Dsb Genes Research Articles

Potential zoonotic role of the tick Amblyomma cf. oblongoguttatum (Ixodida: Ixodidae) in the bacterial transmission of Ehrlichia chaffeensis (Rickettsiales: Anaplasmataceae) in a deciduous tropical forest in Mexico.

Ehrlichia chaffeensis is a bacterium belonging to the Anaplasmataceae family. In Mexico, only 2 species have been recorded in association with tick species and humans. The objective of the present study was to detect the presence of bacteria of the genus Ehrlichia in ticks collected from the Chamela-Cuixmala Biosphere Reserve, Jalisco, Mexico. The collected ticks were identified and analyzed individually by polymerase chain reaction to amplify a fragment of the Anaplasmataceae 16S rRNA gene and the Ehrlichia-specific dsb gene. A total of 204 ticks, corresponding to 5 species of Ixodidae and 1 of Argasidae, were collected from 147 mammals of 6 species and 4 orders; 57 ticks collected from vegetation were also included. Among the total ticks collected, 1.47% (3/204) was positive for Ehrlichia sp. DNA was obtained using the primers EHR 16SD and EHR 16SR for 16S rRNA and DSB-330 and DSB-728 for dsb. The positive samples corresponded to a larva (Amblyomma sp.) associated with Didelphis virginiana and 2 nymphs (Amblyomma cf. oblongoguttatum) infesting Nasua narica. None of the ticks collected from the vegetation tested positive for Ehrlichia sp. DNA on the basis of the 16S rRNA and dsb genes. The sequences from the larvae of Amblyomma sp. and the nymphs of A. cf. oblongoguttatum were similar to those of E. chaffeensis. The phylogenetic analysis inferred with maximum likelihood corroborated the identity as E. chaffeensis. Although the role of these tick species as vectors of E. chaffeensis is still undetermined, the presence of infected ticks in the area indicates a potential zoonotic risk.

Phylogenetic analysis of Ehrlichia species isolated from horses and dogs in Iran

This study aimed to determine the prevalence and phylogenetic analysis of Ehrlichia spp. in horses and dogs in Iran. Blood samples were collected from 400 animals, including 200 horses and 200 dogs, from five different provinces in Iran. Polymerase chain reaction (PCR) was used to detect Ehrlichia spp. based on amplification of the 16S rRNA gene. The semi-nested PCR method was used to amplify the dsb, TRP36, and gltA genes. The results showed that 4.5 % of the samples (3 % horses and 6 % dogs) were positive for Ehrlichia sp. The highest prevalence was observed in Kerman and Khuzestan, while the lowest was found in West Azerbaijan, Golestan, and Mazandaran. The study suggests that the populations of dogs and horses in the country should be considered important factors in the epidemiology of ehrlichiosis. Phylogenetic analysis based on the dsb and TRP36 genes revealed that the prevalent species were E. canis and E. ruminantium.

Pleural effusion-related Nocardia otitidiscaviarum, Anaplasma platys and Ehrlichia canis coinfection in a dog.

The coinfections by some microorganisms have been related to severe diseases in humans and animals, where immunosuppressive agents favor opportunistic behavior of other pathogens. A 4-month-old, female mixed-breed dog with a two-week history of inappetence, prostration, emaciation, and respiratory distress was admitted at a veterinary hospital in Brazil. Tachycardia, pale mucous membranes, severe respiratory distress, and a large number of ticks (Rhipicephalus sanguineus s.l.) in different body regions were observed at clinical examination. Hematological examination of dog showed leukocytosis, neutrophilia, mild anemia, and thrombocytopenia, whereas unremarkable values in biochemical tests. Thoracic radiography revealed a pleural effusion image. Blood and the pleural fluid (purulent aspect) samples were subjected to qPCR (16S rRNA and dsb genes) and sequencing, which identified Ehrlichia canis and Anaplasma platys coinfection. An aggregate of coccoid-to-branching or long filamentous microorganisms, surrounded by pyogranulomatous inflammatory reaction was seen at the cytology of the pleural fluid. Bacteriological culture of pleural effusion showed colonies compatible with the genus Nocardia, which revealed gram-positive filamentous organisms with a tendency of fragmentation and were identified as Nocardia otitidiscaviarum in mass spectrometry (MALDI-TOF MS). Therapy of N. otitidiscaviarum isolate using levofloxacin (supported by a previous in vitro susceptibility testing) and doxycycline for E. canis and A. platys resulted in complete resolution of the clinical picture. Here, we report for the first time a triple coinfection by Nocardia otitidiscaviarum, A. platys, and E. canis in a dog with pleural effusion, where debilitating or immunosuppressive conditions induced by A. platys and E. canis coinfection probably contributed to the opportunistic behavior of N. otitidiscaviarum.

Molecular detection and genetic characterization of Ehrlichia canis and Ehrlichia sp. in neotropical primates from Brazil

The Anaplasmataceae family includes obligate, arthropod-transmitted intracellular bacteria that can be zoonotic and potentially fatal. Studies focusing on the interaction between neotropical primates and the agents of this family are scarce. The present study aimed to identify agents of the Anaplasmataceae family in the whole blood of free-living and captive neotropical primates in the State of Mato Grosso, Central-West Brazil. Thirty-eight samples of six nonhuman primate (NHP) species were collected in seven municipalities and analysed through polymerase chain reaction (PCR), nucleotide sequencing, and phylogenetic analysis of the dsb, groEL, 16S rRNA, and gltA genes. DNA fragments similar to those of Ehrlichia canis were detected in Sapajus apella and Ehrlichia chaffeensis from Mico melanurus. The sequences generated in this study and homologous sequences retrieved from GenBank® were used for phylogenetic analyses to characterize the Ehrlichial agents detected in NHPs. The agents were then grouped into clades corresponding to different isolates from the NHP species. In addition, an Anaplasma sp. closely related to Anaplasma marginale was identified in two S. apella individuals. These findings shed light on the susceptibility of neotropical NHPs to Anaplasmataceae agents. These bacteria are known to be transmitted by ticks, which can also serve as possible sources of infection for other animals, including humans.

Molecular characterization of Ehrlichia canis and Babesia vogeli reveals multiple genogroups associated with clinical traits in dogs from urban areas of Colombia

Ehrlichia canis and Babesia vogeli are vector-borne pathogens that infect blood cells and produce the diseases Canine Monocytic Ehrlichiosis (CME) and Babesiosis in dogs. Considering the lack of studies on these pathogens in Colombia, this study aims to determine the molecular prevalence and genetic characterization of E. canis and Babesia spp., in dogs from the Metropolitan Area of Bucaramanga (MAB), Santander, a region with one of the greatest pet densities in Colombia. One hundred eighty-five dogs were surveyed and analyzed through molecular, clinical, and hematological approaches. The molecular detection of E. canis and Babesia spp., was performed by conventional PCR targeting the dsb and 18S rRNA genes, respectively. To identify genogroups, E. canis positive samples underwent a hemi-nested PCR of the trp36 gene, and the PCR products were subsequently sequenced. Molecular analyses showed a prevalence of 13% (24/185; CI 95%, 8.1 – 18.0%) and 1.09% (2/185; CI 95,% -0.43 – 2.6%) for E. canis and B. vogeli respectively, as well as the presence of the genogroups US (USA), BR (Brazil), and CR (Costa Rica), in 62.5, 16.6, and 16.6% of E. canis positive samples, respectively. Values of hematocrit, hemoglobin, platelets, erythrocytes, white blood cell (WBC) count, lymphocytes, and eosinophils showed significant differences between animals infected with the different genogroups of E. canis (p< 0.05). In contrast, hematocrit values, hemoglobin, platelets, red blood cells, and creatine kinase MB isoenzyme (CK-MB) were lower in B. vogeli positive animals. Statistical analysis indicated that E. canis infection was associated with specific socioeconomic sectors as well as with some household features (p< 0.05). In conclusion, our results present evidence of the circulation of multiple genogroups of E. canis in the MAB, which is associated with different geographical origins and clinical traits. Epidemiological analyses suggest a need to increase molecular surveillance and prevention campaigns especially in lower socioeconomic sectors.

Novel Ehrlichia and Hepatozoon genotypes in white-eared opossums (Didelphis albiventris) and associated ticks from Brazil

White-eared opossums (Didelphis albiventris) are well adapted to anthropized areas. The increased contact with domestic animals and humans mediates the transmission of arthropod-borne pathogens. Despite the worldwide occurrence of tick-borne Anaplasmataceae and Hepatozoidae species in a variety of vertebrates, few studies reported serological evidence or molecular detection of theses agentes in marsupials. Up to now, while Ehrlichia/Anaplasma spp. have only been detected in marsupials from Brazil, Hepatozoon spp. have been reported in marsupials from Chile, Australia and Brazil. The present work aimed to investigate, using molecular techniques and blood smear analysis, the presence of Ehrlichia spp., Anaplasma spp., and Hepatozoon sp. in the blood and ticks collected from D. albiventris in urban forest fragments from midwestern Brazil. Between May and December 2017, 43 D. albiventris (27 males and 16 females) were captured for blood and tick collection in the city of Campo Grande, state of Mato Grosso do Sul, midwestern Brazil. Ticks (46 Amblyomma dubitatum nymphs and 24 Amblyomma spp. larvae) were collected from 14 out 43 (32.5%) of the white-eared opossums. Panoptic-stained blood smears were performed using peripheral blood (tail tip) of the captured opossums. DNA extracted from blood and tick samples were subjected to PCR/qPCR assays for Anaplasmataceae agents (rrs, gltA, groEL, sodB, and dsb genes, and 23S-5S intergenic region) and Hepatozoon spp. (18S rRNA gene), followed by Sanger sequencing, BLASTn and phylogenetic analyses. An inclusion resembling Ehrlichia morulae was found in a white-eared opossum's monocyte from a blood smear stained with Panoptic. Five (11.63% [5/43]) white-eared opossums’ blood samples and 7 (25% [7/28]) tick samples (2 pools of Amblyomma spp. larvae and 5 pools of A. dubitatum nymphs) were positive for Anaplasmataceae via a PCR assay targeting the conserved rrs gene. Phylogenetic analysis based on the rrs gene positioned three sequences obtained from opossums and ticks together as a subclade within the Ehrlichia canis clade. However, all samples were negative in a qPCR assay specific for E. canis based on the dsb gene. Phylogenetic analyses positioned the gltA and 23S-5S ITS sequences obtained from opossums’ blood samples in a separate clade from the other validated Ehrlichia species. One (2.3% [1/43]) opossum blood sample was positive for the 18S rRNA gene of Hepatozoon sp. The phylogenetic analysis positioned the Hepatozoon sp. sequence obtained from a D. albiventris specimen in a clade with a sequence previously detected in a black storm petrel (Oceanodroma melania) from Mexico. All the other sequences of Hepatozoon sp. previously detected in marsupials from Brazil were positioned in a separated clade. The present work showed the occurrence of putative novel genotypes of Ehrlichia sp. and Hepatozoon sp. in white-eared opossums and associated A. dubitatum ticks from midwestern Brazil.

Systematic evaluation of TaqMan real-time polymerase chain reaction assays targeting the dsb and gltA loci of Ehrlichia canis in recombinant plasmids and naturally infected dogs.

Background and Aim:Because of the diversity of local genotypes of Ehrlichia canis, genes targeted by TaqMan real-time polymerase chain reaction (RT-PCR) assays should be systematically evaluated. This study evaluated the amplification efficiency, linearity, precision, and sensitivity of two TaqMan RT-PCR assays targeting the dsb and gltA loci of E. canis in recombinant plasmids and naturally infected dogs.Materials and Methods:Thirty blood samples were collected from dogs showing clinical signs of canine monocytic ehrlichiosis at the Kasetsart University Veterinary Teaching Hospital, Bangkok, Thailand. The dsb and gltA genes were amplified by conventional PCRs (cPCRs) on the blood samples and were then sequenced. Meanwhile, RT-PCR was used to detect dsb and gltA genes in 10-fold dilutions of the recombinant plasmids.Results:Both dsb and gltA were amplified with a high degree of linearity (R2≥0.975 and 0.993, respectively) in all dilutions, although the mean percentage of relative standard deviation of gltA was lower, but the difference was non-significant. The detection limits of RT-PCR and cPCR were 10−7 and 10−6, respectively, for both loci. RT-PCR targeting dsb (22/30; 73.3%) and gltA (15/30; 50%) yielded a number of positive results that did not differ significantly (p=0.06). The RT-PCR positive results of the dsb gene (22/30) differed significantly from that of cPCR (11/30) (p=0.004). In contrast, the RT-PCR positive results of the gltA gene (15/30) did not differ significantly from that of cPCR (12/30) (p=0.43). The mean Ct value (30.2) based on dsb RT-PCR of 22 positive cases was higher than that of gltA RT-PCR (Ct=27.4) on 15 positive cases. The Ct values from dsb RT-PCR were >30 in all seven discordant samples that were not detected by the gltA RT-PCR.Conclusion:RT-PCR targeting the dsb gene was more sensitive for detecting E. canis in naturally infected dogs. This study suggested that TaqMan RT-PCR of the dsb gene should be selected for E. canis research in this region.

A novel copper-sensing two-component system for inducing Dsb gene expression in bacteria

In nature, bacteria must sense copper and tightly regulate gene expression to evade copper toxicity. Here, we identify a new copper-responsive two-component system named DsbRS in the important human pathogen Pseudomonas aeruginosa; in this system, DsbS is a sensor histidine kinase, and DsbR, its cognate response regulator, directly induces the transcription of genes involved in protein disulfide bond formation (Dsb) (i.e., the dsbDEG operon and dsbB). In the absence of copper, DsbS acts as a phosphatase toward DsbR, thus blocking the transcription of Dsb genes. In the presence of copper, the metal ion directly binds to the sensor domain of DsbS, and the Cys82 residue plays a critical role in this process. The copper-binding behavior appears to inhibit the phosphatase activity of DsbS, leading to the activation of DsbR. The copper resistance of the dsbRS knock-out mutant is restored by the ectopic expression of the dsbDEG operon, which is a DsbRS major target. Strikingly, cognates of the dsbRS-dsbDEG pair are widely distributed across eubacteria. In addition, a DsbR-binding site, which contains the consensus sequence 5′-TTA-N8-TTAA-3′, is detected in the promoter region of dsbDEG homologs in these species. These findings suggest that the regulation of Dsb genes by DsbRS represents a novel mechanism by which bacterial cells cope with copper stress.

Detection of Ehrlichia sp. in Amblyomma sculptum parasitizing horses from Brazilian Pantanal wetland

Bacteria of the genus Ehrlichia are transmitted by ticks and also are an important cause of infection in wild and domestic mammals. Infection with Ehrlichia spp. has been reported in horses, especially in the USA, Nicaragua and Brazil. In this study, we report the parasitism by Amblyomma sculptum, Rhipicephalus microplus and Dermacentor nitens ticks in horses from a ranch located in south Pantanal wetland. Molecular and serological analyzes to determine infection by Ehrlichia spp. in horses and their respective ticks were carried out. A total of 12 horses were submitted to blood collection to investigate antibodies by indirect immunofluorescence assay (IFA) using Ehrlichia canis crude antigens and to be tested by polymerase chain reaction (PCR) in order to amplify fragments of the 16S rRNA, dsb, groEL and sodB gene of Ehrlichia spp. A total of 164 tick specimens were removed from horses, stored in isopropanol and later identified as D. nitens, A. sculptum and R. microplus. DNA from ticks were extracted and subjected to the same PCR assays to detect Ehrlichia spp. Anti-Ehrlichia spp. antibodies were detected in five/12 (41.7 %) horses by IFA, with antibody titers ranging from 40 to 160. All horse DNA samples were negative for the 16S rRNA, dsb, groEL and sodB of Ehrlichia spp. One A. sculptum female was positive to all target genes of Ehrlichia. This tick was parasitizing an Ehrlichia-seropositive horse with antibody titer of 80. Nucleotide sequences of 16S rRNA, dsb, groEL and sodB genes showed close relationship with different strains of Ehrlichia detected in wild mammals, Amblyomma ticks and horses from Brazil and Argentina. Detection of anti-Ehrlichia sp. antibodies suggests that horses have been exposed to an ehrlichial agent in the Pantanal. Future studies on Ehrlichia infection should be carried out to better elucidate and to bring new information about equine ehrlichiosis, since these animals are important hosts of ticks in the Brazilian Pantanal wetlands.

Genome-wide identification of DNA double-strand break repair genes and transcriptional modulation in response to benzo[α]pyrene in the monogonont rotifer Brachionus spp.

The DNA repair system has evolved from the common ancestor of all life forms and its function is highly conserved within eukaryotes. In this study, to reveal the role of DNA double-strand break repair (DSB) genes in response to benzo[α]pyrene (B[α]P), we first identified DSB genes in relation to homologous recombination and non-homologous end joining events in four Brachionus rotifer spp.: B. calyciflorus, B. koreanus, B. plicatilis, and B. rotundiformis. In all the Brachionus spp., 39 orthologous genes to human DSB repair genes were identified. Furthermore, three genes in B. koreanus, two genes in B. plicatilis, and one gene in B. calyciflorus and B. rotundiformis were present as duplicated genes, indicating that these genes were diversified over speciation in the genus Brachionus. Moreover, we compared DSB repair genes on the gene structures in four monogonont Brachionus rotifers and the bdelloid rotifer Adineta vaga, which possesses highly efficient DNA repair ability. The transcriptional responses of four monogonont Brachionus rotifers in response to B[α]P exposure showed how B[α]P exposure led to DSBs and subsequently recruited DNA DSB repair pathways in the rotifer B. koreanus. Taken together, this study provides a better understanding of the potential role of DSB repair genes in the monogonont rotifer Brachionus spp. in response to B[α]P.

Ehrlichia spp. and Anaplasma spp. in Xenarthra mammals from Brazil, with evidence of novel \u2018Candidatus Anaplasma spp.\u2019

Anaplasmataceae agents are obligatory intracellular Gram-negative α-proteobacteria that are transmitted mostly by arthropod vectors. Although mammals of the Superorder Xenarthra (sloths, anteaters, and armadillos) have been implicated as reservoirs for several zoonotic agents, only few studies have sought to detect Anaplasmataceae agents in this group of mammals. This study aimed to investigate the occurrence and genetic diversity of Anaplasma spp. and Ehrlichia spp. in blood and spleen samples of free-living Xenarthra from four different states in Brazil (São Paulo, Mato Grosso do Sul, Rondônia, and Pará). Nested and conventional PCR screening assays were performed to detect the rrs and dsb genes of Anaplasma spp. and Ehrlichia spp., respectively. The assays were positive in 27.57% (91/330) of the Anaplasma spp. and 24.54% (81/330) of the Ehrlichia spp. Of the 91 positive Anaplasma spp. samples, 56.04% were positive in a conventional PCR assay targeting the 23S–5S intergenic region. Phylogenetic and distance analyses based on the rrs gene allocated Anaplasma sequences from sloths captured in Rondônia and Pará states in a single clade, which was closely related to the A. marginale, A. ovis, and A. capra clades. The sequences detected in southern anteaters from São Paulo were allocated in a clade closely related to sequences of Anaplasma spp. detected in Nasua nasua, Leopardus pardalis, and Cerdocyon thous in Brazil. These sequences were positioned close to A. odocoilei sequences. Genotype analysis corroborated previous findings and demonstrated the circulation of two distinct Anaplasma genotypes in animals from north and southeast Brazil. The first genotype was new. The second was previously detected in N. nasua in Mato Grosso do Sul state. The intergenic region analyses also demonstrated two distinct genotypes of Anaplasma. The sequences detected in Xenarthra from Pará and Rondônia states were closely related to those in A. marginale, A. ovis, and A. capra. Anaplasma spp. sequences detected in Xenarthra from São Paulo and were allocated close to those in A. phagocytophilum. The analyses based on the dsb gene grouped the Ehrlichia spp. sequences with sequences of E. canis (São Paulo, Mato Grosso do Sul, and Pará) and E. minasensis (Rondônia and Pará). The data indicate the occurrence of E. canis and E. minasensis and two possible new Candidatus species of Anaplasma spp. in free-living mammals of the Superorder Xenarthra in Brazil.

The co-infection with Ehrlichia minasensis, Anaplasma marginale and Anaplasma platys is not associated with anemia in beef cattle in the Brazilian Pantanal.

The Anaplasmataceae family is composed of obligatory intracellular Gram-negative bacteria transmitted by arthropod vectors. In Brazil, with the exception of Anaplasma marginale, little is known about the occurrence of other Anaplasma and Ehrlichia species infecting cattle. The present study aimed at investigating the occurrence of Anaplasma spp. and Ehrlichia spp. in beef cattle (Bos indicus) sampled in the Brazilian Pantanal, an area prone to periodic flooding and endemic for bovine trypanosomiasis. Blood samples from 400 cattle were collected and screened by PCR assays based on rrs and dsb genes from Anaplasma spp. and Ehrlichia spp., respectively. Positive samples for Anaplasma spp. were subjected to qPCR assays based on the msp-2 gene and nPCR based on the groEL gene. As a result, 4.75% (19/400) and 48.12% (167/347) were positive for Anaplasma platys and Ehrlichia minasensis, respectively. Besides, positivity of 56.75% (227/400) for A. marginale and seropositivity of 90.75% (363/400) for Trypanosoma vivax were found. A high rate of co-infection was observed (67.25%), from which the co-infection by A. marginale and E. minasensis was more frequently found in calves than cows. Interestingly, none of the animals presenting co-infection showed anemia or other clinical signs. The present study showed, for the first time, the occurrence of A. platys and E. minasensis in beef cattle in the southern Pantanal, as well as a high rate of co-infection by A. marginale, E. minasensis and T. vivax in the sampled animals.

Uncommon Ehrlichia canis infection associated with morulae in neutrophils from naturally infected dogs in Brazil.

Ehrlichia and Anaplasma species are the most common tick-borne disease (TBD) pathogens in dogs worldwide. Ehrlichia canis, the aetiological agent of the Canine Monocytic Ehrlichiosis (CME), is known to replicate within the cytoplasm of mononuclear cells into clusters of organisms called morulae. However, detection of morulae in neutrophils is commonly observed in dogs infected by Ehrlichia ewingii or Anaplasma phagocytophilum. We report uncommon clinical cases of canine ehrlichiosis presenting morulae compatible with E.ewingii and A.phagocytophilum in dogs from two distinct regions of Brazil. Eight dogs were admitted to two veterinary teaching hospitals from Brazil, showing clinical or haematological signs suggestive of TBD. Blood or peritoneal fluid was withdrawn for haematological and cytologic analysis. All samples were evaluated by PCR assays for Ehrlichia and Anaplasma using genus-specific primers for dsb, 16S rRNA and groEL genes, followed by sequencing. Samples were also evaluated by nested PCR assays for the 16S rRNA gene of E. ewingii and groEL gene of A.phagocytophilum and Anaplasma platys. Seven dogs revealed thrombocytopenia, six dogs had monocytosis and five presented lymphopenia and anaemia. All dogs showed morulae structures compatible with Ehrlichia spp. in neutrophils and were PCR-positive for the dsb and 16S rRNA gene fragments of Ehrlichia, with sequences showing 100% identity with multiple E.canis sequences deposited in the GenBank™. Sequencing of 16S rRNA and groEL gene fragments from one PCR-positive dog showed 100% identity with A.platys. Overall, our data suggest that in endemic regions for E.canis, that is Brazil, the presence of morulae in neutrophils may indicate infection by this bacterium. Herein, morulae were also found in neutrophils present in the peritoneal fluid of a dog. Also, this is the first report of E.canis and Hepatozoon canis co-infection in neutrophils from naturally infected dogs confirmed by DNA sequencing.

Emergence of a Novel Ehrlichia minasensis Strain, Harboring the Major Immunogenic Glycoprotein trp36 with Unique Tandem Repeat and C-Terminal Region Sequences, in Haemaphysalis hystricis Ticks Removed from Free-Ranging Sheep in Hainan Province, China.

Ehrlichia minasensis, a recently described Ehrlichia species that is the most closely related to, but clearly distinct from, Ehrlichia canis, has been circulating in not only bovines, cervids, and dogs but also several tick species from Canada, Brazil, France, Pakistan, Ethiopia, and Israel. However, there are no reports of E. minasensis in China. The purpose of this study was to explore whether E. minasensis is present naturally in ticks in China. Through PCR targeting of the genus-conserved dsb gene, E. minasensis DNA was detected in Haemaphysalis hystricis ticks removed from free-ranging sheep in Hainan Province, South China in 2017. The partial sequence of the dsb, 16S rRNA, and groEL genes demonstrated that the Hainan strain shared 99% identity with the dsb gene of E. minasensis strain UFMG-EV (GenBank: JX629808), with the 16S rRNA of E. minasensis isolate E-2650 (MH500005) and with the groEL gene of E. minasensis strain UFMG-EV (JX629806), respectively. Moreover, sequence analysis of the major immunogenic tandem repeat protein (trp36) revealed that the Hainan strain harbored a unique tandem repeat sequence (APEAAPVSAPEAAPVSAPVS) and a C-terminal region that differed from those of other known E. minasensis strains. Additionally, phylogenetic analysis based on the entire amino acid sequence of trp36 revealed that the Hainan strain was closely related to a recently described E. minasensis strain from Brazil, of which the sister clade contained different strains of E. canis. The discovery of this novel Hainan strain in H. hystricis ticks represents the first known natural presence of E. minasensis in South China, highlighting the need for its constant surveillance.

Novel Ehrlichia sp. detected in Magellanic penguins (Sphenicus magellanicus) and in the seabird tick Ixodes uriae from Magdalena Island, southern Chile

Ehrlichia spp. are obligatory intracellular microorganisms that infect hematopoietic, endothelial or blood cells of mammals. Ticks are the only vectors of these agents in nature. To date, the role of birds and their associated ticks as reservoirs of ehrlichiae remains almost unexplored. In this study, we performed a molecular screening for bacteria of Anaplasmataceae family in samples of spleen (n = 72) and lung (n = 17), recovered from 72 carcasses of Magellanic penguins (Spheniscus magellanicus) in Brazil and Chile. One apparently unengorged tick (Ixodes uriae) was also collected while wandering upon one of the carcasses and submitted to molecular analyses as well. Through conventional and nested PCR protocols three genes (16S rRNA, dsb and groEL) of a new Ehrlichia sp. were partially characterized upon organs of three penguins and in the tick coming from Magdalena Island (Chile). First matches after BLASTn comparisons showed that our sequences share 99.4% (16S rRNA), 94.6% (groEL) and 79.3% (dsb) of identity with “Candidatus Ehrlichia ornithorhynchi”, Ehrlichia sp. NS101 and Ehrlichia canis CCZ, respectively. Matrixes of genetic distance including other representatives of the Ehrlichia genus point a 99.4%, 94.0%, and 80.0% of identity with 16S rRNA, groEL and dsb genes from Ehrlichia sp. It25, Ehrlichia sp. NS101, and Ehrlichia chaffeensis San Louis, respectively. A Bayesian phylogenetic analysis of Anaplasmataceae 16S rRNA gene places the detected Ehrlichia sp. into a group with Ehrlichia sp. BAT and Ehrlichia sp. Natal. Although depicting different topologies, Bayesian unrooted phylogenetic trees constructed for groEL and dsb genes position this Ehrlichia sp. into well-supported branches, which reinforces the finding of a new taxon. For the moment, any pathogenic effect of this new Ehrlichia sp. on penguins is still unknown. However, this fact becomes important to assess from a conservation point of view since populations of Magellanic penguins are currently threatened and in an ongoing decrease.

Two different genogroups of Ehrlichia canis from dogs in Thailand using immunodominant protein genes

Ehrlichia canis is the causative agent of canine monocytic ehrlichiosis (CME). While there is a high prevalence of CME in Thailand, genetic diversity of E. canis is still poorly defined. This study examined the molecular characteristics of E. canis using PCR and phylogenetic analysis of the dsb, gp19 and gp36 genes. DNA was extracted from 220 whole blood samples of naturally infected dogs, and all had clinical signs compatible with tick-borne diseases. Of these, 16.4% (36/220) provided positive E. canis DNA via the dsb and gp19 genes. However, only 13 out of the 36 samples (36.1%) were positive for the gp36 gene. Sequences of the dsb gene had very high identity (99–100%) with previously deposited E. canis sequences. Sequences of the gp19 gene were similar to those from US and Taiwanese genogroups (98.8–99.5% identity). Elucidation of genetic characteristics of E. canis based on the gp36 gene displayed 91.4–99.1% shared identity. There were 426–429 bp of a 5′ end pre-repeat tandem region, a 27 bp repetition with variable numbers of a tandem repeat (TR) region of 9 amino acid sequences (TEDSVSAPA), and a variable 3′ end region with sequence length depending on the isolate (72–93 bp). Phylogenetic trees of E. canis, particularly using the gp36 amino acid sequences, showed that the Thai strains fell into two phylogenetic clades contained within other worldwide E. canis strains. Alignment and phylogenetic analysis suggested that E. canis strains from Thailand could be divided into two genogroups, the US and Taiwanese genogroups. This study provides the first characterization of the dsb and gp19 genes of E. canis in Thailand, the results support the conclusion that the gp36 is a potential target for genotyping and elucidation of phylogenetic relationships among E. canis strains.

Identificación de bacterias de la familia Anaplasmataceae en un albergue canino del municipio de Caldas, Antioquia

Objetivo. Fue detectar la circulación de microorganismos de la familia Anaplasmataceae en un alberge canino del municipio de Caldas. Materiales y métodos. Se realizó un estudio descriptivo en 46 perros (Canis lupus familiaris) para evaluar por técnicas moleculares o por inmunodiagnóstico la presencia de Ehrlichia spp. y Anaplasma spp., en un albergue canino del município de Caldas. Se hizo extracción de ADN en muestras de sangre entera con EDTA y se utilizaron cebadores contra los genes dsb y groEl, para detectar secuencias de Ehrlichia y Anaplasma respectivamente y análisis filogenéticos de las secuencias obtenidas. Por otro lado se usó el SNAP 4DX® para el serodiagnóstico. Resultados. En las pruebas moleculares se obtuvieron cinco muestras positivas para Ehrlichia y tres para Anamaplasma. En las pruebas serológicas, tres individuos fueron positivos para Ehrlichia spp., tres para Anaplasma spp. y negativos para los demás ensayos medidos por el SNAP (Dirofilaria immitis y Borrelia burgdorferi). Conclusiones. Se demostró la circulación de Ehrlichia canis y Anaplasma platys en un albergue del municipio de Caldas, Antioquia.

Ehrlichia sp. infection in carthorses of low-income owners, Southern Brazil.

Although well established in dogs, Ehrlichia sp. infection has been scarcely reported in horses. The aim was to perform a comprehensive serological and molecular survey for the detection of Ehrlichia spp. in carthorses from Southern Brazil. Blood samples from 190 carthorses from Paraná State were sampled. Horses were also tested for Borrelia burgdorferi and Anaplasma phagocytophilum. Anti-Ehrlichia sp. antibodies were detected by a commercial rapid ELISA, and immunofluorescence antibody assays (IFA) with E. chaffeensis and E. canis as crude antigens. The molecular and phylogenetic analysis of Ehrlichia sp. was based on 16S rRNA and dsb genes. A total of 52 (27.4%), 4 (2.1%), and 3 (1.6%) horses were positive for Ehrlichia spp., Anaplasma spp. and Borrelia burgdorferi, respectively, by the commercial rapid ELISA. Thirty-eight (20.0%) and 37 (19.5%) horses showed anti-E. chaffeensis and anti-E. canis antibodies by IFA, respectively. One blood sample that also showed anti-E. chaffeensis antibodies was PCR positive for the 16S rRNA and dsb genes of Ehrlichia spp., showing an identity of>98.0% to the uncultured Ehrlichia sp. previously detected in Brazilian jaguars (Panthera onca). Anti-Ehrlichia sp. antibodies and Ehrlichia DNA were detected in carthorses from Southern Brazil, which may post public health concerns due to intimate contact with low-income owners. This is the first report of a natural infection of this bacteria in horses from South America. Clinical signs and the tick vector remain unknown.

Molecular characterization of Ehrlichia canis infecting dogs, Buenos Aires

Canine monocytic ehrlichiosis (CME) is a worldwide potentially fatal tick-borne rickettsial disease of dogs caused by Ehrlichia canis and transmitted by Rhipicephalus sanguineus sensu lato. CME diagnosis includes indirect (serology) and direct (e.g. blood smears and PCR) methods. PCR is more sensitive and specific than direct microscopic examination and positive PCR results confirm infection, whereas positive serologic test results only confirm exposure.The aim of the present study was to perform a molecular characterization of E. canis from canine samples of the Metropolitan Area of Buenos Aires. We studied 223 blood samples of dogs submitted to our institute for CME diagnoses. The samples were initially screened for Anaplasmataceae family by PCR, resulting in 30 positive dogs (13.4%). Subsequently, positive DNAs were analyzed by nested PCR 16S rRNA specific for E. canis or Anaplasma platys, resulting in 15 (6.7%) and 16 (7.2%) positive dogs, respectively.For molecular characterization, samples positive for E. canis were subjected to amplification of a fragment of the dsb and p28 genes. The nucleotide sequences obtained for the dsb fragment resulted in 100% identity with others E. canis found in dogs from different regions of worldwide. The nucleotide sequences obtained for p28 gene resulted in 100% of identity with each other and closely with E. canis str. Jaboticabal (Brazil). Identity with others sequences of E. canis ranged from 76.9 to 79.7%.The occurrence of canine cases molecularly confirmed in Metropolitan Area of Buenos Aires highlights the need for more studies in order to understand epidemiological factors associated with CME, especially the disease transmission dynamic in South America given the existence of two lineages of R. sanguineus sensu lato with different vectorial capacity for transmission of E. canis.

Minimum infection rate of Ehrlichia minasensis in Rhipicephalus microplus and Amblyomma sculptum ticks in Brazil

A new genotype phylogenetically close to Ehrlichia canis named as Ehrlichia minasensis was identified infecting cattle and deer in Canada, as well as Rhipicephalus microplus ticks and cattle in Brazil. Although it was detected in R. microplus, little is known about the epidemiology of this ehrlichiosis, especially in other tick species. This study evaluated the minimum infection rate of E. minasensis in Amblyomma sculptum and R. microplus ticks from locations where naturally infected cattle were previously detected. Overall, 45 engorged R. microplus ticks after molting [43 pools of adults (13.4%), and 2 pools of nymphs (4%)], and 42 engorged females post-oviposition (30.6%) (p=0.008) were positive by PCR for Ehrlichia sp. using the dsb, 16S rRNA and TRP36 genes, making a total of 87 R. microplus samples positive for Ehrlichia spp. (17.1%, IC 95% 14.01-20.75%). The partial sequences generated in the present study were 99–100% similar to the dsb DNA sequence of E. minasensis genotypes UFMG-EV and UFMT–BV, respectively, 100% similar to the 16S rRNA sequence of the E. minasensis genotype BOV2010 from Canada, and 99% similar to the TRP36 sequence of the Ehrlichia sp. UFMT-BV. The results of this study confirm the occurrence of transstadial transmission of this agent in R. microplus ticks and highlight the importance of R. microplus in the epidemiology and transmission of ehrlichiosis in cattle. No A. sculptum ticks were positive by PCR for E. minasensis.