Phagocytophilum Strains Research Articles

Anaplasma phagocytophilum in urban and peri-urban passerine birds in Ile-de-France

Wild animals in general, birds in particular, play a key role in transporting ticks and propagating tick-borne pathogens. Several studies have confirmed the infection of birds with Anaplasma phagocytophilum, with overall prevalence varying widely from country to country and/or study to study. This zoonotic bacterium, transmitted mainly by ticks of the genus Ixodes, is responsible for granulocytic anaplasmosis in humans (HGA) and domestic animals (cats, dogs, horses). The disease is also called tick-borne fever (TBF) in ruminants. Extremely rare in the USA, TBF is very common in Europe, where it causes economic losses in livestock. Conversely, HGA is well established in the USA whereas only a few less severe cases have been observed in Europe. Current typing techniques support the existence of multiple variants with differences in virulence/pathogenicity and tropism for certain tick and host species. However, epidemiological cycles remain difficult to characterize in Europe. Several studies describe a cycle apparently involving only birds in Europe, but no such study has been conducted in mainland France. Our objectives were to search for A. phagocytophilum in passerine birds in the Ile-de-France region and to explore their diversity using groEL and ankA gene typing and multilocus sequence typing (MLST). Various tissues (spleen, liver, and skin) were collected from cadavers of 680 passerines between March and December 2021. The presence of A. phagocytophilum was detected by qPCR Taqman targeting the msp2 gene. Three blackbirds (Turdus merula) were found positive, representing detection rates of 0.4 % in all birds tested and 3.3 % in blackbirds. The higher frequency of detection in blackbirds could be at least partially explained by their lifestyle, as they feed on the ground. Analysis of the results of groEL and ankA typing and MLST from positive blackbirds support the hypothesis that the avian A. phagocytophilum strains in Ile-de-France are distinct from those found in mammals, and that they form their own cluster in Europe.

Molecular investigation of Anaplasma phagocytophilum and related strains among sheep flocks from different parts of Türkiye; with a note of phylogenetic analyses of Anaplasma phagocytophilum- like 1

Anaplasma phagocytophilum is a vector-borne zoonotic pathogen and can infect various vertebrate hosts, especially cattle, sheep, goats, horses, and dogs. Molecular-based studies have revealed that the agent has a high genetic diversity and closely related strains circulate in hosts. In this study, 618 sheep blood samples obtained from different geographic regions of Türkiye were researched for A.phagocytophilum and related strains with PCR, RFLP, and DNA sequence analyses. The DNA of these pathogens was detected in 110 (17.79%) samples. RFLP assay showed that all positive samples were infected with A.phagocytophilum-like 1, whereas A.phagocytophilum–like 2 and A.phagocytophilum were not detected. Partial parts of 16 S rRNA gene of seven randomly selected positive samples were sequenced. The phylogenetic analyses of these isolates revealed that at least two A.phagocytophilum-like 1 isolates circulate among hosts in Türkiye and around the world. A.phagocytophilum-related strains have been reported in molecular-based studies over the last few years, but there is a lack of data on the vector competence, epidemiology, clinical symptoms, and genetic diversity of these pathogens. Therefore, large-scale molecular studies are still needed to obtain detailed data on the above-mentioned topics.

Diversity of Anaplasma phagocytophilum Strains from Roe Deer (Capreolus capreolus) and Red Deer (Cervus elaphus) in Poland.

The Gram-negative bacterium Anaplasma phagocytophilum is an intracellular pathogen and an etiological agent of human and animal anaplasmosis. Its natural reservoir comprises free-ranging ungulates, including roe deer (Capreolus capreolus) and red deer (Cervus elaphus). These two species of deer also constitute the largest group of game animals in Poland. The aim of the study was to genotype and perform a phylogenetic analysis of A. phagocytophilum strains from roe deer and red deer. Samples were subjected to PCR amplification, sequencing, and phylogenetic analysis of strain-specific genetic markers (groEL, ankA). Five haplotypes of the groEL gene from A. phagocytophilum and seven haplotypes of ankA were obtained. The phylogenetic analysis classified the groEL into ecotypes I and II. Sequences of the ankA gene were classified into clusters I, II, and III. Strains of A. phagocytophilum from red deer were in the same ecotype and cluster as strains isolated from humans. Strains of A. phagocytophilum from roe deer represented ecotypes (I, II) and clusters (II, III) that were different from those isolated from red deer, and these strains did not show similarity to bacteria from humans. However, roe deer can harbor nonspecific strains of A. phagocytophilum more characteristic to red deer. It appears that the genetic variants from red deer can be pathogenic to humans, but the significance of the variants from roe deer requires more study.

MOLECULAR CHARACTERIZATION OF ANAPLASMA PHAGOCYTOPHILUM IN IXODID TICKS IN KAYSERİ REGION IN TURKEY

The study was conducted to investigate the presence of Anaplasma phagocytophilum in ixodid ticks collected from cattle in Kayseri region of Turkey using Real Time PCR and to characterize positive isolates based on 16S rRNA gene region. DNA was extracted from 265 adult ticks. Nested PCR analyses were performed using Anaplasma phagocytophilum-specific primers amplifying a 641 bp fragment of 16S rRNA gene region. Real Time PCR analysis revealed positive results in one H. marginatum and R. turanicus sample. DNA sequences were submitted to GenBank and analyzed by pairwise and multiple sequence alignments with other A. phagocytophilum strains in GenBank to investigate the phylogeny. The phylogenetic analysis revealed that A. phagocytophilum isolates collected from H. marginatum and R. turanicus samples in Kayseri region clustered into three main groups (A, B, and C) with previously reported isolates from the world. A and B groups showed high homology, whereas C group had an average genetic variation of 0.2%. The average genetic differences between A and B groups were 10.8±2.0 and 13.0±2.7% between A and C groups, while the average genetic difference between B and C groups was 13.8±2.8%. In conclusion, this study provides scientific data on molecular prevalence and genetic characteristics of A. phagocytophilum in tick samples in Turkey.

Temporal patterns of gene expression in response to inoculation with a virulent Anaplasma phagocytophilum strain in sheep

The aim of this study was to characterize the gene expression of host immune- and cellular responses to a Norwegian virulent strain of Anaplasma phagocytophilum, the cause of tick-borne fever in sheep. Ten sheep were intravenously inoculated with a live virulent strain of A. phagocytophilum. Clinical-, observational-, hematological data as well as bacterial load, flow cytometric cell count data from peripheral blood mononuclear cells and host’s gene expression post infection was analysed. The transcriptomic data were assessed for pre-set time points over the course of 22 days following the inoculation. Briefly, all inoculated sheep responded with clinical signs of infection 3 days post inoculation and onwards with maximum bacterial load observed on day 6, consistent with tick-borne fever. On days, 3–8, the innate immune responses and effector processes such as IFN1 signaling pathways and cytokine mediated signaling pathways were observed. Several pathways associated with the adaptive immune responses, namely T-cell activation, humoral immune responses, B-cell activation, and T- and B-cell differentiation dominated on the days of 8, 10 and 14. Flow-cytometric analysis of the PBMCs showed a reduction in CD4+CD25+ cells on day 10 and 14 post-inoculation and a skewed CD4:CD8 ratio indicating a reduced activation and proliferation of CD4-T-cells. The genes of important co-stimulatory molecules such as CD28 and CD40LG, important in T- and B-cell activation and proliferation, did not significantly change or experienced downregulation throughout the study. The absence of upregulation of several co-stimulatory molecules might be one possible explanation for the low activation and proliferation of CD4-T-cells during A. phagocytophilum infection, indicating a suboptimal CD4-T-cell response. The upregulation of T-BET, EOMES and IFN-γ on days 8–14 post inoculation, indicates a favoured CD4 Th1- and CD8-response. The dynamics and interaction between CD4+CD25+ and co-stimulatory molecules such as CD28, CD80, CD40 and CD40LG during infection with A. phagocytophilum in sheep needs further investigation in the future.

Strains of Anaplasma phagocytophilum from horses in Ohio are related to isolates from humans in the northeastern USA.

The tick-borne obligatory intracellular bacterium Anaplasma phagocytophilum infects granulocytes of humans as well as domesticated and wild animals, causing a febrile disease collectively called granulocytic anaplasmosis. The host species specificity and zoonotic potential of A. phagocytophilum strains remain unclear. In the USA, human granulocytic anaplasmosis (HGA) is primarily reported in Wisconsin and Minnesota, the northeastern coastal states, and northern California. In horses, A. phagocytophilum causes equine granulocytic anaplasmosis (EGA). Although EGA has been reported in the same US regions as HGA, horses with a definitive diagnosis of EGA have not been reported in Ohio, where the incidence of HGA is low. In this study, samples from archived blood smears or whole blood from three horses from three different counties in Ohio that presented in 2018, 2020, and 2021 with clinical signs and laboratory findings consistent with EGA were evaluated by PCR and sequencing. Sequences of ankA (encoding ankyrin A) of the three A. phagocytophilum strains were identical and related to strains isolated from humans in the northeastern USA yet distinct from those in California or Europe based on phylogenetic analysis. Each Ohio strain contains unique A. phagocytophilum p44 genes encoding the immunodominant major outer membrane proteins, underscoring the antigenic diversity of A. phagocytophilum strains that cause EGA in Ohio. From 2018 to 2023, 14 additional cases of EGA were identified by serodiagnosis in Ohio and nearby locations in Pennsylvania, pointing to a heretofore unrecognized prevalence of EGA and the risk of transmission of anaplasmosis to humans and animals in this region.IMPORTANCEThe tick-borne obligatory intracellular bacterium Anaplasma phagocytophilum infects humans as well as domesticated and wild animals, causing a febrile disease collectively called granulocytic anaplasmosis. The epidemiology and the host species specificity and zoonotic potential of A. phagocytophilum strains remain unclear. In this study, ankA (encoding ankyrin A) and p44 gene sequences of A. phagocytophilum were determined in clinical specimens from horses in Ohio and compared with those found in A. phagocytophilum strains from various hosts and geographic regions. With increasing numbers of seropositive horses, the study points out the unrecognized prevalence and uncharacterized strains of A. phagocytophilum infection in horses and the importance of A. phagocytophilum molecular testing for the prevention of equine and human granulocytic anaplasmosis.

Granulocytic anaplasmosis in cats from central Europe and molecular characterization of feline Anaplasma phagocytophilum strains by ankA gene, groEL gene and multilocus sequence typing

BackgroundAnaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It is transmitted by ticks of the Ixodes ricinus complex and causes febrile illness called granulocytic anaplasmosis primarily in humans, horses, dogs, sheep, cattle and goats. In comparison, clinically apparent disease has been described rarely in cats especially compared to dogs and horses. It is currently unknown whether cats are less susceptible to A. phagocytophilum or whether granulocytic anaplasmosis might be underdiagnosed in cats.MethodsTo address this question, we examined clinical signs and laboratory findings in seven A. phagocytophilum infected cats from Germany and Switzerland. We then genetically characterized feline A. phagocytophilum strains and compared them to those from other hosts showing clinically apparent disease. For this purpose, ankA-based, groEL-based and multilocus sequence typing (MLST) were applied. Furthermore, the concordance between these typing methods was assessed.ResultsFever, lethargy and anorexia were the most common clinical signs in cats suffering from granulocytic anaplasmosis. The most frequent laboratory finding was thrombocytopenia. All three typing methods consistently indicated that the A. phagocytophilum strains found infecting cats are the same as those that cause disease in humans, dogs and horses. In general, the three typing methods applied exhibited high concordance.ConclusionsThe genetic characterization of the feline A. phagocytophilum strains indicates that strain divergence is not the explanation for the fact that granulocytic anaplasmosis is much less frequently diagnosed in cats than in dogs and horses. Otherwise, it may be possible that cats are less susceptible to the same strains than dogs and horse are. However, due to the unspecific clinical signs, it should be considered that granulocytic anaplasmosis may be under-diagnosed in cats.Graphical

Anaplasma phagocytophilum in European bison (Bison bonasus) and their ticks from Lithuania and Poland

The increasing population of European bison (Bison bonasus) can contribute to the prevalence of zoonotic pathogens. The aim of the present study was to assess the presence of A. phagocytophilum infection in European bison tissues as well as ticks removed from European bison in Lithuania and Poland. A further objective of this work was to compare the detected A. phagocytophilum strains. A total of 85 tissue samples (spleen) of European bison and 560 ticks belonging to two species, Ixodes ricinus (n = 408) and Dermacentor reticulatus (n = 152) were tested. DNA of A. phagocytophilum was detected based on RT-PCR in 40% of the European bison samples, 8.8% of the I. ricinus and 5.9% of the D. reticulatus ticks. Analysis of the obtained partial 16S rRNA gene sequences of A. phagocytophilum revealed the presence of three variants with two polymorphic sites. Furthermore, phylogenetic analysis with partial msp4 gene sequences grouped A. phagocytophilum variants into three clusters. This study revealed that the groEL gene sequences of A. phagocytophilum from European bison and their ticks grouped into ecotype I and only one sequence from Lithuanian European bison belonged to ecotype II. The results of the present study indicated that European bison may play a role as a natural reservoir of A. phagocytophilum.

Wild deer (Pudupuda) from Chile harbor a novel ecotype of Anaplasmaphagocytophilum

BackgroundDeer species play an important role in the enzootic cycles of several Anaplasma species. While in the Northern Hemisphere ticks of genus Ixodes are well recognized vectors of these intracellular bacteria, less is known regarding the biological cycles of Anaplasma spp. in South America.MethodsUsing PCR protocols and Sanger sequencing, we assessed the presence of Anaplasma spp. in blood and ticks collected on a native deer species (Pudupuda) from southern Chile.ResultsBased on phylogenetic analyses of the 16S rRNA, gltA and groEL genes and calculation of average sequence divergence for groEL, our results bring to light a novel genovariant of Anaplasmaphagocytophilum (named strain “Patagonia”). The strain represents a novel ecotype within the A. phagocytophilum species complex and was detected in both P.puda and their ticks. Using a larger matrix, denser taxon sampling and outgroup, our maximum-likelihood- and Bayesian-inferred phylogenies for groEL provide an accurate picture of the topology of A. phagocytophilum ecotypes and their evolutionary relationships.ConclusionsThis is the first report of an ecotype of A. phagocytophilum in South America. Our results provide novel insight into the genetic diversity and ecology of this complex of bacterial lineages. Further studies should elucidate the enzootic cycle of A. phagocytophilum strain “Patagonia” and assess its pathogenic potential for pudues, domestic animals and humans in the region.Graphical

Molecular Survey of Anaplasma phagocytophilum and Related Strains in Sheep and Goats from Sivas; with a High Prevalence of Anaplasma phagocytophilum-like 1

This study aimed to investigate Anaplasma phagocytophilum and related strains (A. phagocytophilum-like 1 and like 2) in sheep and goats for the first time in Sivas province with molecular techniques. The study material was composed of 247 animal (159 sheep and 88 goats) blood samples from four districts of Sivas province (Sivas City Center, Kangal, Koyulhisar, and Yıldızeli). A. phagocytophilum and related strains were screened with polymerase chain reaction (PCR), PCR-RFLP, and DNA sequence analysis. A. phagocytophilum related strains were found in 44.93% (111/247) of the small ruminants using PCR. The infection rate was 45.91% (73/159) in sheep and 43.18% (38/88) in goats. In this study, 110 samples were positive for only A. phagocytophilum-like 1, while A. phagocytophilum-like 1 and like 2 were mix-infection in one sample. A. phagocytophilum was not detected in sheep or goats. Two randomly selected PCR products were sequenced in both directions, and the consensus sequences were deposited on the GenBank under accession numbers: ON598644 and ON598645. Nucleotide similarity of 99.34-100% was determined between A. phagocytophilum-like 1 isolates obtained in this study and those of A. phagocytophilum-like 1 isolates present in the GenBank database. This study provides the first molecular data on A. phagocytophilum-like 1 and like 2 in Sivas province. Considering the high positive rate of the A. phagocytophilum-like 1 in sheep and goats, there is a paucity of data on clinical symptoms and vector species of the pathogen. Therefore, further studies are needed to investigate the vector tick species and clinical symptoms of the pathogen in the host.

Comparative Whole Genome Analysis of an Anaplasma phagocytophilum Strain Isolated from Norwegian Sheep.

Anaplasma phagocytophilum is a Gram-negative obligate intracellular tick-borne alphaproteobacteria (family Anaplasmatacea, order Rickettsiales) with a worldwide distribution. In Norway, tick borne fever (TBF), caused by A. phagocytophilum, presents a major challenge in sheep farming. Despite the abundance of its tick vector, Ixodes ricinus, and A. phagocytophilum infections in wild and domestic animals, reports of infections in humans are low compared with cases in the U.S. Although A. phagocytophilum is genetically diverse and complex infections (co-infection and superinfection) in ruminants and other animals are common, the underlying genetic basis of intra-species interactions and host-specificity remains unexplored. Here, we performed whole genome comparative analysis of a newly cultured Norwegian A. phagocytophilum isolate from sheep (ApSheep_NorV1) with 27 other A. phagocytophilum genome sequences derived from human and animal infections worldwide. Although the compared strains are syntenic, there is remarkable genetic diversity between different genomic loci including the pfam01617 superfamily that encodes the major, neutralization-sensitive, surface antigen Msp2/p44. Blast comparisons between the msp2/p44 pseudogene repertoires from all the strains showed high divergence between U. S. and European strains and even between two Norwegian strains. Based on these comparisons, we concluded that in ruminants, complex infections can be attributed to infection with strains that differ in their msp2/p44 repertoires, which has important implications for pathogen evolution and vaccine development. We also present evidence for integration of rickettsial DNA into the genome of ISE6 tick cells.

Prevalence and Genotyping of Anaplasma phagocytophilum Strains from Wild Animals, European Bison (Bison bonasus) and Eurasian Moose (Alces alces) in Poland.

Simple SummaryThe populations of bison and moose, the largest wild ruminants in Poland, are growing every year. These animals return to Polish forests after many years of risk of extinction or, as in the case of European bison, are reintroduced step by step. Unfortunately, they are still rare and require close health surveillance and monitoring. One serious threat to their health and life is the bacterial parasite Anaplasma phagocytophilum. Moose and bison can also be sources of pathogenic bacteria for humans, which can be transferred through tick bites. In line with the World Health Organization (WHO) “OneHealth” program, indicating that animal health affects human health and vice versa, the following study examines the occurrence of Anaplasma phagocytophilum bacteria in European bison and Eurasian moose populations using molecular biology tools. Our results provide useful data regarding Anaplasma phagocytophilum that could be used in future strategies for the diagnosis and treatment of people and animals.Wild large ungulates, like European bison (Bison bonasus) and Eurasian moose (Alces alces), form an important part of the circulation of Anaplasma phagocytophilum, a Gram-negative, intracellular, tick-transmitted bacterium, in the natural environment. Bison and moose tissue samples were subjected to 16S rDNA, groEL and ankA partial gene marker amplification with specific primers using various variants of PCR. Out of 42 examined individuals, Anaplasma sp. were detected in 4/13 Eurasian moose (31%) and 7/29 European bison (24%). In addition, 12 groEL and 5 ankA partial gene positive samples were obtained from the examined animals. The phylogenetic analysis of the groEL partial gene classified samples from European bison to ecotype I, and samples from Eurasian moose to ecotype I and II; the analysis of the ankA partial gene assigned the samples to clusters I and IV. This study extends knowledge about A. phagocytophilum in wild large ungulates in Poland. This is the first report about the occurrence of Anaplasma sp. in one of the largest populations of free living European bison in the world. Our findings confirm that strains of A. phagocytophilum from Bison bonasus and Alces alces may constitute a natural reservoir of pathogenic HGA Anaplasma strains.

Anaplasma phagocytophilum in Marmota himalayana

BackgroundHuman granulocytic anaplasmosis is a tick-borne zoonotic disease caused by Anaplasma phagocytophilum. Coinfections with A. phagocytophilum and other tick-borne pathogens are reported frequently, whereas the relationship between A. phagocytophilum and flea-borne Yersnia pestis is rarely concerned.ResultsA. phagocytophilum and Yersnia pestis were discovered within a Marmota himalayana found dead in the environment, as determined by 16S ribosomal rRNA sequencing. Comparative genomic analyses of marmot-derived A. phagocytophilum isolate demonstrated its similarities and a geographic isolation from other global strains. The 16S rRNA gene and GroEL amino acid sequence identity rates between marmot-derived A. phagocytophilum (JAHLEX000000000) and reference strain HZ (CP000235.1) are 99.73% (1490/1494) and 99.82% (549/550), respectively. 16S rRNA and groESL gene screenings show that A. phagocytophilum is widely distributed in marmots; the bacterium was more common in marmots found dead (24.59%, 15/61) than in captured marmots (19.21%, 29/151). We found a higher Y. pestis isolation rate in dead marmots harboring A. phagocytophilum than in those without it (2 = 4.047, p < 0.05). Marmot-derived A. phagocytophilum was able to live in L929 cells and BALB/c mice but did not propagate well.ConclusionsIn this study, A. phagocytophilum was identified for the first time in Marmota himalayana, a predominant Yersinia pestis host. Our results provide initial evidence for M. himalayana being a reservoir for A. phagocytophilum; moreover, we found with the presence of A. phagocytophilum, marmots may be more vulnerable to plague. Humans are at risk for co-infection with both pathogens by exposure to such marmots.

Molecular Survey of Tick-Borne Pathogens Reveals Diversity and Novel Organisms With Veterinary and Public Health Significance in Wildlife From a National Nature Reserve of China.

Wildlife is involved in the maintenance and transmission of various tick-borne pathogens. The objective of the present study was to determine the occurrence and diversity of tick-borne pathogens in free-ranging wild animals collected from Tangjiahe National Nature Reserve of China. Blood or liver samples from 13 wild animals (5 takin, 3 Himalayan goral, 3 Reeves' muntjac, 1 forest musk deer, and 1 wild boar) were collected and screened for piroplasm, Anaplasma spp., Ehrlichia spp., and spotted fever group (SFG) rickettsiae by PCR-based on different gene loci. Three Theileria species, a potential novel Theileria parasite (Theileria sp. T4) and two Anaplasma species were identified in those wildlife. Theileria capreoli was found in Himalayan goral, Reeves' muntjac, and forest musk deer; Theileria luwenshuni, Theileria uilenbergi, and a potential novel, Theileria parasite (Theileria sp. T4), were identified in takin. Meanwhile, Anaplasma bovis was identified in Himalayan goral, takin, Reeves' muntjac, forest musk deer, and wild boar; Anaplasma phagocytophilum and related strains was found in takin, Reeves' muntjac, and forest musk deer. All wildlife included in this study was negative for Babesia, Anaplasma ovis, Anaplasma marginale, Ehrlichia, and SFG rickettsiae. Moreover, coinfection involving Theileria spp. and Anaplasma spp. was observed in eight wild animals. This study provided the first evidence of tick-borne pathogens in free-ranging wild animals from the nature reserve, where contact between domestic and wild animals rarely occurs.

Granulocytic anaplasmosis in captive ring-tailed lemur (Lemur catta) in Poland

BackgroundAnaplasma are obligate intracellular bacteria and aetiological agents of tick-borne diseases of both veterinary and medical interest. The genus Anaplasma comprises six species: Anaplasma marginale, Anaplasma centrale, Anaplasma ovis, Anaplasma phagocytophilum, Anaplasma bovis and Anaplasma platys. They can infect humans, carnivores, ruminants, rodents, insectivores, birds and reptiles. The aim of this study was to present the first clinical case of granulocytic anaplasmosis in a captive ring-tailed lemur in Poland.Case presentationA 4-year-old female lemur presented anorexia, epistaxis and tick infestation. The microscopic examination of a blood smear revealed morulae in neutrophils. Polymerase chain reaction test and sequencing of obtained PCR product confirmed infection by the GU183908 Anaplasma phagocytophilum strain. Therapeutic protocol included doxycycline (2.5 mg/kg p.o., b.i.d.) for 3 weeks and the lemur recovered within 24 h. ConclusionsThis is the first report on granulocytic anaplasmosis in a ring-tailed lemur in Europe, indicating that A. phagocytophilum infection must also be considered in differential diagnosis in this animal species, especially in individuals with thrombocytopenia associated with Ixodes ricinus parasitism.

A new genetic approach to distinguish strains of Anaplasma phagocytophilum that appear not to cause human disease

Genetic diversity of Anaplasma phagocytophilum was assessed in specimens from 16 infected patients and 16 infected Ixodes scapularis ticks. A region immediately downstream of the 16S rRNA gene, which included the gene encoding SdhC, was sequenced. For the A. phagocytophilum strains from patients no sequence differences were detected in this region. In contrast, significantly fewer ticks had a sequence encoding SdhC that was identical to that of the human strains (11/16 vs. 16/16, p = 0.04). This variation is consistent with the premise that not all A. phagocytophilum strains present in nature are able to cause clinical illness in humans. A strain referred to as A. phagocytophilumVariant-1 that is regarded as non-pathogenic for humans was previously described using a different typing method. Data from the current study suggest that both typing methods are identifying the same non-pathogenic strains.

Disruption of VirB6 Paralogs in Anaplasma phagocytophilum Attenuates Its Growth

Many pathogenic bacteria translocate virulence factors into their eukaryotic hosts by means of type IV secretion systems (T4SS) spanning the inner and outer membranes. Genes encoding components of these systems have been identified within the order Rickettsiales based upon their sequence similarities to other prototypical systems. Anaplasma phagocytophilum strains are obligate intracellular, tick-borne bacteria that are members of this order. The organization of these components at the genomic level was determined in several Anaplasma phagocytophilum strains, showing overall conservation, with the exceptions of the virB2 and virB6 genes. The virB6 loci are characterized by the presence of four virB6 copies (virB6-1 through virB6-4) arranged in tandem within a gene cluster known as the sodB-virB operon. Interestingly, the virB6-4 gene varies significantly in length among different strains due to extensive tandem repeats at the 3' end. To gain an understanding of how these enigmatic virB6 genes function in A. phagocytophilum, we investigated their expression in infected human and tick cells. Our results show that these genes are expressed by A. phagocytophilum replicating in both cell types and that VirB6-3 and VirB6-4 proteins are surface exposed. Analysis of an A. phagocytophilum mutant carrying the Himar1 transposon within the virB6-4 gene demonstrated that the insertion not only disrupted its expression but also exerted a polar effect on the sodB-virB operon. Moreover, the altered expression of genes within this operon was associated with the attenuated in vitro growth of A. phagocytophilum in human and tick cells, indicating the importance of these genes in the physiology of this obligate intracellular bacterium in such different environments.IMPORTANCE Knowledge of the T4SS is derived from model systems, such as Agrobacterium tumefaciens The structure of the T4SS in Rickettsiales differs from the classical arrangement. These differences include missing and duplicated components with structural alterations. Particularly, two sequenced virB6-4 genes encode unusual C-terminal structural extensions resulting in proteins of 4,322 (GenBank accession number AGR79286.1) and 9,935 (GenBank accession number ANC34101.1) amino acids. To understand how the T4SS is used in A. phagocytophilum, we describe the expression of the virB6 paralogs and explore their role as the bacteria replicate within its host cell. Conclusions about the importance of these paralogs for colonization of human and tick cells are supported by the deficient phenotype of an A. phagocytophilum mutant isolated from a sequence-defined transposon insertion library.

Molecular characterization of Anaplasma phagocytophilum infection in the cervids and feeding ticks from Lithuania

Anaplasma phagocytophilum is a bacterial pathogen, which is a major cause of zoonotic disease, anaplasmosis. The main vectors of A. phagocytophilum are ticks of the Ixodes ricinus complex. A. phagocytophilum has a broad geographic distribution and a high degree of biological and clinical diversity. Epidemiological studies in multiple countries have shown that the prevalence of A. phagocytophilum highly depends on the density of ticks and their potential hosts such as the cervids, which are one of the main sources of nutrition for Ixodes ticks. In Lithuania, the cervids are important game animals but their contribution as reservoirs for A. phagocytophilum remains unknown. The objectives of the study were to investigate the prevalence of A. phagocytophilum infections in the cervids and feeding ticks and to characterize the A. phagocytophilum strains obtained from the cervids and ticks based on sequence analysis of msp4 gene. A total of 187 ticks were collected from 44 cervids (roe deer, red deer, and moose) harvested by professional hunters during the hunting seasons of 2010–2013 and 2016–2017 in Lithuania. Blood and spleen samples were collected from 29 animals (27 roe deer and two red deer). A. phagocytophilum DNA was identified in ten (37.04%) of the 27 roe deer. The overall prevalence of A. phagocytophilum in I. ricinus and D. reticulatus ticks was 39.3% (70/178) and 22.2% (2/9) respectively. The sequence analysis of the msp4 gene of A. phagocytophilum revealed nine different sequence types: five msp4 sequence types were detected in ticks and seven in roe deer.

The role of different species of wild ungulates and Ixodes ricinus ticks in the circulation of genetic variants of Anaplasma phagocytophilum in a forest biotope in north-western Poland

The aim of this study was to reveal genetic variants of Anaplasma phagocytophilum strains occurring in different species of wild ungulates and in Ixodes ricinus ticks to check the role of the examined species in the circulation of the revealed variants in nature. The aim was also to determine if the detected variants of A. phagocytophilum are specific for particular game species as well as to examine their identity with other strains, including pathogenic ones. Sequences of the amplified groEL heat shock operon and msp2 gene fragments of A. phagocytophilum were obtained from samples collected between 2005 and 2007 from 14 roe deer (Capreolus capreolus), 13 red deer (Cervus elaphus), 1 fallow deer (Dama dama) and 4 wild boar (Sus scrofa) as well as 13 engorged and 11 questing I. ricinus ticks occurring in the area of Puszcza Wkrzańska Forest in north-western Poland. Analysis of the sequences showed the presence of five and four gene variants of groEL and msp2, respectively. The variants showed high identity with sequences derived from strains pathogenic to humans and/or domestic and companion animals. Cervids seem to play a more important role in the circulation of the detected variants in nature than wild boar. Some of the detected variants are not shared by roe and red deer. The results obtained on the basis of groEL and msp2 sequences are discrepant. Analysis of the groEL operon sequence provides more information on A. phagocytophilum strains than the msp2 gene sequence.

The absence of the drhm gene is not a marker for human-pathogenicity in European Anaplasma phagocytophilum strains

BackgroundAnaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It is transmitted by ticks of the Ixodes ricinus complex and causes febrile illness in humans and animals. The geographical distribution of A. phagocytophilum spans the Americas, Europe, Africa and Asia. However, human disease predominantly occurs in North America but is infrequently reported from Europe and Asia. In North American strains, the absence of the drhm gene has been proposed as marker for pathogenicity in humans whereas no information on the presence or absence of the drhm gene was available for A. phagocytophilum strains circulating in Europe. Therefore, we tested 511 European and 21 North American strains for the presence of drhm and compared the results to two other typing methods: multilocus sequence typing (MLST) and ankA-based typing.ResultsAltogether, 99% (478/484) of the analyzable European and 19% (4/21) of the North American samples from different hosts were drhm-positive. Regarding the strains from human granulocytic anaplasmosis cases, 100% (35/35) of European origin were drhm-positive and 100% (14/14) of North American origin were drhm-negative. Human strains from North America and Europe were both part of MLST cluster 1. North American strains from humans belonged to ankA gene clusters 11 and 12 whereas European strains from humans were found in ankA gene cluster 1. However, the North American ankA gene clusters 11 and 12 were highly identical at the nucleotide level to the European cluster 1 with 97.4% and 95.2% of identity, respectively.ConclusionsThe absence of the drhm gene in A. phagocytophilum does not seem to be associated with pathogenicity for humans per se, because all 35 European strains of human origin were drhm-positive. The epidemiological differences between North America and Europe concerning the incidence of human A. phagocytophilum infection are not explained by strain divergence based on MLST and ankA gene-based typing.