Anaplasma phagocytophilum and A. marginale are the etiologic agents of human granulocytic anaplasmosis and bovine anaplasmosis, respectively. Both diseases can be severe, even fatal, and protective vaccines for each are lacking. We recently identified A. phagocytophilum outer membrane protein A (ApOmpA) as being critical for cellular invasion and is expressed during infection of mammalian but not tick cells. Disrupting ApOmpA-host cell interactions significantly inhibits A. phagocytophilum entry into host cells. ApOmpA and its A. marginale ortholog, AM854 (A. marginale OmpA; AmOmpA) exhibit 44% amino acid identity. The ApOmpA invasin domain is highly conserved between both proteins. In this study, we investigated the differential expression of AmOmpA in mammalian versus tick cell lines; the serological cross-reactivity between AmOmpA and ApOmpA; the potential role of AmOmpA in mediating interactions with mammalian host cells; and if inhibiting the AmOmpA-host cell interaction impairs A. marginale cellular invasion. AmOmpA is expressed throughout infection of mammalian, but not tick cells. Sera from A. marginale infected cows recognized both AmOmpA and ApOmpA. Sera from cows immunized with an A. marginale OM complex that conferred protection also recognized both proteins. Thus, ApOmpA and AmOmpA share cross-reactive B-cell epitopes. To determine if AmOmpA plays a role in promoting A. marginale infection, we assessed the abilities of recombinant AmOmpA to competitively inhibit infection of mammalian host cells. To examine the cross-reactive properties of OmpA, we showed that preincubation of host cells with GST-ApOmpA and pretreatment of A. marginale with anti-GST-ApOmpA significantly inhibit A. marginale infection of host cells; and that pretreatment of A. phagocytophilum with serum from cows immunized with an A. marginale OM complex reduces its infection of host cells. These studies advance understanding of conservation of OmpA-mediated cellular invasion between Anaplasma species and highlight the potential of OmpA as a vaccinogen that could offer protection against human and veterinary anaplasmoses.

Obligate intracellular pathogens must invade host cells to survive and pose a global health risk. As such, internalization is a critical life stage and represents an excellent therapeutic target. Oxidoreductase exploitation is a thematic invasion strategy among obligate intracellular pathogens. Delineating the mechanisms and proteins mediating this exploitation could identify novel therapeutic targets for many important pathogens. Anaplasma phagocytophilum infects neutrophils by an incompletely defined mechanism, resulting in the emerging potentially fatal disease, human granulocytic anaplasmosis. The bacterial adhesin, Asp14, contributes to invasion by virtue of its C-terminus engaging an unknown receptor. Yeast two-hybrid analysis identified protein disulfide isomerase (PDI) as a putative Asp14 binding partner. Co-immunoprecipitation confirmed this interaction and identified the Asp14 C-terminus as critical to it. PDI reductase activity inhibition impaired bacterial infection of, but not binding to, host cells. A. phagocytophilum failed to productively infect myeloid-specific PDI conditional knock-out mice. This is the first demonstration of microbial PDI exploitation in vivo. Infection of PDI inhibited cells was rescued when bacterial, but not host surfaces were reduced with the reducing agent tris(2-carboxyethyl)phosphine (TCEP). Furthermore, TCEP restored bacterial infectivity after Asp14 inhibition using an antibody that reduces infection. Mutational analyses identified Asp14 residues critical for binding PDI. These data demonstrate that Asp14 binds and brings PDI to disulfide bonds within A. phagocytophilum surface protein(s) that it reduces, enabling infection. Targeting the Asp14 C-terminus could benefit approaches to prevent/treat granulocytic anaplasmosis. A similar approach would identify proteins from other obligate intracellular pathogens that could prove to be protective targets.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2018-06-28T17:20:50Z No. of bitstreams: 1 texto completo.pdf: 1170314 bytes, checksum: aef38342ffa69b1c377766d841c96e8c (MD5) / Made available in DSpace on 2018-06-28T17:20:50Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1170314 bytes, checksum: aef38342ffa69b1c377766d841c96e8c (MD5) Previous issue date: 2018-02-23 / Neste estudo foram analisadas 100 amostras de sangue de cães, com trombocitopenia marcada (plaquetas < 100.000/μL) ou com visualização direta de hemoparasitas (ou inclusões compatíveis) no esfregaço sanguíneo, obtidas da rotina do laboratório de análises clínicas do DVT (Departamento de Veterinária), do Hospital Veterinário da Universidade Federal de Viçosa (UFV). As amostras foram submetidas à extração de DNA e posteriormente à reação em cadeia da polimerase de tipo nested (nPCR), para o diagnóstico molecular de A. phagocytophilum e outros hemoparasitas. Do total das amostras, 60 foram positivas para no mínimo um hemoparasita. Foi possível detectar a presença de quatro gêneros de hemoparasitas nas amostras de sangue avaliadas: Ehrlichia, Anaplasma, Babesia e Hepatozoon. Foram amplificadas um total de 26 produtos do primer Ehrlichia spp monocítica, 23 de Ehrlichia spp granulocítica 26 com BAB-rum e 10 utilizando o hsp70. Logo ao analisar os resultados, de um total de 86 amostras foi possível identificar os seguintes hemoparasitas: 23 E. canis (identidade 94%-100%), 7 A. platys (identidade 97%-100%), 11 B. vogeli (identidade 86%-99%) e 15 H. canis (identidade 94%-99%). Nenhuma amostra foi positiva utilizando o primer msp4, específico de A. phagocytophilum, porém, o sequenciamento de uma das amostras amplificadas teve uma identidade de 99% com um fragmento da sequência parcial do gene 16S rRNA de A. phagocytophilum depositada no GenBank. A não amplificação de produtos com um primer específico para A. phagocytophilum pode ser atribuída a diversas hipóteses, mas isto não exclui a possibilidade que a bactéria esteja presente no município de Viçosa ou da microrregião. / In this study were analyzed blood samples from 100 dogs, with marked thrombocytopenia (platelets/μL < 100,000) or with direct visualization of hemoparasites (or compatible inclusions) in the blood smear, obtained from the routine of the clinical analysis laboratory, of the DVT (Veterinary Department), from the Veterinary Hospital da Universidade Federal de Viçosa (UFV). The samples were submitted for DNA extraction and subsequently to polymerase chain reaction of nested type (nPCR), for the molecular diagnosis of A. phagocytophilum and other hemoparasites. From all the samples, 60 were positive for at least one hemoparasite. It was possible to detect the presence of four genera of hemoparasites in the blood samples assessed: Ehrlichia, Anaplasma, Babesia and Hepatozoon. Were amplified a total of 26 products from the monocytic ehrlichia spp primer, 23 from the granulocytic ehrlichia spp primer, 26 with BAB-rum primer and 10 using the hsp70 primer. After analyzing the results, from a total of 85 it was possible to identify the following hemoparasites: 23 E. canis (identity 94%- 100%), 7 A. platys (identity 97%-100%), 11 B. vogeli (identity 86%-99%) and 15 H. canis (94% -99% identity). No samples were positive using the msp4 primer, specific for A. phagocytophilum, however, the sequence of one of the amplified samples had a 99% identity with a partial sequence of the 16S rRNA gene of A. phagocytophilum deposited on GenBank. The non- amplification of products with a specific primer for A. phagocytophilum can be attributed to several hypotheses, but this does not exclude the possibility that the bacterium is present in the municipality of Viçosa or in the microregion.

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are causative agents of Lyme disease and human granulocytic anaplasmosis. Their common vector in Europe are the ticks from the genus Ixodes. In our work, we focused on interaction of innate immune cells with the causative agent of Lyme diseases, that are insubstitutable in their function in the early phase of the disease. Anaplasma phagocytophilum is hard to cultivate, the only possibility is to cultivate it in cell cultures. Successful cultivation of Anaplasma phagocytophilum acquired from patients in our geographic area is crucial for following experiments and for diagnostics too. In our experiments, we used validated cell cultures of HL-60 cells, canine monocytes DH82 and murine monocytes P388D1. During our studies of interaction of the causative agent of Lyme diseases with cells, we used two strains of different species Borrelia. Borrelia garinii M192 and Borrelia burgdorferi sensu stricto B31. These strains vary in virulence. The strain M192 is virulent, but the strain B31 lost its virulence by passages. We specialised in study of morphological changes using light microscopy (observation of dyed and fixed preparates and observation in dark field), eventually by transmision electron microscopy. During our experiments, we concluded that HL-60...

Globally, vector borne diseases cause more than a million deaths each year and more than a billion infections in humans. Ticks are of big medicinal importance since they can transmit pathogens that can cause serious infections. Some recently discovered pathogens that can cause infections in humans are Anaplasma phagocytophilum (A. phagocytophilum) that can cause human granulocytic anaplasmosis (HGA) and Candidatus Neoehrlichia mikurensis (N. mikurensis) that can cause Neoehrlichiosis. It is still widely unknown how prevalent these pathogens are, if ticks can be infected with both of these pathogens and if these pathogens can be transovarially transmitted from adult female to egg and larvae. This study aims to screen for these pathogens in collected ticks from southern Sweden and to detect eventual co-infections and transovarial transmission. A real-time qPCR assay targeting the 16S rRNA gene of N. mikurensis and other Anaplasmataceae was applied on 1356 Ixodes ricinus (I. ricinus) ticks collected from 5 sites in southern Sweden. Positive samples were subjected to Sanger sequencing. A. phagocytophilum occurred in 4.64 % of the ticks, N. mikurensis occurred in 1.33 % of the ticks and also Rickettsia was found to occur in 6.27 % of the ticks. No co-infection was detected. Some samples of tick larvae showed positive results after qPCR, indicating transovarial transmission, but none of the sequences were readable.

Ixodes genties erkės yra krauju mintantys nariuotakojai, visų – stuburinių gyvūnų, taip pat žmogaus parazitai. Ixodes ricinus erkės perneša daugybę ligų sukėlėjų, tokių kaip: erkinio encefalito (erkinio encefalito virusas), Laimo ligos (Borrelia burgdorferi sensu lato bakterijos), bebeziozės sukėlėjus (Babesia microti ir Babesia divergens). Taip pat platina ir Anaplasma phagocytophilum bakterijas. Šiuo metu erkių platinamos ligos yra dažniausios vektorių sukeliamos infekcijos Europoje. Pastaraisiais metais išpopuliarėjo molekulinės biologijos metodai, paremti patogenų nukleino rūgščių polimerazės grandinine reakcija ir RLB hibridizacija. Šie metodai laikomi tinkamiausiais, jautriausiais ir specifiškiausiais erkių platinamų ligų sukėlėjų diagnostikai. Šiame darbe naudoti molekuliniai metodai erkių platinamų ligų sukėlėjų: erkinio encefalito viruso, Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato ir Babesia spp., nustatymui iš 2200 erkių, surinktų keturiuose Lietuvos rajonuose. Tyrimų metu nustatyti šeši erkinio encefalito viruso infekcijų atvejai, iš kurių penki teigiami mėginiai nustatyti Radviliškio raj. surinktose erkėse ir vienas – Utenos raj. Anaplasma phagocytophilum bakterijų infekcijos yra dažniausiai pasitaikančios infekcijos Lietuvos erkėse. Tyrimų metu nustatyti 37 (3 %) teigiami Anaplasma phagocytophilum mėginiai, iš kurių 17 (46 %) nustatyti Kėdainių raj. surinktose erkėse, 15 (32 %) – Klaipėdos, 5 (14 %) – Radviliškio ir 3 (8 %) – Utenos raj. Tarp šių... [toliau žr. visą tekstą] / Ticks of genus Ixodes that infect livestock, deer, dogs, and a wide variety of other species including humans. Ixodes ricinus can also transmit numerous diseases including tick-borne encephalitis, Lyme diseases (Borrelia burgdorferi sensu lato infections), babesiosis (Babesia microti and Babesia divergens infections). It can also spread Anaplasma phagocytophilum bacteria. Now tick-borne infections are the most frequent human vector-borne infections in Europe, the incidence of these infections has been on rise, and new infections have emerged. In recent years, molecular detection methods based on PCR amplification of the nucleic acids of pathogens and Reverse Line blot hybridization have been showed to be effective, sensitive and specific methods for diagnosis of tick-borne diseases. In this work were used molecular detection methods for tick-borne pathogens: tick-borne encephalitis virus (TBEV), Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, and Babesia spp., diagnostics in 2200 ticks collected in four Lithuanian regions forests. During investigation were identified six cases of TBEV, which five positive samples were from ticks collected in Radviliškis region and one from Utenos region forest. Anaplasma phagocytophilum bacteria infections were most frequent in Lithuanian ticks, during researches were identified 37 (3 %) cases (17 (46 %) in Kėdainių region, 15 (32 %) – Klaipėdos, 5 (14 %) – Radviliškio, and 3 (8 %) – Utenos region). In these 81 % were in adult... [to full text]

No description available.

The present thesis examines questions related to the temporal origin of the Flavivirus genus and the ecology of tick-borne pathogens. In the first study, we date the origin and divergence time of the Flavivirus genus. It has been argued that the first flaviviruses originated after the last glacial maximum. This has been contradicted by recent analyses estimating that the tick-borne flaviviruses emerged at least before 16,000 years ago. It has also been argued that the Powassan virus was introduced into North America at the time between the opening and splitting of the Beringian land bridge. Supported by tip date and biogeographical calibration, our results suggest that this genus originated circa 120,000 (156,100–322,700) years ago if the Tamana bat virus is included in the genus, or circa 85,000 (63,700–109,600) years ago excluding the Tamana bat virus. In the second study we estimate the prevalence of tick-borne encephalitis virus (TBEV) in host-seeking Ixodes ricinus from 29 localities in Sweden and compare our data with those of neighbouring countries. Nymphs and adult ticks were screened for TBEV using a real-time PCR assay. The mean TBEV prevalence for all tick stages combined was 0.26% for Sweden and 0.28% for all Scandinavian countries, excluding Iceland. The low prevalence of TBEV in nature may partly be explained by the fact that TBEV occurs in spatially small foci and that the inclusion of ticks from non-infected foci will reduce the prevalence estimate. In the third and fourth study, we conducted the first large-scale investigations to estimate the prevalence and geographical distribution of Anaplasma spp. and Rickettsia spp. in host-seeking larvae, nymphs and adults of I. ricinus ticks in Sweden. Ticks were collected from several localities in central and southern Sweden and were subsequently screened for the presence of Anaplasma spp. and Rickettsia spp. using a real-time PCR assay. For all active tick stages combined, the mean prevalence of Anaplasma spp. and Rickettsia spp. in I. ricinus in Sweden was estimated to 1.1% and 4.8%, respectively. It was also shown that A. phagocytophilum and R. helvetica are the main Anaplasma and Rickettsia species occurring in Sweden.

No description available.

Depuis les années 90, les études réalisées au Canada ont permis d’identifier de nouvelles zones endémiques de l’agent de la maladie de Lyme, Borrelia burgdorferi, ou de sa tique vectrice, Ixodes scapularis. Ces régions représentent des zones privilégiées pour étudier le cycle de transmission dans son contexte environnemental. L’objectif principal de ce projet est d’étudier les relations spirochètes – tiques - hôtes et les facteurs environnementaux impliqués dans le cycle de transmission à partir des données du vecteur et de l’agent pathogène recueilli dans le Sud-Ouest du Québec de 2007 à 2008. Tout d’abord sera décrite la saisonnalité des tiques et des associations entre le vecteur et les hôtes rongeurs capturés. En effet, l’identification de la saisonnalité spécifique à chaque stade de la tique I. scapularis permet d’établir quels seront les mois propices pour effectuer les futures collectes de tiques. La saisonnalité synchrone des tiques immatures en quête peut également être un indicateur de la nature ou des souches de B. burgdorferi retrouvées. L’association des tiques immatures à différentes espèces ou à différentes classes d’hôtes (c.-à-d. âge, sexe, statut reproductif) a également été explorée. Nos résultats montrent que les souris du genre Peromyscus, principalement les mâles adultes, contribuent significativement à la survie et au développement du complexe I. scapularis - B. burgdorferi. Les tamias et les écureuils contribuent aussi à la survie et au développement des populations de la tique I. scapularis. Ensuite les associations entre le vecteur et les hôtes cervidés ont été examinées en tenant compte des facteurs environnementaux associés à leur niveau d’infestation. Ceci a permis d’évaluer l’utilisation des cerfs à titre de sentinelles pour le vecteur et les agents pathogènes. D’après nos résultats, bien qu’ils soient des sentinelles efficaces pour détecter Anaplasma phagocytophilum, les cerfs semblent des sentinelles inefficaces pour détecter les zones d’établissement du complexe I. scapularis-B. burgdorferi. Enfin, une analyse de l’impact de la diversité des hôtes et de l’habitat sur l’abondance de la tique I. scapularis et la prévalence de B. burgdorferi a été effectuée et ce, en tenant compte d’autres facteurs environnementaux. Ces analyses ont permis de déterminer les facteurs critiques pour l’établissement du complexe I. scapularis – B. burgdorferi et d’explorer la contribution relative de diverses espèces d’hôtes. D’après nos études, la diversité de la communauté d’hôte et la diversité de l’habitat influencent le complexe I. scapularis - B. burgdorferi. De plus, le climat (la température et les précipitations) joue un rôle significatif dans l’établissement, la survie et le développement des populations d’I. scapularis. Ce projet de recherche a permis d’explorer et d’identifier divers facteurs environnementaux biotiques et abiotiques influençant l’établissement du complexe I. scapularis - B. burgdorferi dans le Sud-Ouest du Québec. Ceux-ci pourraient être utilisés à titre d’indicateurs environnementaux du risque de la maladie de Lyme au Québec et possiblement ailleurs au Canada. / Since the 90s in Canada, studies have identified new endemic areas for the agent of Lyme disease, Borrelia burgdorferi and the tick vector, Ixodes scapularis. These newly endemic areas for complex I. scapularis - B. burgdorferi are prime areas to study the transmission cycle in its environmental context. The main objective of this project is to study the spirochete-tick-host relationships and environmental factors involved in the transmission cycle from the data of the vector and the pathogen collected in southwestern Quebec, 2007 to 2008. First, a description of the seasonality of ticks and associations between the vector and captured rodent hosts was performed. The identification of specific seasonality at each stage of the tick I. scapularis establishes months conducive to make the future collections of ticks. Synchronous seasonality of questing immature ticks may also be an indicator of the nature or strain of B. burgdorferi found. In addition, the association of immature ticks in different species or different classes of hosts (i.e. age, sex, reproductive status) was explored. The Peromyscus mice and adult males in particular contributed significantly to the survival and development of complex I. scapularis-B. burgdorferi. Chipmunks and squirrels also contributed to the survival and development of I. scapularis populations. Second, the associations between vector and examined deer was described taking into account the environmental factors associated with the level of infestation and the use of deer as sentinels for the vector and the pathogen was evaluated. According to our results, the deer are effective sentinels to detect Anaplasma phagocytophilum, but seem ineffective or imprecise to detect endemic areas for complex I. scapularis-B. burgdorferi. Finally, an analysis of the impact of the host diversity and habitat on the abundance of I. scapularis and prevalence of B. burgdorferi considering other environmental factors was performed. These analyzes were used to determine the critical factors in the establishment of complex I. scapularis-B. burgorferi and explore the relative contribution of various host species. According to our studies, the diversity of the host community and habitat influences the complex I. scapularis-B. burgdorferi. On the other hand, the climate (temperature and precipitation) plays a significant role in the establishment, survival and development of the I. scapularis populations. This study allowed us to explore and identify various biotic and abiotic environmental factors influencing the establishment of complex I. scapularis-B. burgdorferi in southwestern Quebec. These environmental factors could be used as environmental indicators of the risk of Lyme disease in Quebec and possibly elsewhere in Canada.