Erik Georg Granquist

Anaplasma phagocytophilum—a widespread multi-host pathogen with highly adaptive strategies

The bacterium Anaplasma phagocytophilum has for decades been known to cause the disease tick-borne fever (TBF) in domestic ruminants in Ixodes ricinus-infested areas in northern Europe. In recent years, the bacterium has been found associated with Ixodes-tick species more or less worldwide on the northern hemisphere. A. phagocytophilum has a broad host range and may cause severe disease in several mammalian species, including humans. However, the clinical symptoms vary from subclinical to fatal conditions, and considerable underreporting of clinical incidents is suspected in both human and veterinary medicine. Several variants of A. phagocytophilum have been genetically characterized. Identification and stratification into phylogenetic subfamilies has been based on cell culturing, experimental infections, PCR, and sequencing techniques. However, few genome sequences have been completed so far, thus observations on biological, ecological, and pathological differences between genotypes of the bacterium, have yet to be elucidated by molecular and experimental infection studies. The natural transmission cycles of various A. phagocytophilum variants, the involvement of their respective hosts and vectors involved, in particular the zoonotic potential, have to be unraveled. A. phagocytophilum is able to persist between seasons of tick activity in several mammalian species and movement of hosts and infected ticks on migrating animals or birds may spread the bacterium. In the present review, we focus on the ecology and epidemiology of A. phagocytophilum, especially the role of wildlife in contribution to the spread and sustainability of the infection in domestic livestock and humans.

Dynamic Transmission of Numerous Anaplasma phagocytophilum Genotypes among Lambs in an Infected Sheep Flock in an Area of Anaplasmosis Endemicity

ABSTRACT The transmission dynamics of Anaplasma phagocytophilum strains circulating within juvenile members of a sheep flock grazing on an Ixodes ricinus-infested pasture in southern Norway were monitored. PCR-based detection of the bacterial p44 fragments in the blood of 16 lambs sampled weekly for 16 weeks following their release into pasture revealed rickettsemia in all animals, with an increasing proportion of infected animals as the survey progressed. Comparison of partial msp4 sequences obtained from infected blood samples revealed 24 distinct genotypes, some of which were repeatedly encountered, occurring in up to six sheep over a 14-week period, whereas others were observed only once. Individual sheep were infected by up to five distinct genotypes, with a specific genotype being encountered for between one and three consecutive weeks, and in some sheep, genotypes detected early in the study were also present in later samples. In general, detection of A. phagocytophilum by PCR correlated well with the observation of infected neutrophils in blood smears. Together these results reveal a previously unrecognized diversity of A. phagocytophilum strains simultaneously circulating within an infected population in an area of endemicity and are consistent with a remarkably dynamic transmission of strains among infected animals.

Outer Membrane Protein Sequence Variation in Lambs Experimentally Infected with Anaplasma phagocytophilum

ABSTRACT Anaplasma phagocytophilum has long been known to cause tick-borne fever in ruminants and has been identified more recently as the causative agent of the emerging disease human granulocytic anaplasmosis. The related organism Anaplasma marginale uses gene conversion of the expression site for two major outer membrane proteins (OMPs) to generate extensive sequence and antigenic variation in these OMPs. This is thought to present a continuously varying repertoire of epitopes to the mammalian host and allow disease persistence. Recent genomic and structural data on human strains of A. phagocytophilum, together with animal studies in model systems, have implicated an orthologous OMP of A. phagocytophilum in a similar mechanism of variation. However, to date there has been little investigation of the mechanisms of antigenic variation or disease persistence in hosts naturally infected with field strains of A. phagocytophilum. Approximately 300,000 lambs in Norway suffer severe disease caused by A. phagocytophilum annually. We show here the persistent and cyclic nature of infection in these animals that is accompanied by loosely programmed sequence variation of the major OMP expression site in each rickettsemic peak. These data will allow analysis of interactions between A. phagocytophilum and the host immune system in naturally occurring persistent infections and provide an important comparison with enduring infections of cattle caused by A. marginale.

Anaplasma phagocytophilum variants in sympatric red deer (Cervus elaphus) and sheep in southern Norway

Infections by the ixodid tick-transmitted bacterial pathogen Anaplasma phagocytophilum are common in domestic ruminants and cervids in the coastal areas of southern Norway. Previous experimental work has shown that A. phagocytophilum strains recovered from red deer (Cervus elaphus) are infective in lambs, but epidemiological links between infections in red deer and sheep have yet to be established. To address this shortfall, the present study explores the genotypic relatedness between A. phagocytophilum strains infecting sympatric red deer and sheep. Blood from 32 lambs grazing on tick-infested pasture, and blood and tissues from 8 red deer shot in proximity to these pastures were collected during the summer and autumn of 2007. The presence of A. phagocytophilum in these samples was determined by PCR-based methods, and genotyping of detected strains was performed using comparative sequence analysis of 16S rDNA and msp4 fragments. A. phagocytophilum DNA was detected in 12 lambs and 7 red deer, 11 and 4 individuals of which 16S rDNA and msp4 sequence data were obtained from, respectively. A total of 9 genotypes were delineated, and only different individuals of the same host species were infected with indistinguishable A. phagocytophilum genotypes. Although 3 of the red deer-infecting genotypes belonged to a cluster of exclusively deer-associated strains phylogenetically remote from those commonly encountered in sheep, one red deer-infecting genotype, although unique, clustered tightly with genotypes associated with a wide range of hosts including sheep.

Variant-specific and diminishing immune responses towards the highly variable MSP2(P44) outer membrane protein of Anaplasma phagocytophilum during persistent infection in lambs.

Anaplasma phagocytophilum is the causative agent of tick-borne fever in small ruminants and has been identified as the zoonotic agent of human granulocytic anaplasmosis. The Norwegian strains of the rickettsia are naturally persistent in lambs and represent a suitable experimental system for analyzing the mechanisms of persistence. Variation of the outer membrane protein MSP2(P44) by recombination of variable pseudogene segments into an expression site is believed to play a key role in persistence of the organism. The goal of the present study was to analyze the dynamics of the immune response towards A. phagocytophilum and MSP2(P44) during persistent infection of lambs. Responses to the hypervariable region of MSP2(P44) were detected shortly after appearance of the respective variants in cyclic rickettsemic peaks, consistent with a process of antigenic variation. In addition, there was a diminishing antibody response to MSP2(P44) and to other A. phagocytophilum antigens overall with time of infection, that was not associated with clearance of the infection.

Variant -and individual dependent nature of persistent Anaplasma phagocytophilum infection

BackgroundAnaplasma phagocytophilum is the causative agent of tick-borne fever in ruminants and human granulocytotropic anaplasmosis (HGA). The bacterium is able to survive for several months in immune-competent sheep by modifying important cellular and humoral defence mechanisms. Little is known about how different strains of A. phagocytophilum propagate in their natural hosts during persistent infection.MethodsTwo groups of five lambs were infected with each of two 16S rRNA gene variants of A. phagocytophilum, i.e. 16S variant 1 which is identical to GenBank no M73220 and 16S variant 2 which is identical to GenBank no AF336220, respectively. The lambs were infected intravenously and followed by blood sampling for six months. A. phagocytophilum infection in the peripheral blood was detected by absolute quantitative real-time PCR.ResultsBoth 16S rRNA gene variants of A. phagocytophilum established persistent infection for at least six months and showed cyclic bacteraemias, but variant 1 introduced more frequent periods of bacteraemia and higher number of organisms than 16S rRNA gene variant 2 in the peripheral blood.ConclusionOrganisms were available from blood more or less constantly during the persistent infection and there were individual differences in cyclic activity of A. phagocytophilum in the infected animals. Two 16S rRNA gene variants of A. phagocytophilum show differences in cyclic activity during persistent infection in lambs.

Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum

BackgroundAnaplasma phagocytophilum is an intracellular organism in the Order Rickettsiales that infects diverse animal species and is causing an emerging disease in humans, dogs and horses. Different strains have very different cell tropisms and virulence. For example, in the U.S., strains have been described that infect ruminants but not dogs or rodents. An intriguing question is how the strains of A. phagocytophilum differ and what different genome loci are involved in cell tropisms and/or virulence. Type IV secretion systems (T4SS) are responsible for translocation of substrates across the cell membrane by mechanisms that require contact with the recipient cell. They are especially important in organisms such as the Rickettsiales which require T4SS to aid colonization and survival within both mammalian and tick vector cells. We determined the structure of the T4SS in 7 strains from the U.S. and Europe and revised the sequence of the repetitive virB6 locus of the human HZ strain.ResultsAlthough in all strains the T4SS conforms to the previously described split loci for vir genes, there is great diversity within these loci among strains. This is particularly evident in the virB2 and virB6 which are postulated to encode the secretion channel and proteins exposed on the bacterial surface. VirB6-4 has an unusual highly repetitive structure and can have a molecular weight greater than 500,000. For many of the virs, phylogenetic trees position A. phagocytophilum strains infecting ruminants in the U.S. and Europe distant from strains infecting humans and dogs in the U.S.ConclusionsOur study reveals evidence of gene duplication and considerable diversity of T4SS components in strains infecting different animals. The diversity in virB2 is in both the total number of copies, which varied from 8 to 15 in the herein characterized strains, and in the sequence of each copy. The diversity in virB6 is in the sequence of each of the 4 copies in the single locus and the presence of varying numbers of repetitive units in virB6-3 and virB6-4. These data suggest that the T4SS should be investigated further for a potential role in strain virulence of A. phagocytophilum.

A comparative study of clinical manifestations, haematological and serological responses after experimental infection with Anaplasma phagocytophilum in two Norwegian sheep breeds

BackgroundIt has been questioned if the old native Norwegian sheep breed, Old Norse Sheep (also called Norwegian Feral Sheep), normally distributed on coastal areas where ticks are abundant, is more protected against tick-borne infections than other Norwegian breeds due to a continuously high selection pressure on pasture. The aim of the present study was to test this hypothesis in an experimental infection study.MethodsFive-months-old lambs of two Norwegian sheep breeds, Norwegian White (NW) sheep and Old Norse (ON) sheep, were experimentally infected with a 16S rRNA genetic variant of Anaplasma phagocytophilum (similar to GenBank accession number M73220). The experiment was repeated for two subsequent years, 2008 and 2009, with the use of 16 lambs of each breed annually. Ten lambs of each breed were inoculated intravenously each year with 0.4 ml A. phagocytophilum-infected blood containing approximately 0.5 × 106 infected neutrophils/ml. Six lambs of each breed were used as uninfected controls. Half of the primary inoculated lambs in each breed were re-challenged with the same infectious dose at nine (2008) and twelve (2009) weeks after the first challenge. The clinical, haematological and serological responses to A. phagocytophilum infection were compared in the two sheep breeds.ResultsThe present study indicates a difference in fever response and infection rate between breeds of Norwegian sheep after experimental infection with A. phagocytophilum.ConclusionAlthough clinical response seems to be less in ON-lambs compared to NW-lambs, further studies including more animals are needed to evaluate if the ON-breed is more protected against tick-borne infections than other Norwegian breeds.

A morphological and molecular study of Anaplasma phagocytophilum transmission events at the time of Ixodes ricinus tick bite

BackgroundAnaplasma phagocytophilum is the causative agent of human granulocytic anaplasmosis (HGA) in humans and tick-borne fever (TBF) in ruminants. The bacterium invades and replicates in phagocytes, especially in polymorphonuclear granulocytes.MethodsIn the present study, skin biopsies and ticks (Ixodes ricinus) were collected from tick feeding lesions on 38 grazing lambs between two and three weeks after access to pastures. The histopathological changes associated with tick bites and A. phagocytophilum infection, were described. In addition the skin biopsies were examined by immunohistochemistry. Furthermore, samples from blood, skin biopsies and ticks were examined by serology, PCR amplification of msp2 (p44), genotyping of rrs (16S rRNA) variants, and compared with the results obtained from histological and immunohistochemical investigations.ResultsTick bites were associated with chronic and hyperplastic inflammatory skin lesions in this study. A. phagocytophilum present in skin lesions were mainly associated with neutrophils and macrophages. Bacteria were occasionally observed in the Tunica media and Tunica adventitia of small vessels, but were rarely found in association with endothelial cells. PCR and genotyping of organisms present in blood, ticks and skin biopsies suggested a haematogenous and a local spread of organisms at the tick attachment sites.ConclusionsThe present study describes different aspects of A. phagocytophilum infection at the site of tick bite, and indicates that A. phagocytophilum rarely associates with endothelium during the early pathogenesis of infection.

An Emerging Tick-Borne Disease of Humans Is Caused by a Subset of Strains with Conserved Genome Structure

The prevalence of tick-borne diseases is increasing worldwide. One such emerging disease is human anaplasmosis. The causative organism, Anaplasma phagocytophilum, is known to infect multiple animal species and cause human fatalities in the U.S., Europe and Asia. Although long known to infect ruminants, it is unclear why there are increasing numbers of human infections. We analyzed the genome sequences of strains infecting humans, animals and ticks from diverse geographic locations. Despite extensive variability amongst these strains, those infecting humans had conserved genome structure including the pfam01617 superfamily that encodes the major, neutralization-sensitive, surface antigen. These data provide potential targets to identify human-infective strains and have significance for understanding the selective pressures that lead to emergence of disease in new species.