Anaplasma Phagocytophilum Strains Research Articles

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.

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

Host–pathogen associations revealed by genotyping of European strains of Anaplasma phagocytophilum to describe natural endemic cycles

BackgroundThe zoonotic intracellular alpha-proteobacterium Anaplasma phagocytophilum is a tick-transmitted pathogen. The associations between vertebrate reservoirs and vectors are described as wide-ranging, and it was previously shown that the pathogenicity of A. phagocytophilum differs depending on the combination of pathogen variant and infected host species. This leads to the question of whether there are variations in particular gene loci associated with different virulence. Therefore, this study aims at clarifying existing host-variant combinations and detecting possible reservoir hosts. To understand these interactions, a complex toolset for molecular epidemiology, phylogeny and network theory was applied.MethodsSequences of up to four gene loci (msp4, msp2, groEL and 16S rRNA) were evaluated for different isolates from variable host species, including, for example, dogs, cattle and deer. Variant typing was conducted for each gene locus individually, and combinations of different gene loci were analysed to gain more detailed information about the genetic plasticity of A. phagocytophilum. Results were displayed as minimum spanning nets and correlation nets.ResultsThe highest diversity of variants for all gene loci was observed in roe deer. In cattle, a reduced number of variants for 16S rRNA [only 16S-20(W) and 16S-22(Y)] but multiple variants of msp4 and groEL were found. For dogs, two msp4 variants [m4-20 and m4-2(B/C)] were found to be linked to different variants of the other three gene loci, creating two main combinations of gene loci variants. Cattle are placed centrally in the minimum spanning net analyses, indicating a crucial role in the transmission cycles by possibly bridging the vector-wildlife cycle to infections of humans and domestic animals. The minimum spanning nets confirmed previously described epidemiological cycles of the bacterium in Europe, showing separation of variants originating from wildlife animals only and a set of variants shared by wild and domestic animals.ConclusionsIn this comprehensive study of 1280 sequences, we found a high number of gene variants only occurring in specific hosts. Additionally, different hosts show unique but also shared variant combinations. The use of our four gene loci expand the knowledge of host–pathogen interactions and may be a starting point to predict future spread and infection risks of A. phagocytophilum in Europe.Graphical abstract

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.

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.

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.

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.

Severe Anaplasma phagocytophilum and Babesia divergens Concomitant Infection in Imported Captive Reindeer (Rangifer tarandus)

Abstract Tick-borne diseases are highly prevalent in domestic and wild ruminants and they may be distributed in wide geographical ranges by animal transportation. The aim of the current study was to investigate the presence of European strains of Babesia spp. and/or Anaplasma spp. in oversea imported reindeer specimens. Imported specimens (n=7) were hospitalized with visible tick infestation (Ixodes ricinus) and signs of cachexia, anemia, and hemoglobinuria. Using blood smears, PCR, and BLAST comparisons, it was confirmed that the animals were infected with a French strain of Anaplasma phagocytophilum and Babesia divergens which is considered to be absent in the USA. We conclude that oversea importation of reindeers must be followed with a routine check for geographically-specific strains of pathogens from the place of origin. This monitoring process must be dynamic and according to recent reports of tick-borne pathogens.

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.

Co-infection, reinfection and superinfection with Anaplasma phagocytophilum strains in a cattle herd based on ankA 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 in humans and animals. We used multilocus sequence typing (MLST) and ankA gene-based typing to study the molecular epidemiology of the A. phagocytophilum strains circulating in a German cattle herd over one pasture season. The aim was to investigate whether co-infection with two distinct variants, reinfection with the same and/or superinfection by a different strain occurred during one pasture season. Eight genetic loci were sequenced in 47 PCR-positive samples from 15 animals.ResultsFive different sequence types (ST) and four ankA alleles were detected in the cattle herd. Three different ST caused clinically overt tick-borne fever in primary infected animals. The concordance between ST and ankA allele was 100%. Therefore, the housekeeping genes used for MLST and the highly variable ankA gene were concatenated to increase resolution. Co-infection could be proven because samples of chronologically close collection dates were included. Co-infecting A. phagocytophilum strains differed by 14 to 18 single nucleotide polymorphisms (SNPs). Most superinfecting variants varied by 14 SNPs from the previous strain and appeared in median after a free interval of 31 days. Thus, it is unlikely that superinfecting strains arose by in-animal evolution. Immunity against re- or superinfection was assumed because the cattle developed clinical signs only during primary infection.ConclusionsThe tick-pathogen-vertebrate host interaction is probably much more complex than previously thought taking into account the frequently occurring events of co-infection, reinfection and superinfection. This complex situation could not be easily simulated in an experimental infection and underlines the value of field studies.

Genetic characterization of Anaplasma phagocytophilum strains from goats (Capra aegagrus hircus) and water buffalo (Bubalus bubalis) by 16S rRNA gene, ankA gene and multilocus sequence typing

Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It is transmitted by ticks and causes tick-borne fever in domestic ruminants such as sheep, cattle and goats. However, in contrast to sheep and cattle little is known about the clinical course of infection in goats. We report here on three cases of symptomatic infection with A. phagocytophilum in two goats (Capra aegagrus hircus) and one water buffalo (Bubalus bubalis). The animals showed symptoms and laboratory findings similar to sheep and cattle. To our knowledge, this is the first report on the symptomatic infection of water buffalos with A. phagocytophilum. The infecting strains were genetically characterized by 16S rRNA gene, ankA gene and multilocus sequence typing (MLST). Four other strains from asymptomatically infected goats were also included. The ankA sequences from five goats were part of the formerly described ankA gene clusters I and IV that are known to contain A. phagocytophilum strains from sheep and cattle. However, the sequences from one goat and from the water buffalo belonged to ankA gene cluster II that was formerly described to be restricted to roe deer. A similar observation was made for MLST as three goats clustered with sequences from sheep and cattle, whereas three other goats and the water buffalo were found to be part of the roe deer cluster. However, since most of the strains from sheep and cattle were distinct from the roe deer strains, roe deer might not represent major reservoir hosts for tick-borne fever in domestic ruminants. When differing parts of the 16S rRNA gene were used for typing the results were conflicting. This shows that the use of a standardized typing method such as MLST is highly desirable to generate easily comparable results.

Prevalence and genetic variability of Anaplasma phagocytophilum in wild rodents from the Italian alps

BackgroundHuman granulocytic anaplasmosis is a zoonotic bacterial disease with increasing relevance for public health in Europe. The understanding of its sylvatic cycle and identification of competent reservoir hosts are essential for improving disease risk models and planning preventative measures.ResultsIn 2012 we collected single ear biopsy punches from 964 live-trapped rodents in the Province of Trento, Italy. Genetic screening for Anaplasma phagocytophilum (AP) was carried out by PCR amplification of a fragment of the 16S rRNA gene. Fifty-two (5.4%) samples tested positive: 49/245 (20%) from the bank vole (Myodes glareolus) and 3/685 (0.4%) samples collected from the yellow-necked mouse (Apodemus flavicollis). From these 52 positive samples, we generated 38 groEL and 39 msp4 sequences. Phylogenetic analysis confirmed the existence of a distinct rodent strain of AP.ConclusionsOur results confirm the circulation of a specific strain of AP in rodents in our study area; moreover, they provide further evidence of the marginal role of A. flavicollis compared to M. glareolus as a reservoir host for this pathogen.

Molecular detection of Anaplasma spp. in pangolins (Manis javanica) and wild boars (Sus scrofa) in Peninsular Malaysia

Anaplasma spp. infects a wide variety of wildlife and domestic animals. This study describes the identification of a novel species of Anaplasma (Candidatus Anaplasma pangolinii) from pangolins (Manis javanica) and Anaplasma bovis from wild boars (Sus scrofa) in Malaysia. Based on 16S rRNA gene sequences, Candidatus Anaplasma pangolinii is identified in a distinct branch within the family Anaplasmataceae, exhibiting the closest sequence similarity with the type strains of Anaplasma bovis (97.7%) and Anaplasma phagocytophilum (97.6%). The sequence also aligned closely (99.9%) with that of an Anaplasma spp. (strain AnAj360) detected from Amblyomma javanense ticks. The nearly full length sequence of the 16S rRNA gene derived from two wild boars in this study demonstrated the highest sequence similarity (99.7%) to the A. bovis type strain. Partial 16S rRNA gene fragments of A. bovis were also detected from a small population of Haemaphysalis bispinosa cattle ticks in this study. Our finding suggests a possible spread of two Anaplasma species in the Malaysian wildlife and ticks. The zoonotic potential of the Anaplasma species identified in this study is yet to be determined.

A putative marker for human pathogenic strains of Anaplasma phagocytophilum correlates with geography and host, but not human tropism

Anaplasma phagocytophilum is an Ixodes species tick-transmitted bacterium that is capable of infecting a variety of host species, although there is a diversity of bacterial strains with differing host tropism. Recent analysis of A. phagocytophilum strains suggested that “drhm”, a gene locus designated “distantly related to human marker” (drhm), which was predicted to be an integral membrane protein with possible transporter functions was not present in available canine and human isolates. By assessing 117 strains from 14 host species from across the US, we extended this analysis. Phylogenetic clades were associated with geography, but not host species. Additionally, a virulent clade that lacks drhm and infects dogs, horses, and humans in northeastern US was identified.

Wildlife reservoirs for vector-borne canine, feline and zoonotic infections in Austria.

Austria's mammalian wildlife comprises a large variety of species, acting and interacting in different ways as reservoir and intermediate and definitive hosts for different pathogens that can be transmitted to pets and/or humans. Foxes and other wild canids are responsible for maintaining zoonotic agents, e.g. Echinococcus multilocularis, as well as pet-relevant pathogens, e.g. Hepatozoon canis. Together with the canids, and less commonly felids, rodents play a major role as intermediate and paratenic hosts. They carry viruses such as tick-borne encephalitis virus (TBEV), bacteria including Borrelia spp., protozoa such as Toxoplasma gondii, and helminths such as Toxocara canis. The role of wild ungulates, especially ruminants, as reservoirs for zoonotic disease on the other hand seems to be negligible, although the deer filaroid Onchocerca jakutensis has been described to infect humans. Deer may also harbour certain Anaplasma phagocytophilum strains with so far unclear potential to infect humans. The major role of deer as reservoirs is for ticks, mainly adults, thus maintaining the life cycle of these vectors and their distribution. Wild boar seem to be an exception among the ungulates as, in their interaction with the fox, they can introduce food-borne zoonotic agents such as Trichinella britovi and Alaria alata into the human food chain.