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Background: Equine granulocytic anaplasmosis (EGA) is a tick-borne disease caused by Anaplasma (A.) phagocytophilum. In Germany, this pathogen is transmitted primarily by Ixodes ricinus. There is limited knowledge about its prevalence in horses in Germany. The aim of this retrospective study was to analyze the results of serological and molecular testing for A. phagocytophilum in horses which were done in a commercial laboratory in Germany over fourteen years. Additionally, risk factors were evaluated, and hematological abnormalities were addressed in horses with positive PCR results., Methods: This retrospective study examined results of direct (Polymerase chain reaction [PCR]) and indirect (immunofluorescence antibody test [IFAT]) detection methods for A. phagocytophilum in horses on samples provided by German veterinarians and processed by the commercial laboratory LABOKLIN from 2008 to 2021. In horses with positive test results, a Complete Blood Count (CBC) and Serum Amyloid A (SAA) were also analyzed where possible., Results: In total, 1217/4834 horses tested positive (PCR: 190/1246 horses, 15.2%; IFAT: 1036/3849 horses, 26.9%). Seasonality and location, as classified by federal state, had a statistically significant impact on PCR results (P < 0.001 for both). In horses with positive PCR results, hematological abnormalities were detected in 112/118 horses (95%), with thrombocytopenia (86%) and anemia (52%) representing the most common findings. The remaining 6/118 horses (5%) showed no hematological abnormalities on CBC. SAA was measured in 35 horses with positive PCR results, which exclusively showed marked elevation., Conclusions: The seasonality of A. phagocytophilum infections confirmed by PCR testing was consistent with known peaks in vector activity in Germany. The high rate of horses with positive PCR results when compared to dogs and cats may be due to a lack of ectoparasite prophylaxis. Infections with A. phagocytophilum should be considered as a differential diagnosis in horses with cytopenia on CBC and SAA elevation, especially in the summer and after any possible tick exposure., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ingo Schaefer reports a relationship with Laboklin Laboratory for Clinical Diagnostics GmbH & Co that includes: employment. Elisabeth Mueller reports a relationship with Laboklin Laboratory for Clinical Diagnostics GmbH & Co that includes: employment. Declaration of interest Elisabeth Müller is the CEO of the commercial laboratory LABOKLIN (Bad Kissingen, Germany) and Ingo Schäfer an employee of the laboratory. This has not influenced the results of our study in any way., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Anaplasma 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., Results: Five 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., Conclusions: The 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.

Background: Anaplasma phagocytophilum is a zoonotic tick-borne intracellular alpha-proteobacterium causing tick-borne fever, which leads to significant economic losses in domestic ruminants in Europe. Its epidemiological cycles are complex and reservoir host species of bovine strains have not yet been identified. Given that little genetic information is available on strains circulating within a defined bovine environment, our objective was to assess the genetic diversity of A. phagocytophilum obtained from the same farms over time., Methods: Blood samplings were performed several times in two European herds. In the French herd, 169 EDTA-blood samples were obtained from 115 cows (32 were sampled two to four times). In the German herd, 20 cows were sampled six times (120 EDTA-blood samples). The presence of A. phagocytophilum DNA was assessed using a qPCR targeting msp2. The positive DNA samples underwent MLST at nine genetic markers (typA, ctrA, msp4, pleD, recG, polA, groEL, gyrA, and ankA). For each locus, sequences were aligned with available bacterial sequences derived from cattle, horse, dog, and roe deer hosts, and concatenated neighbor joining trees were constructed using three to six loci., Results: Around 20% (57/289) of samples were positive. Forty positive samples from 23 French and six German cows (11 of them being positive at two time points) were sequenced. Six loci (typA, ctrA, msp4, pleD, recG, and polA) allowed to build concatenated phylogenetic trees, which led to two distinct groups of bovine variants in the French herd (hereafter called A and B), whereas only group A was detected in the German herd. In 42% of French samples, double chromatogram peaks were encountered in up to four loci. Eleven cows were found infected three weeks to 17 months after first sampling and harboured a new variant belonging to one or the other group., Conclusions: Our results demonstrate the occurrence of two major bovine strain groups and the simultaneous infection of single cows by more than one A. phagocytophilum strain. This challenges the role of cattle as reservoirs for A. phagocytophilum. This role may be facilitated via long-term bacterial persistence in individual cows and active circulation at the herd scale.

Objectives: To describe the clinical signs, laboratory results, therapy and course of disease in dogs with canine granulocytic anaplasmosis in which co-infections had been excluded., Methods: Medical records of dogs naturally infected with Anaplasma phagocytophilum were retrospectively evaluated with regard to clinical signs and laboratory abnormalities at the time of presentation, therapy and course of disease., Results: Nine hundred and seventy-four dogs with clinical signs suspicious for canine granulocytic anaplasmosis were tested for A. phagocytophilum DNA by modified real-time PCR; 72 dogs had a positive result. Nine of the positive dogs were excluded from further evaluation due to other diseases or lack of data. The most common clinical signs in the 63 A. phagocytophilum-positive dogs included in the study were lethargy and reduced activity (83%), fever (67%) and inappetence (63%). Thrombocytopenia was the most common laboratory abnormality (86%), followed by increased liver enzyme activities and hyperbilirubinaemia (77%), anaemia (70%), hypoalbuminaemia (62%) and leucocytosis (27%). Of 36 thrombocytopenic dogs tested for platelet-bound antibodies, 44% were positive. Of the 63 infected dogs, 59 (97%) recovered, two dogs died (epileptic seizures and immune-mediated haemolytic anaemia) and two were lost to follow-up., Clinical Significance: In areas where it is endemic, canine granulocytic anaplasmosis should be considered as a potential cause of acute nonspecific clinical signs or immune-mediated disease if tick exposure cannot be excluded., (© 2017 British Small Animal Veterinary Association.)

Background: Anaplasma phagocytophilum is an obligate intracellular, tick-transmitted bacterium that causes granulocytic anaplasmosis in humans and several mammalian species including domestic ruminants where it is called tick-borne fever (TBF). Different genetic variants exist but their impact with regard to putative differences in host associations and pathogenicity are not yet completely understood., Methods: Natural infections with A. phagocytophilum in a dairy cattle herd in Germany were investigated over one pasture season by using serology, haematology, blood chemistry and polymerase chain reaction (PCR). Sequence analysis of partial 16S rRNA, groEL, msp2 and msp4 genes of A. phagocytophilum was carried out in order to trace possible genetic variants and their relations between cattle, roe deer (Capreolus capreolus) and ticks (Ixodes ricinus) in this area., Results: In total 533 samples from 58 cattle, 310 ticks, three roe deer and one wild boar were examined. Our results show (i) typical clinical symptoms of TBF in first-time infected heifers, such as high fever, reduced milk yield, lower limb oedema and typical haematological and biochemical findings such as severe leukopenia, erythropenia, neutropenia, lymphocytopenia, monocytopenia, a significant increase in creatinine and bilirubin and a significant decrease in serum albumin, γ-GT, GLDH, magnesium and calcium; (ii) a high overall prevalence of A. phagocytophilum infections in this herd as 78.9% (15/19) of the naïve heifers were real-time PCR-positive and 75.9% (44/58) of the entire herd seroconverted; and (iii) a high level of sequence variation in the analysed genes with five variants of the 16S rRNA gene, two variants of the groEL gene, three variants of the msp2 gene and four variants in the msp4 gene with certain combinations of these variants., Conclusions: In cattle particular combinations of the genetic variants of A. phagocytophilum occurred, whereas three roe deer showed different variants altogether. This is indicative for a sympatric circulation of variants in this small geographical region (< 1 km 2 ). Both re- and superinfections with A. phagocytophilum were observed in five cattle showing that infection does not result in sterile immunity. For prevention of clinical cases we suggest pasturing of young, not pregnant heifers to reduce economical losses.

Wild boars (Sus scrofa) have been suggested to be involved in the enzootic cycle of the tick-borne pathogen Anaplasma phagocytophilum. This observation raises the question whether they serve as reservoir hosts for A. phagocytophilum and potentially for other tick-borne pathogens of public health relevance. The aim of this study was to investigate wild boars and their ticks from a forest site in southern Germany for the presence of A. phagocytophilum, Candidatus Neoehrlichia mikurensis, Rickettsia spp., Borrelia burgdorferi sensu lato (s.l.), Borrelia spp. of the relapsing fever group, and Babesia spp. Therefore, 24 wild boars collected from October, 2010, to February, 2013, were investigated by molecular methods. DNA of A. phagocytophilum was detected in three out of 24 (12.5%) wild boars and in four out of 16 (25%) ticks. DNA of none of the other pathogens was found in any wild boar, but Rickettsia spp., B. burgdorferi s.l., and Cand. N. mikurensis were found in one of the investigated ticks each. Sequences of the partial 16S rRNA gene of A. phagocytophilum from one spleen and two ticks showed 100% similarity to GenBank entries from human anaplasmosis cases (accession nos. U02521 and AY886761). The sequence from the third tick was 100% similar to sequences obtained from Ixodes ricinus and roe deer from the same study area previously. Detecting a potentially human pathogenic A. phagocytophilum variant in wild boar confirms previous findings and is of public health interest. To our knowledge, this is the first report of A. phagocytophilum in wild boars in Germany. Whether wild boars support the enzootic cycle of A. phagocytophilum variants involved in human disease requires further attention in future systematic studies.

Canine granulocytic anaplasmosis (CGA) is caused by the rickettsial microorganism Anaplasma phagocytophilum. CGA is typically characterized by fever, thrombocytopenia, lethargy, anorexia, arthropy, and other nonspecific clinical signs. Skin lesions have been described in naturally infected lambs and humans. The pathophysiology of CGA is not entirely clear, and the persistence of the organism after the resolution of clinical signs has been described. The aim of the study was to investigate if A. phagocytophilum can be detected in canine lesional skin biopsies from A. phagocytophilum-seropositive dogs with etiologically unclear skin lesions that improved after the treatment with doxycycline. Paraffin-embedded lesional skin biopsies were allocated into separate groups: biopsies from A. phagocytophilum-seropositive dogs responsive to treatment with doxycycline (n=12), biopsies from A. phagocytophilum-seronegative dogs (n=2), and biopsies in which skin lesions histopathologically resembled a tick bite (n=10). The serological status of the latter group was unknown. Histology of the seropositive and seronegative dog skin lesions did not indicate an etiology. DNA was extracted, and a conventional PCR for partial 16S rRNA gene was performed. Anaplasma phagocytophilum DNA was amplified from 4/12 seropositive dogs' skin biopsies. All sequences were 100% identical to the prototype A. phagocytophilum human strain (GenBank accession number U02521). Anaplasma phagocytophilum was not amplified from the 2 seronegative and 10 suspected tick bite dogs. Serum antibody titers of the PCR-positive dogs ranged from 1:200 to 1:2048. Histopathologically, a mild-to-moderate perivascular to interstitial dermatitis composed of a mixed cellular infiltrate and mild-to-moderate edema was seen in all seropositive dogs. In 8/12 seropositive dogs, vascular changes as vasculopathy, fibrinoid necrosis of the vessel walls, and leukocytoclastic changes were observed. In summary, our results support the hypothesis that the persistence of A. phagocytophilum in the skin may be causative for otherwise unexplained skin lesions in seropositive dogs., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Ehrlichiae are obligate intracytoplasmic Gram-negative, tick-borne bacteria belonging to the Anaplasmataceae family. Ehrlichioses are considered emerging diseases in both humans and animals. Several members of the genus Ehrlichia have been isolated and propagated in vitro. This study describes the continuous propagation of a Brazilian Ehrlichia sp. isolate in IDE8 tick cells, canine DH82 cells and bovine aorta cells. Initially, the organisms were isolated from the haemolymph of a Rhipicephalus (Boophilus) microplus tick into IDE8 cells. Infected IDE8 cells were brought from Brazil to Germany, where the organisms were continuously propagated in IDE8, DH82 and bovine aorta cells. Bovine aorta cells were infected and propagated for 3 months, corresponding to six subcultures, whereas the other two infected cell lines were kept for more than 1 year. During the cultivation period, 36 and 14 subcultures were carried out in IDE8 and DH82 cell cultures, respectively. Reinfection of IDE8 cells with organisms grown in DH82 cells was achieved. Sequence analysis made with a fragment of the 16S rRNA gene showed that this Ehrlicha sp. is closely related to Ehrlichia canis. However, the maximum likelihood phylogenetic tree shows that it falls in a separate phylogenetic clade from E. canis., (© 2013 Blackwell Verlag GmbH.)

Four cows from North-West Germany have been diagnosed with tick-borne fever (TBF) based on the demonstration of morulae in neutrophilic granulocytes in their blood smears, positive signals in real-time PCR specific for Anaplasma phagocytophilum using DNA extracted from their buffy coats, and demonstration of specific antibodies in their sera using a commercially available immunofluorescence assay. Clinical findings included high fever, decreased milk production, lower limb edema with stiff walking, eye and nasal discharge, and depression. These signs developed about a week after the animals had been brought to the pasture for the first time in their life. All cows recovered after 5-15 days, although DNA of A.phagocytophilum could be detected by real-time PCR up to 6 weeks after onset of the disease. Considering the known prevalences of A.phagocytophilum in ticks in Germany and its detection in dogs and horses, we think that underdiagnosing of TBE in cattle is highly likely. Therefore TBF should be taken into account as differential diagnosis in case of high fever and/or a sudden decrease in milk production in pastured animals.

Anaplasma (A.) phagocytophilum is a tick-transmitted obligate intracellular bacterium and has been identified in a wide range of mammalian species, causing febrile disease in some. Few reports show that it can also cause granulocytic anaplasmosis in cats. As data on the occurrence of A. phagocytophilum in cats from Germany is limited, a total of 326 serum and 306 EDTA-blood samples from cats from Germany were screened by direct (Giemsa-stained blood/buffy coat smears, real-time PCR) and indirect (IFAT) methods. Of 274 Giemsa-stained blood smears which could be evaluated none was positive for morulae, but one blood sample (< or =0.1%; 1/306) was positive for A. < or = phagocytophilum-DNA in PCR. Antibodies (cutoff > or = 1:64) were detected in 53 out of 326 samples (16.2%). Altogether, the results show a high seroprevalence rate of anti-A. phagocytophilum antibodies in cats in Germany while the low detection rate of this bacterial agent by direct methods is similar to those of other studies on A. phagocytophilum infections in cats.

The European hedgehog (Erinaceus europaeus) is a common insectivore in most parts of Europe and is frequently infested by the ticks Ixodes ricinus and I. hexagonus. I. ricinus ticks have been found infected with Anaplasma phagocytophilum, an obligate intracellular bacterium, but little is known about the potential of the hedgehog as a reservoir host. In this study, the infection with A. phagocytophilum and the genetic variants involved were investigated in a captive hedgehog population which was kept in a fenced, natural grass and bush garden habitat, and also in its ticks. Additionally hedgehogs from hedgehog caretaking stations were investigated. EDTA blood and ticks were collected from the captive hedgehog population once a month from March to October 2007 and in March and April 2008. All 3 developmental stages of I. ricinus and I. hexagonus occurred on the hedgehogs. After DNA extraction, the samples were screened for A. phagocytophilum with a real-time PCR, and selected samples were further investigated with a nested PCR targeting the partial 16S rRNA gene, followed by sequencing. One hundred thirty-six out of 220 hedgehog blood samples (61.8%) from altogether 48 individuals, 413 out of 563 I. ricinus samples and 90 out of 338 I. hexagonus samples were PCR-positive. Thirty-two hedgehogs were positive more than once, most frequently twice or 3 times, but also up to 9 times. Sequencing of the partial 16S rRNA gene resulted in 6 variants, but one variant ('A') was the most frequent which appeared in 93.8% of the positive hedgehogs. This variant (equaling Frankonia II, GenBank AF136712) has recently been reported from human, equine, and canine granulocytic anaplasmosis cases and thus, its specific association with hedgehogs is an important finding in the epidemiology of A. phagocytophilum in Europe. The high infection rate of both hedgehogs and ticks with A. phagocytophilum and the simultaneous infestation with 2 tick species of all developmental stages suggest that the hedgehog may be a suitable reservoir for at least some variants of A. phagocytophilum., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Previous studies have shown that public parks provide extraordinary habitats for ticks and tick-borne pathogens. Therefore, the aim of this study was to investigate the tick abundance (ticks/100 m(2)) in urban areas and the prevalence of Anaplasma phagocytophilum. Collection of ticks was performed by the flagging method in selected Bavarian public parks in a 2-year survey. DNA from 30 ticks of each developmental stage, sampling site and month was extracted and screened by specific real-time PCR for A. phagocytophilum. Selected positive samples were further genetically differentiated by nested PCR, targeting the 16S rRNA gene. A total of 13,403 Ixodes ricinus were collected with an average tick abundance of 15-53 ticks/100 m(2) in 2009 and 15-35 ticks/100 m(2) in 2010, depending on the sampling site. DNA of A. phagocytophilum was detected in 11.6% (n=146) and 8.5% (n=50) of adult females, 13.3% (n=164) and 9.2% (n=51) of adult males as well as 5% (n=60) and 3.9% (n=29) of nymphs for 2009 (n=3685) and 2010 (n=1884), respectively. 16S rRNA gene analysis revealed 6 different genetic variants, 2 of which were 100% identical to known sequences in humans. The results give strong evidence that the occurrence of A. phagocytophilum in I. ricinus is highly variable in different habitat types due to geographical, climatic, and biological factors as well as different genetic variants of A. phagocytophilum circulated in ticks with a noticeable habitat and host tropism., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Anaplasma phagocytophilum is an obligate intracellular bacterium causing granulocytic anaplasmosis in dogs, horses, and humans and tick-borne fever of ruminants. The bacterium has been detected in a variety of other mammals including wild ruminants without overt clinical signs of disease. Isolates in cell culture have been obtained from humans, dogs, horses, sheep, and ticks, but no strain from wild ruminants exists in cell culture in Europe. From September to November 2010, EDTA blood samples were collected from the jugular vein of 19 shot roe deer from a forest in southern Germany. The presence of specific A. phagocytophilum DNA was demonstrated with a real-time PCR targeting the msp2 gene in all 19 animals. Subsequently, blood cells were used to inoculate the tick cell line IDE8. The first infected IDE8 cells were detected in Giemsa-stained smears 57 days post inoculation. Only one roe deer yielded a positive culture which has been propagated for 9 consecutive passages thus far representing 228 days in culture. Further analysis of the A. phagocytophilum strain was performed by PCR followed by sequencing for the partial 16S rRNA, groEL, msp2, and msp4 genes. Phylogenetic topology of groEL and msp4 sequences placed the roe deer isolate in close proximity to sequences available from roe deer and goats from the neighbouring Alpine regions of Austria and Switzerland, and of msp2 with other ruminant species. This represents the first isolation of A. phagocytophilum in a tick cell line directly from an infected wild ruminant reservoir host, Capreolus capreolus, in Europe. The availability of a cultured A. phagocytophilum strain isolated from roe deer will allow us to study the biological characteristics and the pathogenic potential of this strain as well as to compare its host tropism and its genetic and antigenetic properties with those of other A. phagocytophilum strains from other animal species., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

The main objectives of this prospective study were to establish prevalence of Anaplasma phagocytophilum infections in dogs from Northeast Germany; and to evaluate the hematological parameters of sero- or real-time PCR-positive clinically healthy dogs. The mean prevalence of A. phagocytophilum seropositivity of 522 dogs (258 suspected to have anaplasmosis, 264 healthy) was 43%. There was no difference between sick (46.9%) and healthy dogs (39.8%) (p=0.100). The PCR test was positive in 30 dogs (20 sick, 10 healthy); morulae were found in 12 of them. Twenty-six of 30 dogs tested PCR-positive between May and September (p<0.05). There was no difference with regard to abnormal CBC parameters between seropositive and seronegative clinically healthy dogs. The CBC was within reference range in 10 PCR-positive clinically healthy dogs suggesting a routine examination of blood donors for A. phagocytophilum in endemic areas to minimize the risk of transmission., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The occurrence of genetic variants of Anaplasma phagocytophilum was studied in wild ungulates from the northern and central eastern Alps in Tyrol, Austria. For this purpose, spleen samples collected from 53 game animals during the hunting season 2008/2009 (16 roe deer [Capreolus capreolus], 10 red deer [Cervus elaphus], 16 Alpine chamois [Rupicapra r. rupicapra], 7 Alpine ibex [Capra i. ibex], and 4 European mouflons [Ovis orientalis musimon]) were analyzed. Thirty-five animals originated from the Karwendel mountains, 12 from the Kaunertal area (Ötztal Alps), and the remaining from other mountainous areas in Tyrol. DNA extracts were screened with a real-time polymerase chain reaction targeting the msp2 gene of A. phagocytophilum. A total of 23 (43.4%) samples, from all ungulate species studied, were A. phagocytophilum positive. As of the date of this article, A. phagocytophilum has not been reported in the Alpine ibex. The positive samples were investigated further with polymerase chain reactions for amplification of the partial 16S rRNA, groEL, and msp4 genes. Sequence analysis using forward and reverse primers revealed seven different 16S rRNA gene variants. No variant could be attributed to any particular ungulate species. The groEL gene revealed 11 different variants, which grouped in the phylogenetic analysis into two distinct clusters: one cluster contained the sequences from roe deer, whereas the sequences of the other species formed the second cluster. The msp4 gene showed a high degree of variability in the amplified part with a total of 10 different sequence types. The results show that the wild mountain ungulates were infected to a considerable extent with various variants of A. phagocytophilum. The pathogenicity of the variants and the reservoir competence of the species investigated in this study deserve further attention in future studies.

Central Switzerland is a highly endemic region for tick-borne fever (TBF) in cattle, however, little is known about A. phagocytophilum in goats. In the present study, 72 animals from six goat flocks (373 EDTA blood-samples) in Central Switzerland were analysed for A. phagocytophilum DNA. A real-time PCR targeting the msp2 gene of A. phagocytophilum was performed and in positive samples the partial 165 rRNA, groEL and msp4 gene were amplified for sequence analysis. Four DNA extracts were positive. Different sequence types on basis of the amplified genes were found. For comparison, sequences of A. phagocytophilum from 12 cattle (originating from Switzerland and Southern Germany) were analysed. The 165 rRNA gene sequences from cattle were all identical amongst each other, but the groEL and msp4 gene differed depending on the origin of the cattle samples and differed from the variants from goats. This study clearly provides molecular evidence for the presence of different types of A. phagocytophilum in goat flocks in Switzerland, a fact which deserves more thorough attention in clinical studies.