No description available.

Zugl.: Berlin, Freie Univ., Diss., 2009

Background: Equine granulocytic anaplasmosis (EGA), caused by the organism Anaplasma phagocytophilum, is a tick-borne disease of clinical importance in Southwest Virginia. The disease is recognized worldwide and causes pyrexia, anorexia, limb edema, and lethargy. Diagnosis in endemic areas is often based on clinical signs, but confirmation of infection can be made via detection of morulae on a peripheral blood smear or polymerase chain reaction analysis (PCR) at the time of disease or by serologic detection of antibodies 2-4 weeks post infection. There is growing interest in stall-side methods for diagnosis of various equine diseases which has led to an increased use of the SNAP 4DX Plus Test® for vector-borne diseases. Objectives: Determine seroprevalence of antibodies to A. phagocytophilum in the equine population of Southwest Virginia and changes in seroprevalence compared to samples taken 6 years earlier. Determine the percentage of horses with clinical signs consistent with EGA that were positive for A. phagocytophilum infection and assess common presenting clinical signs, hematologic variables, and confirmatory diagnostic test results. Animals: Seroprevalence was evaluated in horses presented for routine annual Coggins testing in 2013 and 2019-2020. Clinical features of disease and diagnostic test results were evaluated in horses presenting with clinical signs compatible with A. phagocytophilum infection from September 2019-August 2020. Methods: Seroprevalence was determined using the IDEXX SNAP 4DX Plus Test® on serum collected from horses presenting for annual Coggins testing in 2013 and 2019-2020. Samples collected in 2013 had been stored at -7580 degrees F since collection. Age, sex, county of residence, and month of sampling were statistically analyzed in the seroprevalence population. Horses presenting with clinical disease consistent with EGA from September 2019-August 2020 had the following diagnostic tests performed: complete blood count (CBC), blood smear for morulae detection, polymerase chain reaction (PCR) analysis, immunofluorescence antibody testing (IFAT), and the IDEXX SNAP 4DX Plus Test®. Results: Seroprevalence of A. phagocytophilum in the equine population of Southwest Virginia increased from 8.5% in 2013 to 11.2% in 2019-2020, although this increase was not statistically significant. In the 2019-2020 population, month of sampling was significantly associated with presence of antibodies to A. phagocytophilum. Positive samples were more common from November-February than other times of the year. When the two sample time periods were combined, sex was significantly associated with presence of antibodies to A. phagocytophilum with geldings more likely to be seropositive. Within the clinical case population, 35% of horses with clinical signs compatible with equine granulocytic anaplasmosis had confirmed infection. The most common hematologic abnormality in affected horses was thrombocytopenia. PCR analysis was the most sensitive diagnostic test to diagnose infection followed by identification of morulae on blood smears. Conclusions: Seroprevalence of A. phagocytophilum is similar to other endemic areas in the United States and appears to be increasing over time. In active clinical cases, diagnosis is best made via PCR or detection of morulae on a blood smear. The SNAP 4DX Plus Test® was not appropriate for diagnosis of active EGA in acute cases. Seroprevalence of Anaplasma phagocytophilum in the equine population of Southwest Virginia / Master of Science / Equine granulocytic anaplasmosis (EGA) is a common tick-borne disease in the United States and worldwide. The causative bacteria, Anaplasma phagocytophilum, also infects humans, dogs, and various domestic animal species. In horses, signs of disease include fever, decreased appetite, leg swelling, and depression. Diagnostic testing that is both accurate and timely is still lacking. The point-of-care SNAP 4DX Plus Test® used to diagnose vector-borne infectious disease in dogs has been suggested for similar use in horses. The objectives of the current study were to determine seroprevalence of antibodies to A. phagocytophilum in the equine population of Southwest Virginia and to characterize the clinical signs and diagnostic test findings of horse with clinical signs of EGA. Seroprevalence was determined using the SNAP 4DX Plus Test®. Serum samples were obtained from horses presenting for annual Coggins testing in 2019-2020. Samples from 2013 were also tested to determine if seroprevalence had increased. Horses presenting with clinical signs consistent with A. phagocytophilum were examined by a veterinarian and had blood drawn for a complete blood count (CBC), blood smear evaluation, polymerase chain reaction analysis (PCR), immunofluorescent antibody testing (IFAT), and the SNAP 4DX Plus Test®. Seroprevalence in 2019-2020 was 11.2% and 8.5% in 2013. This is similar to other endemic areas in the United States and Europe. In horses sampled from 2019-2020, the month of sampling was significantly associated with presence of antibodies to A. phagocytophilum with most of the positive samples being identified in November through February. Geldings were more likely to be seropositive than mares. Thirty five percent of horses with signs consistent with EGA were confirmed to have the disease. Within this population, PCR analysis and/or detection of morulae on the blood smear were reliable indicators of disease while diagnostic techniques utilizing serology were unreliable. This is the first study to determine seroprevalence of A. phagocytophilum in Southwest Virginia. In the actively infected population, PCR and blood smear evaluation remain the most sensitive methods of diagnosis. While the SNAP 4DX Plus Test® is useful for serologic data collection, it was not appropriate for acute diagnosis of EGA.

Em função da proximidade cada vez maior entre o cavalo e o homem, é de extrema importância ter conhecimentos das doenças que acometem os equinos, que por ventura, podem acometer seres humanos. Dentre muitas doenças, pode-se citar duas, que promovem grandes perdas econômicas aos rebanhos eqüinos, tanto no tratamento desses rebanhos, como com a morte dos mesmos, dificultando a importação e exportação de animais: a babesiose e a erliquiose (anaplasmose), que podem estar ou não associadas, acometendo um animal, concomitantemente. A presente pesquisa teve como objetivo investigar e diagnosticar doença concomitante babesiose (por Babesia equi ou Theileria equi) e Erliquiose (por Erliquia equi ou Anaplasma phagocytophilum), no estado de São Paulo, em rebanhos eqüinos, utilizando as técnicas de Nested PCR (Nested polymerase chain reaction reação em cadeia pela polimerase para diagnóstico de T. equi e A. phagocytophilum) e c-ELISA (competitive enzyme-linked immunosorbent assay para diagnóstico de T. equi) ou ELISA indireto (para diagnóstico de A. phagocytophilum) e comparar os resultados obtidos nas diferentes técnicas em 250 amostras de eqüino (sangue total e soro). Como resultado, obteve-se 38,4%, 46% e 36% de positividade, respectivamente, nos testes de pesquisa de hematozoário, c-ELISA e Nested PCR para Theileria equi e 0%, 3% e 0% de positividade, respectivamente, nos testes de pesquisa de hemoparasita, ELISA indireto e Nested PCR para Anaplasma phagocytophilum, não sendo observada a co-infecção de Babesiose e Anaplasmose no rebanho estudo / Due to the increasing proximity between horse and man, it is of extreme importance to understand the diseases that affect horses which by chance may affect humans. Among many diseases, babesiosis and ehrlichiosis (anaplasmosis) promote high economic losses to horses herds in consequence of costs of treatment and also death, making it difficult to import and export animals: They can or not be linked affecting an animal at the same time. This study aimed to investigate and diagnose concomitant babesiosis (Babesia equi and Theileria equi) and ehrlichiosis (for ehrlichia equipment or Anaplasma phagocytophilum) in equine herds of the state of Sao Paulo, using the techniques of Nested PCR (Nested polymerase chain reaction for the diagnosis of T. equi and A. phagocytophilum) and c-ELISA (competitive enzyme-linked immunosorbent assay for diagnosis of T. equi) or ELISA (for diagnosis of A. phagocytophilum). Also to compare results obtained in these different techniques in 250 samples of horse (whole blood and serum). Results showed 38.4%, 46% and 36% positivity, respectively, in tests for the detection of Theileria equi through hematozoan, c-ELISA and Nested PCR and 0%, 3% and 0% positivity, respectively, in tests for the detection of Anaplasma phagocytophilum through blood parasites, indirect ELISA and Nested PCR. It was not observed co-infection Babesiosis and anaplasmosis in the herd study

Em função da proximidade cada vez maior entre o cavalo e o homem, é de extrema importância ter conhecimentos das doenças que acometem os equinos, que por ventura, podem acometer seres humanos. Dentre muitas doenças, pode-se citar duas, que promovem grandes perdas econômicas aos rebanhos eqüinos, tanto no tratamento desses rebanhos, como com a morte dos mesmos, dificultando a importação e exportação de animais: a babesiose e a erliquiose (anaplasmose), que podem estar ou não associadas, acometendo um animal, concomitantemente. A presente pesquisa teve como objetivo investigar e diagnosticar doença concomitante babesiose (por Babesia equi ou Theileria equi) e Erliquiose (por Erliquia equi ou Anaplasma phagocytophilum), no estado de São Paulo, em rebanhos eqüinos, utilizando as técnicas de Nested PCR (Nested polymerase chain reaction reação em cadeia pela polimerase para diagnóstico de T. equi e A. phagocytophilum) e c-ELISA (competitive enzyme-linked immunosorbent assay para diagnóstico de T. equi) ou ELISA indireto (para diagnóstico de A. phagocytophilum) e comparar os resultados obtidos nas diferentes técnicas em 250 amostras de eqüino (sangue total e soro). Como resultado, obteve-se 38,4%, 46% e 36% de positividade, respectivamente, nos testes de pesquisa de hematozoário, c-ELISA e Nested PCR para Theileria equi e 0%, 3% e 0% de positividade, respectivamente, nos testes de pesquisa de hemoparasita, ELISA indireto e Nested PCR para Anaplasma phagocytophilum, não sendo observada a co-infecção de Babesiose e Anaplasmose no rebanho estudo / Due to the increasing proximity between horse and man, it is of extreme importance to understand the diseases that affect horses which by chance may affect humans. Among many diseases, babesiosis and ehrlichiosis (anaplasmosis) promote high economic losses to horses herds in consequence of costs of treatment and also death, making it difficult to import and export animals: They can or not be linked affecting an animal at the same time. This study aimed to investigate and diagnose concomitant babesiosis (Babesia equi and Theileria equi) and ehrlichiosis (for ehrlichia equipment or Anaplasma phagocytophilum) in equine herds of the state of Sao Paulo, using the techniques of Nested PCR (Nested polymerase chain reaction for the diagnosis of T. equi and A. phagocytophilum) and c-ELISA (competitive enzyme-linked immunosorbent assay for diagnosis of T. equi) or ELISA (for diagnosis of A. phagocytophilum). Also to compare results obtained in these different techniques in 250 samples of horse (whole blood and serum). Results showed 38.4%, 46% and 36% positivity, respectively, in tests for the detection of Theileria equi through hematozoan, c-ELISA and Nested PCR and 0%, 3% and 0% positivity, respectively, in tests for the detection of Anaplasma phagocytophilum through blood parasites, indirect ELISA and Nested PCR. It was not observed co-infection Babesiosis and anaplasmosis in the herd study

Thesis--Auburn University, 2005. / Abstract. Vita. Includes bibliographic references.

No description available.

Anaplasma phagocytophilum est une alpha-protéobactérie, parasite intracellulaire stricte, à localisation intra-granulocytaire et principalement vectorisée par des tiques du genre Ixodes. Elle est notamment l'agent de l'anaplasmose granulocytaire bovine, ou Tick-borne fever, une maladie provoquant d'importantes pertes économiques chez les bovins en Europe. Cette bactérie, peut également infecter un large spectre d'hôtes, tels que des ruminants sauvages ou des rongeurs. Cependant, l'épidémiologie de l'infection par A. phagocytophilum est encore mal connue. Le(s) réservoir(s) des souches de ruminants domestiques en Europe n'est/ne sont notamment pas identifié(s) à l'heure actuelle. Il est donc nécessaire d'approfondir nos connaissances dans ce domaine, notamment afin de lutter plus efficacement contre l'atteinte des bovins. L'objectif de cette thèse était de caractériser la diversité génétique des variants d'A. phagocytophilum circulant chez les ruminants en France. Pour cela, nous avons, dans un premier temps, étudié la circulation des souches d'A. phagocytophilum au sein des ruminants sauvages et domestiques en développant et en utilisant une technique MLVA (Multiple-Locus Variable-number tandem repeat Analysis). Cela nous a permis de suspecter fortement l'existence d'au moins deux cycles épidémiologiques impliquant des variants d'A. phagocytophilum présents chez les ruminants en France. Le premier pourrait impliquer le cerf comme espèce réservoir et les ruminants domestiques (entre autres) comme espèces sensibles, alors que le second impliquerait le chevreuil comme réservoir, sans impact significatif chez les bovins. Dans un deuxième temps, après avoir développé une technique de séquence capture pour A. phagocytophilum, nous avons séquencé et caractérisé le premier génome de cette bactérie issu d'un bovin. La comparaison de ce génome aux neuf autres génomes actuellement disponibles a permis d'identifier quatre protéines uniquement présentes chez la souche bovine, et neuf uniquement chez les deux souches de ruminants domestiques étudiés, ce qui amène à envisager leur implication potentielle dans le tropisme d'hôte de ces souches / Anaplasma phagocytophilum is an obligate intracellular alpha-proteobacterium mainly transmitted by Ixodes ticks. In domestic ruminants, it is the causative agent of tick-borne fever, which causes significant economic losses in Europe. It also infects a large range of hosts, including wild ruminants and rodents. The epidemiology of A. phagocytophilum is not yet fully understood. For example, the reservoir host(s) for European domestic ruminant strains has/have not been identified to date, which doesn't facilitate control of cattle infection. Our objective was to explore the genetic diversity of A. phagocytophilum obtained from ruminants in France. For this purpose, we first studied the circulation of this pathogen in domestic and wild ruminants, using a new MLVA technique. Our results potentially reveal the existence of at least two different epidemiological transmission cycles of A. phagocytophilum. The first cycle may involve red deer as reservoir hosts, and possibly domestic ruminants, as either accidental or longer-term hosts, whereas the second might involve roe deer. In a second study, we have sequenced and characterized the genome of A. phagocytophilum obtained from a cow. Following comparison with nine available genomes, we identified four genes specific to the A. phagocytophilum bovine genome, and nine common to both genomes from domestic ruminants (i.e. a cow and a sheep). These genes could be involved in host tropism of ruminant strains

Anaplasma phagocytophilum est une alpha-protéobactérie à multiplication intracellulaire stricte transmise par les tiques du genre Ixodes sp. Responsable notamment des anaplasmoses granulocytaires bovine et humaine, elle peut également infecter de nombreux mammifères, tels les ruminants sauvages ou les rongeurs. En Europe, plusieurs cycles, encore mal connus, semblent coexister et impliquer des hôtes (réservoirs et victimes) différents. Cela semble être en particulier le cas pour les souches bovines et humaines, conduisant à supposer que les souches bovines ne seraient pas zoonotiques. Du fait de sa localisation intracellulaire in vivo, et plus particulièrement au sein des polynucléaires neutrophiles des hôtes vertébrés, la culture d’A. phagocytophilum au laboratoire expose à d’importantes difficultés méthodologiques. De ce fait, la compréhension des relations entre la bactérie et ses hôtes (mammifères et tiques vectrices) n’a conduit à ce jour qu’à un nombre restreint de publications. Cette thèse s’intéresse à ces interactions, que j’ai abordées sciemment à différents niveaux. Dans un premier temps, l’étude épidémiologique menée en troupeau bovin nous a permis de mettre en évidence une diversité génétique importante parmi les souches y circulant, et nous a amenés à formuler l’hypothèse que les bovins pourraient jouer un rôle en tant que réservoirs de l’infection. Différents mécanismes, notamment l’échappement à la réponse immunitaire de l’hôte, l’absence de protection croisée entre souches et peut-être la sanctuarisation de l’infection dans des cellules niches, permettraient d’expliquer ce rôle de réservoir. L’étude de l’infection des cellules endothéliales, effectuée afin d’explorer leur rôle en tant que cellules niches d’une part et leur implication dans la barrière d’espèce d’autre part, a conduit à envisager que ces cellules pourraient permettre le passage des bactéries vers le courant sanguin, mais a priori pas leur multiplication. Afin d’amorcer l’exploration de la réponse transcriptomique d’A. phagocytophilum lors du changement d’hôte (vertébré vs invertébré), nous avons soumis des cultures de cellules de tiques infectées à un choc thermique, ce qui nous a permis de suggérer qu’un nombre restreint de mécanismes transcriptomiques est mis en jeu en réponse au choc thermique induit par le changement d’hôte vertébré/invertébré. Néanmoins, la capacité d’A. phagocytophilum à répondre à un stress thermique plus important est bien maintenue. Les protéines que nous avons identifiées comme les plus différentiellement exprimées au cours de cette étude pourraient s’avérer jouer un rôle préférentiel dans l’infection du vecteur ou du mammifère. La technique du double-hybride en système de levure, expérimentée dans la dernière partie de ce travail, et qui nous a permis de mettre en évidence trois interacteurs pour APH_0032, protéine de la membrane vacuolaire, pourrait s’avérer intéressante pour étudier les interactions de ces protéines vis-à-vis des banques d’ADNc de vertébré et de tique, mais aussi pour explorer le tropisme tissulaire des souches, puisque notre équipe a précédemment mis en évidence le fait qu’un allèle particulier d’APH_0032 est associé aux avortements bovins. . Ces approches complémentaires posent la question des fondements d’une telle variabilité génétique et d’une telle diversité d’hôtes chez une bactérie intracellulaire obligatoire et ouvrent un grand champ de perspectives / Anaplasma phagocytophilum is an obligate intracellular alphaproteobacterium, mainly transmitted by Ixodes ticks. It is the causative agent of bovine and human granulocytic anaplasmosis and can infect various mammalian species, including rodents and wild ruminants. Several epidemiological cycles may coexist in Europe. In particular, human and bovine strains seem to belong to distinct cycles, which leads to the hypothesis that cattle strain are not zoonotic. Due to its intracellular location in vivo inside granulocyte neutrophils, A. phagocytophilum culture is challenging and leads to several methodological difficulties. This explains why few studies have so far been performed in order to explore the interactions between this bacterium and its host species (mammals and ticks). In order to investigate these interactions at different levels, I performed four complementary studies. First, our epidemiological study in cattle herd highlighted the genetic diversity of strains circulating in the herds and challenges the role of cattle as a reservoir for A. phagocytophilum. The infections of endothelial cells that we performed to study the role of these cells as niche cells and/or determinants of species barrier during A. phagocytophilum infection led us to consider that endothelial cells could host A. phagocytophilum during their transmission from dermis to blood, without allowing their multiplication. For studying A. phagocytophilum transcriptomic reactions during the transmission from tick to vertebrate host, we submitted infected tick cells to heat shocks. Our results suggest that few transcriptomic events are induced during this transmission. Nevertheless, A. phagocytophilum is able to respond to non-physiological heat stress. We identified differentially expressed proteins, which could play an important role during tick or mammal infection. The yeast two hybrid analysis allowed us to detect three host cell interactors to APH_0032, an A. phagocytophilum vacuolar membrane protein. This technique could be applied for studying the molecular interactions involving proteins that where differentially expressed during heat shock, for example. Finally, our four complementary studies raise the question of the basis for such genetic variability and host diversity within an obligate intracellular bacterium and open up a wide field of perspectives

Obligate intracellular pathogens must acquire host cell-derived nutrients to facilitate their survival. One such bacterial pathogen, Anaplasma phagocytophilum, replicates within neutrophils and non-phagocytic cells in a bacterial-modified, host cell-derived vacuole. The bacterium exploits host cell vesicular trafficking pathways to route nutrients to its vacuole and utilizes Rab GTPases, guanine nucleotide-dependent, vesicular trafficking regulators, to do so. We previously discovered that the A. phagocytophilum vacuolar membrane is decorated with a specific subset of Rab GTPases - Rab1, Rab4A, Rab10, Rab11, Rab14, Rab22A and Rab35. Rab1 is exclusively found on the endoplasmic reticulum (ER) and thus its localization suggests that the bacterium intercepts the ER. Rab10, which is found on the ER, trans-Golgi and recycling endosomes, localizes to the vacuolar membrane in a guanine nucleotide-independent and bacterial protein synthesis-dependent manner. This suggests that a bacterial-encoded protein is binding to and recruiting Rab10. In this study, we determined that A. phagocytophilum hijacks two very nutrient-rich sources in the secretory pathway - trans-Golgi- and endoplasmic reticulum-derived vesicles. A. phagocytophilum localizes perinuclearly adjacent to the Golgi apparatus during infection. A. phagocytophilum and Anaplasma marginale, an intravacuolar bovine pathogen, also localize near the smooth ER and rough ER in both mammalian and tick host cells. These results are supported by transmission electron microscopy analyses of infected cells. Membrane markers for the rough ER label the peripheries of A. marginale and A. phagocytophilum organisms in both mammalian and tick host cells, which suggests that they are translocated into the pathogen vacuole. Furthermore, membrane markers for trans-Golgi-derived vesicles, including endogenous Rab10, label the periphery of intravacuolar A. phagocytophilum organisms. Markers for the trans-Golgi and the ER co-fractionate with A. phagocytophilum in density gradient centrifugation studies. siRNA knockdown of Rab10 pronouncedly reduces delivery of trans-Golgi markers into the pathogen-occupied vacuole, significantly reduces infection, and impedes bacterial conversion to the bacterium’s dense-cored form. These results suggest that trans-Golgi recruitment is Rab10 dependent and is critical for bacterial development. We identified an outer membrane A. phagocytophilum moonlighting protein, uridine monophosphate kinase that specifically binds GST-Rab10 in affinity chromatography assays and interacts with Rab10 in vivo. We hypothesize that this surface protein is mediating the interaction of the bacteria with intravacuolar trans-Golgi derived vesicles. This interaction could be critical for the delivery of essential nutrients. Taken together, these data suggest that nutritional virulence mechanisms of A. phagocytophilum and A. marginale target the host secretory pathway. Additionally, they suggest a novel mechanism whereby pathogens translocate nutrient rich vesicles into the pathogen vacuole, thus delivering essential nutrients right to their front door.