[Show abstract][Hide abstract] ABSTRACT: Obligate intracellular pathogenic bacteria evolved to manipulate their host cells with a limited range of proteins constrained by their compact genomes. The harsh environment of a phagocytic defense cell is one that challenges the majority of commensal and pathogenic bacteria; yet, these are the obligatory vertebrate homes for important pathogenic species in the Anaplasmataceae family. Survival requires that the parasite fundamentally alter the native functions of the cell to allow its entry, intracellular replication, and transmission to a hematophagous arthropod. The small genomic repertoires encode several eukaryotic-like proteins, including ankyrin A (AnkA) of Anaplasma phagocytophilum and Ank200 and tandem-repeat containing proteins of Ehrlichia chaffeensis that localize to the host cell nucleus and directly bind DNA. As a model, A. phagocytophilum AnkA appears to directly alter host cell gene expression by recruiting chromatin modifying enzymes such as histone deacetylases and methyltransferases or by acting directly on transcription in cis. While cis binding could feasibly alter limited ranges of genes and cellular functions, the complex and dramatic alterations in transcription observed with infection are difficult to explain on the basis of individually targeted genes. We hypothesize that nucleomodulins can act broadly, even genome-wide, to affect entire chromosomal neighborhoods and topologically associating chromatin domains by recruiting chromatin remodeling complexes or by altering the folding patterns of chromatin that bring distant regulatory regions together to coordinate control of transcriptional reprogramming. This review focuses on the A. phagocytophilum nucleomodulin AnkA, how it impacts host cell transcriptional responses, and current investigations that seek to determine how these multifunctional eukaryotic-like proteins facilitate epigenetic alterations and cellular reprogramming at the chromosomal level.
[Show abstract][Hide abstract] ABSTRACT: The present study aims to detect and characterize by molecular techniques, the presence of tick-borne pathogens in wild captive carnivore blood samples from Brazil. Blood was collected from 76 Brazilian felids, 23 exotic felids, 3 European wolves (Canis lupus), and 97 Brazilian canids maintained in captivity in zoos located in São Paulo and Mato Grosso states, Brazil. DNA of each sample was used in PCR reactions for Ehrlichia, Anaplasma, and Rickettsia identification. The blood from 10/100 (10%) of canids (1 European wolf, 3 bush dogs, and 6 crab-eating foxes) and from 21/99 (21%) felids (4 pumas, 6 little spotted cats, 4 ocelots, 3 jaguarundis, 1 tiger, and 3 lions) contained fragments of 16S rRNA gene of Ehrlichia spp. Fragments of Anaplasma spp. groESL and 16S rRNA genes were detected in the blood of 1/100 (1%) canids (1 bush dog) and in 4/99 (3%) felids (4 little spotted cats), respectively. Rickettsia species infections were not identified. The present work showed that new strains of Ehrlichia and Anaplasma spp. circulate among wild carnivores in Brazil.
Ticks and Tick-borne Diseases 06/2012; 3(4):247-53. · 2.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Spotted fever is a disease caused by bacteria from the genus Rickettsia of the spotted fever group (SFG). Rickettsia rickettsii is likely the main agent of Brazilian spotted fever (BSF). With the objective of gathering information on the circulation of SFG rickettsiae in Londrina, Parana state, ticks from dogs and horses and also blood from dogs, horses and humans were collected in a neighbourhood of the city which presented potential for circulation of rickettsiae between hosts and vectors. Amblyomma cajennense, Dermacentor nitens, and Rhipicephalus sanguineus ticks were subjected to Polymerase Chain Reaction targeting a fragment of the Rickettsia gltA gene. This specific gene encodes the enzyme citrate synthase of Rickettsia spp., and results on all ticks were negative. Human and animal sera were tested by Indirect Immunofluorescence Assay in which R. rickettsii and R. parkeri were used as antigens. Sera from 4.7% human, 2.7% canine and 38.5% equine were positive for R. rickettsii. For R. parkeri, 0.9% human, 2.7% canine and 11.5% equine samples were positive. All samples reactive to R. parkeri also reacted to R. rickettsii. An epidemiological questionnaire was applied, but there were no statistically significant results. Comparison of our serological results with previous studies in Brazil, among BSF endemic and non-endemic areas, indicates that there is no established rickettsial infection in the study area, a statement corroborated with our molecular analysis. Nonetheless, as humans of the present study are highly exposed to tick infestations, health education within the population is needed to obtain efficient tick control.
Zoonoses and Public Health 09/2011; 58(6):416-23. · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Anaplasma phagocytophilum is the zoonotic cause of granulocytic anaplasmosis. We hypothesized that immune response, specifically gamma interferon (IFN-γ), plays a role in disease severity. To test this, horses were infected and IFNG expression was pharmacologically downregulated using corticosteroids. Eight horses were infected with A. phagocytophilum; 4 received dexamethasone on days 4 to 8 of infection. Clinical signs, hematologic parameters, and transcription of cytokine/chemokine genes were compared among treated and untreated horses. Infection was quantitated by msp2 real-time PCR and microscopy. As anticipated, there was significantly greater leukopenia, thrombocytopenia, and anemia in infected versus uninfected horses. The A. phagocytophilum load was higher for dexamethasone-treated horses. Dexamethasone reduced IFNG transcription by day 12 and IL-8 and IL-18 by days 7 to 9 and increased IL-4 on day 7. The ratio of IL-10 to IFNG was increased by dexamethasone on day 9. There were no hematologic differences between the infected horses. Dexamethasone suppression of proinflammatory response resulted in delayed infection-induced limb edema and decreased icterus, anorexia, and reluctance to move between days 6 and 9 and lower fever on day 7. These results underscore the utility of the equine model of granulocytic anaplasmosis and suggest that Th1 proinflammatory response plays a role in worsening disease severity and that disease severity can be decreased by modulating proinflammatory response. A role for Th1 response and macrophage activation in hematologic derangements elicited by A. phagocytophilum is not supported by these data and remains unproven.
[Show abstract][Hide abstract] ABSTRACT: Neurological manifestations of Lyme disease (or neuroborreliosis) occur variably and while it is clear that Borrelia burgdorferi can invade the nervous system, how it does so is not well understood. Pathogen penetration through the blood brain barrier (BBB) is often influenced by calcium signaling in the endothelial cells triggered by extracellular host-pathogen interactions. We examined the traversal of B. burgdorferi across the human BBB using in vitro model systems constructed of human brain microvascular endothelial cells (HBMEC) grown on Costar Transwell inserts. Pretreatment of the cell monolayers with BAPTA-AM (an intracellular calcium chelator) or phospholipase C (PLC) inhibitor U73122 inhibited B. burgdorferi transmigration. By 5 h, BAPTA-AM significantly inhibited (82-99%; p <0.017) spirochete traversal of HBMEC compared to DMSO controls. Spirochete traversal was almost totally blocked (> or =99%; p <0.017) after pretreatment with the PLC-beta inhibitor U73122 as a result of barrier tightening based on electric cell-substrate impedance sensing (ECIS). The data suggest a role for calcium signaling in CNS spirochete invasion through endothelial cell barriers.
Clinical Microbiology and Infection 05/2009; 15(5):422-6. · 4.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fifty-five dogs with suspected tickborne disease were tested by immunofluorescence assay and PCR for Anaplasma phagocytophilum infection. Thirty (54.5 per cent) of the dogs were seropositive and five of them fulfilled the serological criteria for an active infection, with either seroconversion or a fourfold increase in antibody titres. Fragments of DNA of the expected size were detected by PCR in two seropositive and three seronegative dogs. However, direct amplicon sequencing failed to identify active A phagocytophilum infections, but revealed the presence of Anaplasma platys DNA in the PCR-positive animals.
The Veterinary record 03/2009; 164(6):168-71. · 1.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The recent detection of Anaplasma phagocytophilum in Portugal stimulated further research on the agent's enzootic cycle, which usually involves rodents. Thus a total 322 rodents belonging to five species, including 30 Apodemus sylvaticus (wood mouse), 65 Mus musculus (house mouse), 194 M. spretus (algerian mouse), 5 Rattus norvegicus (brown rat) and 28 R. rattus (black rat), were studied by indirect immunofluorescent assay (IFA) and/or polymerase chain reaction (PCR) for A. phagocytophilum exposure in four sampling areas of mainland and two areas of Madeira Island, Portugal. Overall, 3.6% (7/194) of M. spretus presented with IFA-positive results. Seropositive mice were detected in all three mainland sampling areas where this species was captured, with prevalence of 5.2% (5/96) and 5.0% (1/20) for the Ixodes-areas of Arrábida and Mafra, and 1.3% (1/78) for Mértola, a difference that was not statistically significant (p > 0.05). The majority of IFA-positive mice were detected in spring when considering either Arrábida alone (p = 0.026) or all M. spretus sampling areas together (p = 0.021), although the significance of this association was not evident after Bonferroni correction. Nevertheless, neither the seropositive M. spretus, nor additional samples of 10% seronegative rodents from mainland, and 16% of rodents collected in Madeira Island showed evidence of A. phagocytophilum active infections when spleen and/or lung samples were tested by PCR. Either the M. spretus results represents residual antibodies from past A. phagocytophilum infections, present infections with limited bacteremia, or cross-reactions with closely related agents deserves more investigation.
[Show abstract][Hide abstract] ABSTRACT: A retrospective study to detect Anaplasma phagocytophilum antibodies by indirect immunofluorescence (IFA) and Western blot (WB) assay was conducted in 367 potentially exposed patients from Portugal. The study included 26 patients with confirmed Lyme borreliosis (LB), 77 with suspected LB, 264 seronegative patients studied for possible tick-transmitted LB and boutonneuse fever (LB/BF) infection, and 96 healthy blood donors. Overall, patients with LB and suspected LB (n = 2 [7.7%] and n = 6 [7.8%], respectively) were more often seropositive (n = 8 [7.8%]; P < 0.001), whereas only 1 (0.4%; P = 0.046) patient in the LB/BF seronegative group had confirmed disease. This study is the first evidence of human exposure to A. phagocytophilum or an antigenically similar bacterium in Portugal, and suggests that LB patients are significantly more likely to contact A. phagocytophilum.
Annals of the New York Academy of Sciences 10/2006; 1078:100-5. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The manifestations of Lyme disease, caused by Ixodes spp. tick-transmitted Borrelia burgdorferi, range from skin infection to bloodstream invasion into the heart, joints and nervous system. The febrile infection human granulocytic anaplasmosis is caused by a neutrophilic rickettsia called Anaplasma phagocytophilum, also transmitted by Ixodes ticks. Previous studies suggest that co-infection with A. phagocytophilum contributes to increased spirochetal loads and severity of Lyme disease. However, a common link between these tick-transmitted pathogens is dissemination into blood or tissues through blood vessels. Preliminary studies show that B. burgdorferi binds and passes through endothelial barriers in part mediated by host matrix metalloproteases. Since neutrophils infected by A. phagocytophilum are activated to release bioactive metalloproteases and chemokines, we examined the enhanced B. burgdorferi transmigration through vascular barriers with co-infection in vitro. To test whether endothelial transmigration is enhanced with co-infection, B. burgdorferi and A. phagocytophilum-infected neutrophils were co-incubated with EA.hy926 cells (HUVEC-derived) and human brain microvascular endothelial cells in Transwell cultures. Transmigration of B. burgdorferi through endothelial cell barriers was determined and endothelial barrier integrity was measured by transendothelial electrical resistivity. More B. burgdorferi crossed both human BMEC and EA.hy926 cells in the presence of A. phagocytophilum-infected neutrophils than with uninfected neutrophils without affecting endothelial cell integrity. Such a mechanism may contribute to increased blood and tissue spirochete loads.
International Journal for Parasitology 06/2006; 36(5):601-5. · 3.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Biological and geographic heterogeneity of anthropozoonosis caused by Anaplasma phagocytophilum is poorly understood. Seven North American A. phagocytophilum strains were compared by PFGE. The average genome size was 1.58 Mbp, and restriction patterns were identical. New World strains of A. phagocytophilum have a large genome and a high degree of genetic uniformity.
Journal of Clinical Microbiology 08/2003; 41(7):3392-4. · 4.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Based on seroprevalence studies and tick infection rates, tick-borne human granulocytic ehrlichiosis (HGE) is thought to occur in Germany, but to date no clinical case has been detected. Reported here are the first ehrlichial sequences derived from a German horse that fell ill with granulocytic ehrlichiosis. The analysis of three different genes (16S rRNA gene, groESL, and ankA) revealed up to 100% identity with ehrlichial sequences derived from patients with HGE in other countries or from infected ticks in Germany. Thus, the current lack of clinical cases of HGE in Germany is unlikely to result from the absence of pathogenic granulocytic ehrlichiae strains in German ticks.
European Journal of Clinical Microbiology 06/2003; 22(5):303-5. · 3.02 Impact Factor