Related Articles

Background

A major challenge in sheep farming during the grazing season along the coast of south-western Norway is tick-borne fever (TBF) caused by the bacteria Anaplasma phagocytophilum that is transmitted by the tick Ixodes ricinus.

Methods

A study was carried out in 2007 and 2008 to examine the prevalence of A. phagocytophilum infection and effect on weaning weight in lambs. The study included 1208 lambs from farms in Sunndal Ram Circle in Møre and Romsdal County in Mid-Norway, where ticks are frequently observed. All lambs were blood sampled and serum was analyzed by an indirect fluorescent antibody assay (IFA) to determine an antibody status (positive or negative) to A. phagocytophilum infection. Weight and weight gain and possible effect of infection were analyzed using ANOVA and the MIXED procedure in SAS.

Results

The overall prevalence of infection with A. phagocytophilum was 55%. A lower weaning weight of 3% (1.34 kg, p < 0.01) was estimated in lambs seropositive to an A. phagocytophilum infection compared to seronegative lambs at an average age of 137 days.

Conclusions

The results show that A. phagocytophilum infection has an effect on lamb weight gain. The study also support previous findings that A. phagocytophilum infection is widespread in areas where ticks are prevalent, even in flocks treated prophylactic with acaricides.

Backgroud

Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila) causes the disease tick-borne fever (TBF) in domestic ruminants and has for decades been one of the main scourges for the sheep industry in the coastal areas of Norway. Current control strategies are based on reduction of tick infestation by chemical acaricides.

Methods

In the present study, we investigated if frequent pour-on applications of pyrethroids would reduce tick infestion rate and seroprevalence of A. phagocytophilum infection in sheep. Forty lambs, one month old, of the Norwegian White Sheep breed were used. The lambs belonged to the experimental sheep flock at the Department of Production Animal Clinical Sciences. None of the lambs had been on I. ricinus infested pasture before turnout (day 0). All lambs were twins and twenty lambs were treated with a pour-on pyrethroid (Bayticol®, Bayer A/S, DK-2300) with a dose of 5 ml on days 0, 14, 28, 42, 56, 70, 84, 98, 112 and 128. Twenty lambs were untreated controls. The lambs were collected every fourteen days on pasture for treatment. In addition, the lambs were examined for ticks, blood sampled, weighed, and rectal temperature was recorded.

Results and conclusion

A significant reduction in tick infestion rate was detected on treated lambs. However, the present results indicate that frequent acaricide treatment does not reduce the seroprevalence to A. phagocytophilum on tick-infested pasture.

Background

Equine Granulocytic Anaplasmosis (EGA) is caused by Anaplasma phagocytophilum, a tick-transmitted, obligate intracellular bacterium. In Europe, it is transmitted by Ixodes ricinus. A large number of genetic variants of A. phagocytophilum circulate in nature and have been found in ticks and different animals. Attempts have been made to assign certain genetic variants to certain host species or pathologies, but have not been successful so far. The purpose of this study was to investigate the causing agent A. phagocytophilum of 14 cases of EGA in naturally infected horses with molecular methods on the basis of 4 partial genes (16S rRNA, groEL, msp2, and msp4).

Results

All DNA extracts of EDTA-blood samples of the horses gave bands of the correct nucleotide size in all four genotyping PCRs. Sequence analysis revealed 4 different variants in the partial 16S rRNA, groEL gene and msp2 genes, and 3 in the msp4 gene. One 16S rRNA gene variant involved in 11 of the 14 cases was identical to the "prototype" variant causing disease in humans in the amplified part [GenBank: U02521]. Phylogenetic analysis revealed as expected for the groEL gene that sequences from horses clustered separately from roe deer. Sequences of the partial msp2 gene from this study formed a separate cluster from ruminant variants in Europe and from all US variants.

Conclusions

The results show that more than one variant of A. phagocytophilum seems to be involved in EGA in Germany. The comparative genetic analysis of the variants involved points towards different natural cycles in the epidemiology of A. phagocytophilum, possibly involving different reservoir hosts or host adaptation, rather than a strict species separation.

Anaplasma phagocytophilum is a Gram-negative, tick-transmitted, obligate intracellular bacterium that elicits acute febrile diseases in humans and domestic animals. In contrast to the United States, human granulocytic anaplasmosis seems to be a rare disease in Europe despite the initial recognition of A. phagocytophilum as the causative agent of tick-borne fever in European sheep and cattle. Considerable strain variation has been suggested to occur within this species, because isolates from humans and animals differed in their pathogenicity for heterologous hosts. In order to explain host preference and epidemiological diversity, molecular characterization of A. phagocytophilum strains has been undertaken. Most often the 16S rRNA gene was used, but it might be not informative enough to delineate distinct genotypes of A. phagocytophilum. Previously, we have shown that A. phagocytophilum strains infecting Ixodes ricinus ticks are highly diverse in their ankA genes. Therefore, we sequenced the 16S rRNA and ankA genes of 194 A. phagocytophilum strains from humans and several animal species. Whereas the phylogenetic analysis using 16S rRNA gene sequences was not meaningful, we showed that distinct host species correlate with A. phagocytophilum ankA gene clusters.

Anaplasma phagocytophilum DNA is detected in Portuguese Ixodes ricinus and I. ventalloi ticks.

A total of 278 Ixodes ticks, collected from Madeira Island and Setúbal District, mainland Portugal, were examined by polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum. Six (4%) of 142 Ixodes ricinus nymphs collected in Madeira Island and 1 nymph and 1 male (2%) of 93 I. ventalloi collected in Setúbal District tested positive for A. phagocytophilum msp2 genes or rrs. Infection was not detected among 43 I. ricinus on mainland Portugal. All PCR products were confirmed by nucleotide sequencing to be identical or to be most closely related to A. phagocytophilum. To our knowledge, this is the first evidence of A. phagocytophilum in ticks from Setúbal District, mainland Portugal, and the first documentation of Anaplasma infection in I. ventalloi. Moreover, these findings confirm the persistence of A. phagocytophilum in Madeira Island's I. ricinus.

Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis (HGA), shares the same enzootic life cycle as Borrelia burgdorferi, the causative agent of Lyme disease. Although La Crosse, WI, is a well-recognized Lyme disease focus with an abundance of Ixodes scapularis vector ticks and the first documentation of HGA occurred in patients from northwestern Wisconsin, local transmission of A. phagocytophilum has not to date been documented. In this study, we evaluated DNA extracted from 201 ticks captured locally by a real-time PCR that targeted a unique region within msp2, and 24 samples (12%) yielded positive results. The PCR also detected A. phagocytophilum DNA in blood samples obtained from 53 patients with clinical abnormalities consistent with HGA, and sequencing confirmed that the DNA was recovered from the Ap-ha variant of A. phagocytophilum, associated exclusively with human infection. The findings therefore confirmed that the upper Midwestern focus for HGA endemicity now includes the regions immediately surrounding La Crosse, WI. The results also validated the utility of the real-time msp2 PCR test for confirming acute HGA in the clinical setting.

Borrelia burgdorferi, the agent of Lyme disease, and Anaplasma phagocytophilum, the agent of human anaplasmosis, are both transmitted by Ixodes sp. ticks and may occasionally coinfect a host. The population distributions of tick-transmitted B. burgdorferi infection were assessed using quantitative PCR targeting the flaB gene of B. burgdorferi in the ear, heart base, quadriceps muscle, skin, and tibiotarsal joint tissue of C3H mice previously infected with A. phagocytophilum. Population distributions of Anaplasma infection were assessed by targeting the p44 gene. A. phagocytophilum in blood and serologic response to both agents were evaluated. Spirochete numbers were increased in the ears, heart base, and skin of coinfected mice, but Anaplasma numbers remained constant. Antibody response to A. phagocytophilum, but not B. burgdorferi, was decreased in coinfected mice. These results suggest that coinfection with A. phagocytophilum and B. burgdorferi modulates pathogen burden and host antibody responses. This may be explained by the ability of A. phagocytophilum to functionally impair neutrophils, important cells in the early defense against B. burgdorferi infection.

In Germany humans with acute granulocytic ehrlichiosis have not yet been described. Here, we characterized three different genes of Anaplasma phagocytophilum strains infecting German Ixodes ricinus ticks in order to test whether they differ from strains in other European countries and the United States. A total of 1,022 I. ricinus ticks were investigated for infection with A. phagocytophilum by nested PCR and sequence analysis. Forty-two (4.1%) ticks were infected. For all positive ticks, parts of the 16S rRNA and groESL genes were sequenced. The complete coding sequence of the ankA gene could be determined in 24 samples. The 16S rRNA and groESL gene sequences were as much as 100% identical to known sequences. Fifteen ankA sequences were ≥99.37% identical to sequences derived from humans with granulocytic ehrlichiosis in Europe and from a horse with granulocytic ehrlichiosis in Germany. Thus, German I. ricinus ticks most likely harbor A. phagocytophilum strains that can cause disease in humans. Nine additional sequences were clearly different from known ankA sequences. Because these newly described sequences have never been obtained from diseased humans or animals, their biological significance is currently unknown. Based on this unexpected sequence heterogeneity, we propose to use the ankA gene for further phylogenetic analyses of A. phagocytophilum and to investigate the biology and pathogenicity of strains that differ in the ankA gene.

Background

Anaplasma 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.

Methods

Two 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.

Results

Both 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.

Conclusion

Organisms 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.

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 analysing 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 analyse 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.

Abstract

Granulocytic anaplasmosis (GA) is an emerging tick-transmitted disease that persists in rodent- Ixodes ricinus-complex tick cycles across the Holarctic. Although the putative reservoir for anaplasmosis in the western United States is the dusky-footed woodrat (Neotoma fuscipes), this rodent was not shown reservoir-competent because of failure of infection from woodrats to other animals via ticks. Redwood chipmunks are common in habitats where Anaplasma phagocytophilum is common, have high PCR- and seroprevalence, and are infested with a diversity of Ixodes spp. ticks. Experimental infection of seven wild-caught A. phagocytophilum-negative redwood chipmunks induced persistent periods of recurrent rickettsemia during the persistent phase of infection. Of three animals for which xenodiagnosis was attempted, all successfully infected pools of I. pacificus larvae during the primary rickettsemia. We show that chipmunks are reservoir-competent for GA and may be important for maintaining infection in nature.

Ecological changes are recognized as an important driver behind the emergence of infectious diseases. The prevalence of infection in ticks depends upon ecological factors that are rarely taken into account simultaneously. Our objective was to investigate the influences of forest fragmentation, vegetation, adult tick hosts, and habitat on the infection prevalence of three tick-borne bacteria, Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and Rickettsia sp. of the spotted fever group, in questing Ixodes ricinus ticks, taking into account tick characteristics. Samples of questing nymphs and adults were taken from 61 pastures and neighboring woodlands in central France. The ticks were tested by PCR of pools of nymphs and individual adults. The individual infection prevalence was modeled using multivariate regression. The highest infection prevalences were found in adult females collected in woodland sites for B. burgdorferi sensu lato and A. phagocytophilum (16.1% and 10.7%, respectively) and in pasture sites for Rickettsia sp. (8.7%). The infection prevalence in nymphs was lower than 6%. B. burgdorferi sensu lato was more prevalent in woodlands than in pastures. Forest fragmentation favored B. burgdorferi sensu lato and A. phagocytophilum prevalence in woodlands, and in pastures, the B. burgdorferi sensu lato prevalence was favored by shrubby vegetation. Both results are probably because large amounts of edges or shrubs increase the abundance of small vertebrates as reservoir hosts. The Rickettsia sp. prevalence was maximal on pasture with medium forest fragmentation. Female ticks were more infected by B. burgdorferi sensu lato than males and nymphs in woodland sites, which suggests an interaction between the ticks and the bacteria. This study confirms the complexity of the tick-borne pathogen ecology. The findings support the importance of small vertebrates as reservoir hosts and make a case for further studies in Europe on the link between the composition of the reservoir host community and the infection prevalence in ticks.

The msp2 and p44 genes encode polymorphic major outer membrane proteins that are considered unique to the intraerythrocytic agent of Anaplasma marginale and the intragranulocytic agent of Anaplasma phagocytophilum, respectively. In the present study, however, we found an msp2 gene in A. phagocytophilum that was remarkably conserved among A. phagocytophilum strains from human granulocytic anaplasmosis (HGA) patients, ticks, and a horse from various regions in the United States, but the gene was different in a sheep isolate from the United Kingdom. The msp2 gene in the A. phagocytophilum strain HZ genome was a single-copy gene and was located downstream of two Ehrlichia chaffeensis omp-1 homologs and a decarboxylase gene (ubiD). The msp2 gene was expressed by A. phagocytophilum in the blood from HGA patients NY36 and NY37 and by A. phagocytophilum isolates from these patients cultured in HL-60 cells at 37°C. The msp2 gene was also expressed in a DBA/2 mouse infected by attaching ticks infected with strain NTN-1 and in a horse experimentally infected by attaching strain HZ-infected ticks. However, the transcript of the msp2 gene was undetectable in A. phagocytophilum strain HZ in SCID mice and Ixodes scapularis ticks infected with strain NTN-1. These results indicate that msp2 is functional in various strains of A. phagocytophilum, and relative expression ratios of msp2 to p44 vary in different infected hosts. These findings may be important in understanding roles that Msp2 proteins play in granulocytic ehrlichia infection and evolution of the polymorphic major outer membrane protein gene families in Anaplasma species.

Ticks act as vectors of many pathogens of domestic animals and humans. Anaplasma phagocytophilum in Europe is transmitted by the ixodid tick vector Ixodes ricinus. A. phagocytophilum causes a disease with diverse clinical signs in various hosts. A great genetic diversity of the groESL operon of A. phagocytophilum has been found in ticks elsewhere. In Slovenia, the variety of the groESL operon was conducted only on deer samples. In this study, the prevalence of infected ticks was estimated and the diversity of A. phagocytophilum was evaluated. On 8 locations in Slovenia, 1924 and 5049 (6973) I. ricinus ticks were collected from vegetation in the years 2005 and 2006, respectively. All three feeding stages of the tick's life cycle were examined. The prevalence of ticks infected with A. phagocytophilum in the year 2005 and in the year 2006 was 0.31% and 0.63%, respectively, and it did not differ considerably between locations. The similarity among the sequences of groESL ranged from 95.6% to 99.8%. They clustered in two genetic lineages along with A. phagocytophilum from Slovenian deer. One sequence formed a separate cluster. According to our study, the prevalence of A. phagocytophilum in ticks is comparable to the findings in other studies in Europe, and it does not vary considerably between locations and tick stages. According to groESL operon analysis, two genetic lineages have been confirmed and one proposed. Further studies on other genes would be useful to obtain more information on genetic diversity of A. phagocytophilum in ticks in Slovenia.

Background

Anaplasma phagocytophilum causes human granulocytic anaplasmosis (HGA) in humans, which has been recognized as an emerging tick-borne disease in the United States and Europe. Although about 65 cases of HGA have been reported in Europe, some of them do not fulfill the criteria for confirmed HGA. Confirmation of HGA requires A. phagocytophilum isolation from blood, and/or identification of morulae in granulocytes and/or positive PCR results with subsequent sequencing of the amplicons to demonstrate specific rickettsial DNA. Seroconversion or at least fourfold increase in antibody titers to A. phagocytophilum has been used as criteria for confirmed HGA also.

Case presentation

Infection with A. phagocytophilum was confirmed by PCR in a patient in Sicily, Italy, who had negative serology for A. phagocytophilum. A fragment of A. phagocytophilum 16S rDNA was amplified by two independent laboratories and sequenced from two separate patient's blood samples. The 16S rDNA sequence was identical in both samples and identical to the sequence of the A. phagocytophilum strain USG3 originally obtained from a dog.

Conclusion

Infection with A. phagocytophilum was confirmed in a patient without a detectable antibody response against the pathogen. The results reported herein documented the first case of confirmed HGA in Sicily, Italy. These results suggested the possibility of human infections with A. phagocytophilum strains that result in clinical symptoms and laboratory findings confirmatory of HGA but without detectable antibodies against the pathogen.

Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis (HGA), one of the major tick-borne zoonoses in the United States. The surface of A. phagocytophilum plays a crucial role in subverting the hostile host cell environment. However, except for the P44/Msp2 outer membrane protein family, the surface components of A. phagocytophilum are largely unknown. To identify the major surface proteins of A. phagocytophilum, a membrane-impermeable, cleavable biotin reagent, sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-Biotin), was used to label intact bacteria. The biotinylated bacterial surface proteins were isolated by streptavidin agarose affinity purification and then separated by electrophoresis, followed by capillary liquid chromatography-nanospray tandem mass spectrometry analysis. Among the major proteins captured by affinity purification were five A. phagocytophilum proteins, Omp85, hypothetical proteins APH_0404 (designated Asp62) and APH_0405 (designated Asp55), P44 family proteins, and Omp-1A. The surface exposure of Asp62 and Asp55 was verified by immunofluorescence microscopy. Recombinant Asp62 and Asp55 proteins were recognized by an HGA patient serum. Anti-Asp62 and anti-Asp55 peptide sera partially neutralized A. phagocytophilum infection of HL-60 cells in vitro. We found that the Asp62 and Asp55 genes were cotranscribed and conserved among members of the family Anaplasmataceae. With the exception of P44-18, all of the proteins were newly revealed major surface-exposed proteins whose study should facilitate understanding the interaction between A. phagocytophilum and the host. These proteins may serve as targets for development of chemotherapy, diagnostics, and vaccines.

Human granulocytic anaplasmosis (HGA), caused by Anaplasma phagocytophilum, is an emerging tick-borne zoonotic disease throughout the world. The first HGA cases in China were documented in 2008, and the greatest challenge posed by the disease is rapid and accurate diagnosis during the acute phage of illness. In this study, we successfully cloned and expressed an A. phagocytophilum immunoreactive surface protein (major surface protein 2 [MSP2]) and demonstrated that this recombinant protein has natural immunogenicity by Western blotting and enzyme-linked immunosorbent assay (ELISA) using human HGA-positive sera and reference rabbit HGA-positive sera. The rabbit antisera against the recombinant protein also reacted actively with the natural antigen of A. phagocytophilum by immunofluorescence assay (IFA). No cross-reaction was observed between the recombinant protein and rabbit antisera against 10 common members of the order Rickettsiales by ELISA when the sera were diluted more than 1:100. We concluded that the recombinant MSP2 protein exhibited excellent antigenicity and specificity, results that should lay the foundation for the development of a simple and rapid diagnostic reagent and a vaccination for anaplasmosis.

Anaplasma phagocytophilum, the agent of human anaplasmosis, persists in ticks and mammals. We show that A. phagocytophilum induces the phosphorylation of actin in an Ixodes ricinus tick cell line and Ixodes scapularis ticks, to alter the ratio of monomeric/filamentous (G/F) actin. A. phagocytophilum–induced actin phosphorylation was dependent on Ixodes p21-activated kinase (IPAK1)–mediated signaling. A. phagocytophilum stimulated IPAK1 activity via the G protein–coupled receptor Gβγ subunits, which mediated phosphoinositide 3-kinase (PI3K) activation. Disruption of Ixodes gβγ, pi3k, and pak1 reduced actin phosphorylation and bacterial acquisition by ticks. A. phagocytophilum–induced actin phosphorylation resulted in increased nuclear G actin and phosphorylated actin. The latter, in association with RNA polymerase II (RNAPII), enhanced binding of TATA box–binding protein to RNAPII and selectively promoted expression of salp16, a gene crucial for A. phagocytophilum survival. These data define a mechanism that A. phagocytophilum uses to selectively alter arthropod gene expression for its benefit and suggest new strategies to interfere with the life cycle of this intracellular pathogen, and perhaps other Rickettsia-related microbes of medical importance.

A total of 60 sheep were exposed to Anaplasma phagocytophilum infection on an enclosed area of Ixodes ricinus-infested pasture in North Wales, United Kingdom, and rapidly acquired acute A. phagocytophilum infections detectable by PCR and blood smear examination. Of the ticks that had engorged in the previous instar on infected sheep, 52% of adult ticks and 28% of nymphs were PCR positive; a significant, 10-fold increase in prevalence compared to that of ticks that engorged on sheep preinfection was observed (P = 0.015). The likelihood that ticks were PCR positive, after feeding on the sheep and molting to the next instar, increased marginally with increasing numbers of infected neutrophils per milliliter of blood of their sheep host (P = 0.068) and increased significantly when they were collected from sheep carrying higher numbers of adult female ticks (P = 0.017), but increasing numbers of feeding nymphs had a significant negative effect on transmission (P = 0.049). The numbers of circulating neutrophils and of infected neutrophils also varied significantly with the numbers of ticks feeding on the sheep when the blood was collected. Our study suggests that ruminants are efficient reservoirs of A. phagocytophilum during the acute and post-acute phases of infection. The risk of ruminant-derived infections may, however, be strongly affected by variations in tick densities, which may influence transmission from acutely infected animals via effects on the numbers of infected cells in the blood and possibly by within-skin modulation of infection.

Anaplasma phagocytophilum is the agent of human anaplasmosis, the second most common tick-borne illness in the United States. This pathogen, which is closely related to obligate intracellular organisms in the genera Rickettsia, Ehrlichia, and Anaplasma, persists in ticks and mammalian hosts; however, the mechanisms for survival in the arthropod are not known. We now show that A. phagocytophilum induces expression of the Ixodes scapularis salp16 gene in the arthropod salivary glands during vector engorgement. RNA interference–mediated silencing of salp16 gene expression interfered with the survival of A. phagocytophilum that entered ticks fed on A. phagocytophilum–infected mice. A. phagocytophilum migrated normally from A. phagocytophilum–infected mice to the gut of engorging salp16-deficient ticks, but up to 90% of the bacteria that entered the ticks were not able to successfully infect I. scapularis salivary glands. These data demonstrate the specific requirement of a pathogen for a tick salivary protein to persist within the arthropod and provide a paradigm for understanding how Rickettsia-like pathogens are maintained within vectors.

Background

Ixodes ricinus, a competent vector of several pathogens, is the tick species most frequently reported to bite humans in Europe. The majority of human cases of Lyme borreliosis (LB) and tick-borne encephalitis (TBE) occur in the north-eastern region of Italy. The aims of this study were to detect the occurrence of endemic and emergent pathogens in north-eastern Italy using adult tick screening, and to identify areas at risk of pathogen transmission. Based on our results, different strategies for tick collection and pathogen screening and their relative costs were evaluated and discussed.

Methods

From 2006 to 2008 adult ticks were collected in 31 sites and molecularly screened for the detection of pathogens previously reported in the same area (i.e., LB agents, TBE virus, Anaplasma phagocytophilum, Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis"). Based on the results of this survey, three sampling strategies were evaluated a-posteriori, and the impact of each strategy on the final results and the overall cost reductions were analyzed. The strategies were as follows: tick collection throughout the year and testing of female ticks only (strategy A); collection from April to June and testing of all adult ticks (strategy B); collection from April to June and testing of female ticks only (strategy C).

Results

Eleven pathogens were detected in 77 out of 193 ticks collected in 14 sites. The most common microorganisms detected were Borrelia burgdorferi sensu lato (17.6%), Rickettsia helvetica (13.1%), and "Ca. N. mikurensis" (10.5%). Within the B. burgdorferi complex, four genotypes (i.e., B. valaisiana, B. garinii, B. afzelii, and B. burgdorferi sensu stricto) were found. Less prevalent pathogens included R. monacensis (3.7%), TBE virus (2.1%), A. phagocytophilum (1.5%), Bartonella spp. (1%), and Babesia EU1 (0.5%). Co-infections by more than one pathogen were diagnosed in 22% of infected ticks. The prevalences of infection assessed using the three alternative strategies were in accordance with the initial results, with 13, 11, and 10 out of 14 sites showing occurrence of at least one pathogen, respectively. The strategies A, B, and C proposed herein would allow to reduce the original costs of sampling and laboratory analyses by one third, half, and two thirds, respectively. Strategy B was demonstrated to represent the most cost-effective choice, offering a substantial reduction of costs, as well as reliable results.

Conclusions

Monitoring of tick-borne diseases is expensive, particularly in areas where several zoonotic pathogens co-occur. Cost-effectiveness studies can support the choice of the best monitoring strategy, which should take into account the ecology of the area under investigation, as well as the available budget.

Serologic and molecular evidence of Anaplasma phagocytophilum has been demonstrated in white-tailed deer (WTD; Odocoileus virginianus), and deer are an important host for the tick vector Ixodes scapularis. In this study, we describe experimental infection of WTD with A. phagocytophilum. We inoculated four WTD with a human isolate of A. phagocytophilum propagated in tick cells. Two additional deer served as negative controls. All inoculated deer developed antibodies (titers, ≥64) to A. phagocytophilum, as determined by an indirect fluorescent antibody test, between 14 and 24 days postinfection [p.i.]), and two deer maintained reciprocal titers of ≥64 through the end of the 66-day study. Although morulae were not observed in granulocytes and A. phagocytophilum was not reisolated via tick cell culture of blood, 16S reverse transcriptase nested PCR (RT-nPCR) results indicated that A. phagocytophilum circulated in peripheral blood of three deer through at least 17 days p.i. and was present in two deer at 38 days p.i. Femoral bone marrow from one deer was RT-nPCR positive for A. phagocytophilum at 66 days p.i. There was no indication of clinical disease. These data confirm that WTD are susceptible to infection with a human isolate of A. phagocytophilum and verify that WTD produce detectable antibodies upon exposure to the organism. Because adults are the predominant life stage of I. scapularis found on deer and because adult I. scapularis ticks do not transmit A. phagocytophilum transovarially, it is unlikely that WTD are a significant source of A. phagocytophilum for immature ticks even though deer have a high probability of natural infection. However, the susceptibility and immunologic response of WTD to A. phagocytophilum render them suitable candidates as natural sentinels for this zoonotic tick-borne organism.

Mast cells are sentinels for infection. Upon exposure to pathogens, they release their stores of proinflammatory cytokines, chemokines, and histamine. Mast cells are also important for the control of certain tick-borne infections. Anaplasma phagocytophilum is an obligate intracellular tick-transmitted bacterium that infects neutrophils to cause the emerging disease granulocytic anaplasmosis. A. phagocytophilum adhesion to and infection of neutrophils depend on sialylated and α1,3-fucosylated glycans. We investigated the hypotheses that A. phagocytophilum invades mast cells and inhibits mast cell activation. We demonstrate that A. phagocytophilum binds and/or infects murine bone marrow-derived mast cells (BMMCs), murine peritoneal mast cells, and human skin-derived mast cells. A. phagocytophilum infection of BMMCs depends on α1,3-fucosylated, but not sialylated, glycans. A. phagocytophilum binding to and invasion of BMMCs do not elicit proinflammatory cytokine secretion. Moreover, A. phagocytophilum-infected cells are inhibited in the release of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), IL-13, and histamine following stimulation with IgE or antigen. Thus, A. phagocytophilum mitigates mast cell activation. These findings potentially represent a novel means by which A. phagocytophilum usurps host defense mechanisms and shed light on the interplay between mast cells and vector-borne bacterial pathogens.

Recent advances in climate research together with a better understanding of tick–pathogen interactions, the distribution of ticks and the diagnosis of tick-borne pathogens raise questions about the impact of environmental factors on tick abundance and spread and the prevalence and transmission of tick-borne pathogens. While undoubtedly climate plays a role in the changes in distribution and seasonal abundance of ticks, it is always difficult to disentangle factors impacting on the abundance of tick hosts from those exerted by human habits. All together, climate, host abundance, and social factors may explain the upsurge of epidemics transmitted by ticks to humans. Herein we focused on tick-borne pathogens that affect humans with epidemic potential. Borrelia burgdorferi s.l. (Lyme disease), Anaplasma phagocytophilum (human granulocytic anaplasmosis), and tick-borne encephalitis virus (tick-borne encephalitis) are transmitted by Ixodes spp. Crimean–Congo hemorrhagic fever virus (Crimean–Congo hemorrhagic fever) is transmitted by Hyalomma spp. In this review, we discussed how vector tick species occupy the habitat as a function of different climatic factors, and how these factors impact on tick survival and seasonality. How molecular events at the tick–pathogen interface impact on pathogen transmission is also discussed. Results from statistically and biologically derived models are compared to show that while statistical models are able to outline basic information about tick distributions, biologically derived models are necessary to evaluate pathogen transmission rates and understand the effect of climatic variables and host abundance patterns on pathogen transmission. The results of these studies could be used to build early alert systems able to identify the main factors driving the subtle changes in tick distribution and seasonality and the prevalence of tick-borne pathogens.

Summary: Anaplasma phagocytophilum persists in nature by cycling between mammals and ticks. Human infection by the bite of an infected tick leads to a potentially fatal emerging disease called human granulocytic anaplasmosis. A. phagocytophilum is an obligatory intracellular bacterium that replicates inside mammalian granulocytes and the salivary gland and midgut cells of ticks. A. phagocytophilum evolved the remarkable ability to hijack the regulatory system of host cells. A. phagocytophilum alters vesicular traffic to create an intracellular membrane-bound compartment that allows replication in seclusion from lysosomes. The bacterium downregulates or actively inhibits a number of innate immune responses of mammalian host cells, and it upregulates cellular cholesterol uptake to acquire cholesterol for survival. It also upregulates several genes critical for the infection of ticks, and it prolongs tick survival at freezing temperatures. Several host factors that exacerbate infection have been identified, including interleukin-8 (IL-8) and cholesterol. Host factors that overcome infection include IL-12 and gamma interferon (IFN-γ). Two bacterial type IV secretion effectors and several bacterial proteins that associate with inclusion membranes have been identified. An understanding of the molecular mechanisms underlying A. phagocytophilum infection will foster the development of creative ideas to prevent or treat this emerging tick-borne disease.