. Even though Ehrlichia sp. strain Córdoba was clustered with the three Ehrlichia strains from Brazil, the genetic similarity was too low to consider them as the same taxonomic entity. Blood samples of dogs were positive to Anaplasma platys. The association of these two novel strains with A. tigrinum has epidemiological relevance because adult stages of this tick species are common parasite of dogs in rural and peri-urban areas and they are aggressive to humans. The presence of these two novel Ehrlichia strains implies a potential epidemiological risk in Argentina because the species of the genus Ehrlichia are known to be pathogenic to both domestic mammals and humans.

Molecular epidemiology of the emerging zoonosis agent Anaplasma phagocytophilum (Foggie, 1949) in dogs and ixodid ticks in Brazil.

BackgroundAnaplasma phagocytophilum is an emerging pathogen of humans, dogs and other animals, and it is transmitted by ixodid ticks. The objective of the current study was a) detect A. phagocytophilum in dogs and ixodid ticks using real-time Polymerase Chain Reaction (qPCR); and b) Determine important variables associated to host, environment and potential tick vectors that are related to the presence of A. phagocytophilum in dogs domiciled in Rio de Janeiro, Brazil.MethodsWe tested blood samples from 398 dogs and samples from 235 ticks, including 194 Rhipicephalus sanguineus sensu lato, 15 Amblyomma cajennense, 8 Amblyomma ovale and 18 pools of Amblyomma sp. nymphs. A semi-structured questionnaire was applied by interviewing each dog owner. Deoxyribonucleic acid obtained from ticks and dog buffy coat samples were amplified by qPCR (msp2 gene). The sequencing of 16S rRNA and groESL heat shock operon genes and a phylogenetic analysis was performed. The multiple logistic regression model was created as a function of testing positive dogs for A. phagocytophilum.ResultsAmong the 398 blood samples from dogs, 6.03% were positive for A. phagocytophilum. Anaplasma phagocytophilum was detected in one A. cajennense female tick and in five R. sanguineus sensu lato ticks (four males and one female). The partial sequences of the 16S rRNA, and groESL genes obtained were highly similar to strains of A. phagocytophilum isolated from wild birds from Brazil and human pathogenic strains. The tick species collected in positive dogs were R. sanguineus sensu lato and A. cajennense, with A.cajennense being predominant. Tick infestation history (OR = 2.86, CI = 1.98-14.87), dog size (OR = 2.41, IC: 1.51-12.67), the access to forest areas (OR = 3:51, CI: 1.52-16.32), hygiene conditions of the environment in which the dogs lived (OR = 4.35, CI: 1.86-18.63) and Amblyomma sp. infestation (OR = 6.12; CI: 2.11-28.15) were associated with A. phagocytophilum infection in dogs.ConclusionsThis is the first report of A. phagocytophilum in ixodid ticks from Brazil. The detection of A. phagocitophylum in A. cajennense, an aggressive feeder on a wide variety of hosts, including humans, is considered a public health concern.

Anaplasma infections in ticks and reservoir host from Slovakia

Anaplasma phagocytophilum is a worldwide distributed bacterium with a significant medical and veterinary importance. It grows within the phagosome of infected neutrophils and is responsible for human granulocytic anaplasmosis (HGA), tick-borne fever (TBF) of small ruminants and cattle, canine and equine granulocytic anaplasmosis, but infects also a great variety of wildlife species. Wild ungulates and rodents are considered reservoirs of infection in natural foci. The objective of this study was to determine the spectrum of animal species involved in the circulation of A. phagocytophilum in Slovakia and to analyze the variability of obtained nucleotide sequences, in order to determine whether genotypes from Slovakia cluster according to host-species or geographical location.Several animal species and vector ticks were screened for the presence of members of the family Anaplasmataceae using PCR based methods. Additional data on the molecular evidence of Anaplasma ovis and Candidatus Neoehrlichia mikurensis are presented. These pathogens were detected in tested sheep flocks and rodents with the mean infection rates of 8.16% and 10.75%, respectively. A. phagocytophilum was genotyped by 16S rRNA and groEL gene sequencing. Bacterial DNA was confirmed in questing ixodid ticks, in domesticated canine, wild rodents and several species of wild ungulates.In European isolates, 16S rRNA gene does not seem to be an appropriate locus for the analyses of heterogeneity as it is too conservative. Similarly, 16S rRNA isolates from our study did not reveal any polymorphisms. All isolates were identical in overlapped region and showed identity with sequences from ticks, horses or ruminants previously isolated elsewhere in the world. On the other hand, the groESL heat shock operon is widely used for determination of diversity and the analyses have already revealed considerable degree of heterogeneity.Tested ungulates were infected with A. phagocytophilum to a considerable extent. High proportions of red and roe deer tested positive and the rates of infection reached over 60.0%. GroEL sequences from canine, wild ungulates and ticks from Slovakia clustered within a clade together with isolates from horses, humans, wild ungulates and ticks from Slovakia or elsewhere in the world. Sequences from rodents clustered apart from those obtained from wild ungulates, ticks and humans. These results suggest that European rodents do not harbour A. phagocytophilum strains with strong zoonotic potential such as those from United States.

Anaplasmaspp. in Wild Mammals andIxodes ricinusfrom the North of Spain

Our objectives were to investigate the presence of Anaplasma spp. infection in red deer, wild boars, and Ixodes ricinus removed from deer surveyed in La Rioja, as well as to analyze the presence of Anaplasma spp. in I. ricinus from different Spanish regions--ours included. A total of 21 deer and 13 wild boar blood samples as well as 295 I. ricinus removed from deer, vegetation, or asymptomatic people were tested by polymerase chain reaction targeting Anaplasma spp. 16S rRNA gene and groESL heat shock operon. Twelve deer blood samples were found to be infected with Anaplasma centrale (n = 7) or Anaplasma phagocytophilum (n = 5). No wild boar blood samples gave positive polymerase chain reaction results. Further, A. phagocytophilum was detected in 12 out of 89 I. ricinus removed from deer and in 18 out of 168 I. ricinus collected over vegetation in the North of Spain. Anaplasma spp. was not detected in any of the 38 I. ricinus removed from people. Nucleotide sequences for 16S rRNA gene showed substancial heterogeneity. The etiological agent of human anaplasmosis was found in two deer blood samples, an adult tick from deer, and a nymph from vegetation. The 16S rRNA sequences for 12 out of 35 samples matched the sequence of the Ap-variant 1 strain previously described in the United States, and the remaining 19 positive samples (deer blood and I. ricinus) showed variations with unknown significance. Although the groEL DNA sequences varied among analyzed strains, the deduced amino acid sequences did not change for any of them. This study suggests that deer population from La Rioja harbors strains of A. phagocytophilum similar to that pathogen for humans and other of unknown pathogenicity. Further, it seems that the Ap-variant 1 is circulating among I. ricinus ticks from the North of Spain more frequently than the A. phagocytophilum strain associated to human anaplasmosis.

A Newly DiscoveredAnaplasma phagocytophilumVariant in Rodents from Southeastern China

Anaplasma phagocytophilum was detected by polymerase chain reaction in 13 (14.1%) of 92 rodents captured from a mountainous area of Zhejiang Province in southeastern China. The nucleotide sequences of 1442-bp, nearly entire 16S rRNA gene amplified from these rodents, had 100% identity, but varied from all known corresponding sequences of A. phagocytophilum deposited in GenBank. To further identify and classify the variant, fragments of 357-bp partial citrate synthase gene (gltA), 849-bp major surface protein 4 gene (msp4), and 443-bp groESL heat-shock operon gene, were amplified and analyzed. The nucleotide sequences of the partial gltA gene amplified from the rodents were identical to each other, but distinct from previously reported A. phagocytophilum sequences,as were msp4 and groESL. These findings indicate that the newly discovered agent represents a novel A. phagocytophilum variant.

Cloning and Molecular Characterization of groESL Heat-Shock Operon in Methylotrophic Bacterium Methylovorus Sp. Strain SS1 DSM 11726

The groESL bicistronic operon of a restricted facultative methylotrophic bacterium Methylovorus sp. strain SS1 DSM 11726 was cloned and characterized. It was found to consist of two ORFs encoding proteins with molecular masses of 11,395 and 57,396 daltons, which showed a high degree of homology to other bacterial GroES and GroEL proteins. The genes were clustered in the transcription order groES-groEL. Northern blot analyses suggested that the groESL operon is transcribed as a bicistronic 2.2-kb mRNA, the steady-state level of which was markedly increased by temperature elevation. Primer extension analysis demonstrated one potential transcription start site preceding the groESL operon, which is located 100 bp upstream of the groES start codon. The transcription start site was preceded by a putative promoter region highly homologous to the consensus sequences of Escherichia coli sigma 32-type heat shock promoter, which functioned under both normal and heat shock conditions in E. coli. Heat shock mRNA was maximally produced by Methylovorus sp. strain SS1 approximately 10 min after increasing the temperature from 30 to 42 degrees C. The groESL operon was also induced by hydrogen peroxide or salt shock.

PCR detection of granulocytic ehrlichiae in Ixodes ricinus ticks and wild small mammals in western Switzerland.

The presence of granulocytic ehrlichiae was demonstrated by PCR in Ixodes ricinus ticks and wild small mammals in Switzerland in two areas of endemicity for bovine ehrlichiosis. Six ticks (three females and three nymphs) (1.4%) of 417 I. ricinus ticks collected by flagging vegetation contained ehrlichial DNA. A total of 201 small mammals from five species, wood mouse (Apodemus sylvaticus), yellow-necked mouse (Apodemus flavicollis), earth vole (Pitymys subterraneus), bank vole (Clethrionomys glareolus), and common shrew (Sorex araneus), were trapped. The analysis of I. ricinus ticks [corrected] collected on 116 small mammals showed that nine C. glareolus voles and two A. sylvaticus mice hosted infected tick larvae. In these rodents, granulocytic ehrlichia infection was also detected in blood, spleen, liver, and ear samples. Further examinations of 190 small mammals without ticks or with noninfected ticks showed the presence of ehrlichial DNA in spleen and other tissues from six additional C. glareolus, three A. flavicollis, and one S. araneus mammals. This study suggests that A. sylvaticus, A. flavicollis, S. araneus, and particularly C. glareolus are likely to be natural reservoirs for granulocytic ehrlichiae. Partial 16S rRNA gene sequences of granulocytic ehrlichiae from ticks and rodents showed a high degree of homology (99 to 100%) with granulocytic ehrlichiae isolated from humans. In contrast, groESL heat shock operon sequence analysis showed a strong divergence (approximately 5%) between the sequences in samples derived from rodents and those derived from samples from questing ticks or from other published ehrlichia sequences. Dual infections with granulocytic ehrlichia and Borrelia burgdorferi were found in ticks and small mammals.

Dusky-footed wood rats (Neotoma fuscipes) as reservoirs of granulocytic Ehrlichiae (Rickettsiales: Ehrlichieae) in northern California.

Dusky-footed wood rats (Neotoma fuscipes) and Peromyscus sp. mice (P. maniculatus and P. truei) were collected from one site in Placer County, one site in Santa Cruz County, and two sites in Sonoma County in northern California. Serum or plasma samples from 260 rodents were tested for antibodies to the agent of human granulocytic ehrlichiosis. Of these, samples from 25 wood rats (34% of those tested) and 10 (8%) Peromyscus sp. mice were found to be seropositive, but only those from one site. PCR assays targeting the groESL heat shock operon were conducted on all seropositive specimens and a subset of seronegative blood specimens. Ehrlichial DNA was identified in 17 (68%) of the 25 seropositive wood rat blood samples and in 1 of the 10 (10%) Peromyscus sp. specimens. None of 40 seronegative blood samples was PCR positive. Both seropositive and PCR-positive animals were collected during each trapping period. One male tick out of 84 Ixodes pacificus adults collected was PCR positive; samples of Dermacentor occidentalis nymphs and adults were negative. Nucleotide sequences of amplicons from three wood rat blood specimens and from the single PCR-positive tick differed by one and two bases, respectively, from a sequence previously obtained from Ehrlichia equi. At one site in Sonoma County, wood rats had a concurrent high prevalence of seropositivity and PCR positivity, while other sigmodontine rodents collected at the site were only occasionally infected. We suggest that dusky-footed wood rats serve as reservoirs of granulocytic ehrlichial agents in certain areas of northern California. The tick species involved in the transmission of granulocytic ehrlichiae among wood rats remains unknown.

Cloning, sequencing, and functional expression in Escherichia coli of chaperonin (groESL) genes from Vibrio cholerae.

Using a series of oligonucleotides synthesized on the basis of conserved nucleotide motifs in heat-shock genes, the groESL heat-shock operon from a Vibrio cholerae TSI-4 strain has been cloned and sequenced, revealing that the presence of two open reading frames (ORFs) of 291 nucleotides and 1,632 nucleotides separated by 54 nucleotides. The first ORF encoded a polypeptide of 97 amino acids, GroES homologue, and the second ORF encoded a polypeptide of 544 amino acids, GroEL homologue. A comparison of the deduced amino acid sequences revealed that the primary structures of the V. cholerae GroES and GroEL proteins showed significant homology with those of the GroES and GroEL proteins of other bacteria. Complementation experiments were performed using Escherichia coli groE mutants which have the temperature-sensitive growth phenotype. The results showed that the groES and groEL from V. cholerae were expressed in E. coli, and groE mutants harboring V. cholerae groESL genes regained growth ability at high temperature. The evolutionary analysis indicates a closer relationship between V. cholerae chaperonins and those of the Haemophilus and Yersinia species.

Detection of Ehrlichia risticii, the agent of Potomac horse fever, in freshwater stream snails (Pleuroceridae: Juga spp.) from northern California.

Ehrlichia DNA was identified by nested PCR in operculate snails (Pleuroceridae: Juga spp.) collected from stream water in a northern California pasture in which Potomac horse fever (PHF) is enzootic. Sequencing of PCR-amplified DNA from a suite of genes (the 16S rRNA, groESL heat shock operon, 51-kDa major antigen genes) indicated that the source organism closely resembled Ehrlichia risticii, the causative agent of PHF. The minimum percentage of Juga spp. harboring the organism in the population studied was 3.5% (2 of 57 snails). No ehrlichia DNA was found in tissues of 123 lymnaeid, physid, and planorbid snails collected at the same site. These data suggest that pleurocerid stream snails may play a role in the life cycle of E. risticii in northern California.

PCR amplification and comparison of nucleotide sequences from the groESL heat shock operon of Ehrlichia species.

Degenerate PCR primers derived from conserved regions of the eubacterial groESL heat shock operon were used to amplify groESL sequences of Ehrlichia equi, Ehrlichia phagocytophila, the agent of human granulocytic ehrlichiosis (HGE), Ehrlichia canis, Bartonella henselae, and Rickettsia rickettsii. The groESL nucleotide sequences were less conserved than the previously determined 16S rRNA gene sequences of these bacteria. A phylogenetic tree derived from deduced GroEL amino acid sequences was similar to trees based on 16S rRNA gene sequences. Nucleotide sequences obtained from clinical samples containing E. equi, E. phagocytophila, or the HGE agent were very similar (99.9 to 99.0% identity), and the deduced amino acid sequences were identical. Some divergence was evident between nucleotide sequences amplified from samples originating from the United States (E. equi and the HGE agent) and sequences from the European species, E. phagocytophila. A single pair of PCR primers derived from these sequences was used to detect E. chaffeensis and HGE agent DNA in blood samples from human patients with ehrlichiosis.