Detection of Anaplasma and Ehrlichia bacteria in humans, wildlife, and ticks in the Amazon rainforest.

Tick-borne bacteria of the genera Ehrlichia and Anaplasma cause several emerging human infectious diseases worldwide. In this study, we conduct an extensive survey for Ehrlichia and Anaplasma infections in the rainforests of the Amazon biome of French Guiana. Through molecular genetics and metagenomics reconstruction, we observe a high indigenous biodiversity of infections circulating among humans, wildlife, and ticks inhabiting these ecosystems. Molecular typing identifies these infections as highly endemic, with a majority of new strains and putative species specific to French Guiana. They are detected in unusual rainforest wild animals, suggesting they have distinctive sylvatic transmission cycles. They also present potential health hazards, as revealed by the detection of Candidatus Anaplasma sparouinense in human red blood cells and that of a new close relative of the human pathogen Ehrlichia ewingii, Candidatus Ehrlichia cajennense, in the tick species that most frequently bite humans in South America. The genome assembly of three new putative species obtained from human, sloth, and tick metagenomes further reveals the presence of major homologs of Ehrlichia and Anaplasma virulence factors. These observations converge to classify health hazards associated with Ehrlichia and Anaplasma infections in the Amazon biome as distinct from those in the Northern Hemisphere.

Duration of tick attachment necessary for transmission of Anaplasma phagocytophilum by Ixodes scapularis (Acari: Ixodidae) nymphs.

This study assessed the duration of tick attachment necessary for a successful transmission of Anaplasma phagocytophilum by an infected I. scapularis nymph. Individual nymphs were placed upon BALB/c mice and allowed to feed for predetermined time intervals of 4 to 72 h. Ticks removed from mice at predetermined intervals were tested by PCR for verification of infection and evaluation of the bacterial load. The success of pathogen transmission to mice was assessed by blood-PCR at 7, 14 and 21 days postinfestation, and IFA at 21 days postinfestation. Anaplasma phagocytophilum infection was documented in 10-30 % of mice, from which ticks were removed within the first 20 h of feeding. However, transmission success was ≥70% if ticks remained attached for 36 h or longer. Notably, none of the PCR-positive mice that were exposed to infected ticks for 4 to 8 h and only half of PCR-positive mice exposed for 24 h developed antibodies within 3 weeks postinfestation. On the other hand, all mice with detectable bacteremia after being infested for 36 h seroconverted. This suggests that although some of the ticks removed prior to 24 h of attachment succeed in injecting a small amount of A. phagocytophilum, this amount is insufficient for stimulating humoral immunity and perhaps for establishing disseminated infection in BALB/c mice. Although A. phagocytophilum may be present in salivary glands of unfed I. scapularis nymphs, the amount of A. phagocytophilum initially contained in saliva appears insufficient to cause sustainable infection in a host. Replication and, maybe, reactivation of the agent for 12-24 h in a feeding tick is required before a mouse can be consistently infected.

Ehrlichiosis and anaplasmosis subcommittee report to the Tick-borne Disease Working Group.

Ehrlichioses and anaplasmosis have undergone dramatic increases in incidence, and the geographic ranges of their occurrence and vectors have also expanded. There is marked underreporting of these diseases owing to deficient physician awareness and knowledge of the illnesses as well as limited access to appropriate diagnostic tests. Human monocytic ehrlichiosis and anaplasmosis are life threatening diseases with estimated case fatality rates of 2.7 and 0.3%, respectively. However, knowledge of their full range of signs and symptoms is incomplete, and the incidence of subclinical infections is unknown. Currently available laboratory diagnostic methods are poorly utilized, and with the exception of nucleic acid amplification tests are not useful for diagnosis during the acute stage of illness when timely treatment is needed. The Ehrlichiosis and Anaplasmosis Subcommittee of the Tick-Borne Disease Working Group recommended active clinical surveillance to determine the true incidence, full clinical spectrum, and risk factors for severe illness, as well as standardized surveillance of ticks for these pathogens, and enhanced education of primary medical caregivers and the public regarding these diseases. The subcommittee identified the needs to develop sensitive, specific acute stage diagnostic tests for local clinical laboratories and point-of-care testing, to develop approaches for utilizing electronic medical records, data mining, and artificial intelligence for assisting early diagnosis and treatment, and to develop adjunctive therapies for severe disease.

Wild ungulate species differ in their contribution to the transmission of Ixodes ricinus-borne pathogens.

BACKGROUND: Several ungulate species are feeding and propagation hosts for the tick Ixodes ricinus as well as hosts to a wide range of zoonotic pathogens. Here, we focus on Anaplasma phagocytophilum and Borrelia burgdorferi (s.l.), two important pathogens for which ungulates are amplifying and dilution hosts, respectively. Ungulate management is one of the main tools to mitigate human health risks associated with these tick-borne pathogens. Across Europe, different species of ungulates are expanding their ranges and increasing in numbers. It is currently unclear if and how the relative contribution to the life-cycle of I. ricinus and the transmission cycles of tick-borne pathogens differ among these species. In this study, we aimed to identify these relative contributions for five European ungulate species. METHODS: We quantified the tick load and collected ticks and spleen samples from hunted fallow deer (Dama dama, n = 131), moose (Alces alces, n = 15), red deer (Cervus elaphus, n = 61), roe deer (Capreolus capreolus, n = 30) and wild boar (Sus scrofa, n = 87) in south-central Sweden. We investigated the presence of tick-borne pathogens in ticks and spleen samples using real-time PCR. We determined if ungulate species differed in tick load (prevalence and intensity) and in infection prevalence in their tissue as well as in the ticks feeding on them. RESULTS: Wild boar hosted fewer adult female ticks than any of the deer species, indicating that deer are more important as propagation hosts. Among the deer species, moose had the lowest number of female ticks, while there was no difference among the other deer species. Given the low number of infected nymphs, the relative contribution of all ungulate species to the transmission of B. burgdorferi (s.l.) was low. Fallow deer, red deer and roe deer contributed more to the transmission of A. phagocytophilum than wild boar. CONCLUSIONS: The ungulate species clearly differed in their role as a propagation host and in the transmission of B. burgdorferi and A. phagocytophilum. This study provides crucial information for ungulate management as a tool to mitigate zoonotic disease risk and argues for adapting management approaches to the local ungulate species composition and the pathogen(s) of concern.

Incompetence of the Asian Longhorned Tick (Acari: Ixodidae) in Transmitting the Agent of Human Granulocytic Anaplasmosis in the United States.

The Asian longhorned tick, Haemaphysalis longicornis Neumann (Acari: Ixodidae), was recently introduced into the United States and is now established in at least 15 states. Considering its ability for parthenogenetic propagation and propensity for creating high-density populations, there is concern that this tick may become involved in transmission cycles of endemic tick-borne human pathogens. Human granulocytic anaplasmosis (HGA) caused by Anaplasma phagocytophilum is one of the more common tick-borne diseases in the United States, especially in the northeastern and midwestern states. There is considerable geographical overlap between HGA cases and the currently known distribution of H. longicornis, which creates a potential for this tick to encounter A. phagocytophilum while feeding on naturally infected vertebrate hosts. Therefore, we evaluated the ability of H. longicornis to acquire and transmit the agent of HGA under laboratory conditions and compared it to the vector competence of I. scapularis. Haemaphysalis longicornis nymphs acquired the pathogen with the bloodmeal while feeding on infected domestic goats, but transstadial transmission was inefficient and PCR-positive adult ticks were unable to transmit the pathogen to naïve goats. Results of this study indicate that the Asian longhorned tick is not likely to play a significant role in the epidemiology of HGA in the United States.

Epidemiological link between canine monocytic ehrlichiosis caused by Ehrlichia canis and the presence of Rhipicephalus sanguineus sensu stricto in Argentina.

In this work, we analyze data that support an epidemiological link between cases of canine monocytic ehrlichiosis (CME) by Ehrlichia canis and the presence of Rhipicephalus sanguineus sensu stricto as vector in an endemic area for this tick in Argentina. In a blood sample of a 1-year-old toy poodle with CME compatible clinical signs, which showed CME typical morulae in monocytes in Giemsa-stained blood smear, DNA of E. canis was detected by PCR. Further, DNA of E. canis was also detected in a female of R. sanguineus s.s. collected on the infected dog. Rhipicephalus sanguineus s.s. is the only member of the R. sanguineus group that prevails in the study area. The results of this study suggest that R. sanguineus s.s. may play a more important role in the transmission of E. canis than it was assumed so far. The epidemiological link between CME cases and R. sanguineus s.s. as vector in temperate areas of Argentina described in this work contrast previous studies which found that R. sanguineus sensu lato "tropical lineage" (which is absent in the study area) is competent to transmit E. canis but not R. sanguineus s.s.

Multiple Ehrlichia chaffeensis Genes Critical for Its Persistent Infection in a Vertebrate Host Are Identified by Random Mutagenesis Coupled with In Vivo Infection Assessment.

Ehrlichia chaffeensis, a tick-transmitted obligate intracellular rickettsial agent, causes human monocytic ehrlichiosis. In recent reports, we described substantial advances in developing random and targeted gene disruption methods to investigate the functions of E. chaffeensis genes. We reported earlier that the Himar1 transposon-based random mutagenesis is a valuable tool in defining E. chaffeensis genes critical for its persistent growth in vivo in reservoir and incidental hosts. The method also aided in extending studies focused on vaccine development and immunity. Here, we describe the generation and mapping of 55 new mutations. To define the critical nature of the bacterial genes, infection experiments were carried out in the canine host with pools of mutant organisms. Infection evaluation in the physiologically relevant host by molecular assays and by xenodiagnoses allowed the identification of many proteins critical for the pathogen's persistent in vivo growth. Genes encoding proteins involved in biotin biosynthesis, protein synthesis and fatty acid biosynthesis, DNA repair, electron transfer, and a component of a multidrug resistance (MDR) efflux pump were concluded to be essential for the pathogen's in vivo growth. Three known immunodominant membrane proteins, i.e., two 28-kDa outer membrane proteins (P28/OMP) and a 120-kDa surface protein, were also recognized as necessary for the pathogen's obligate intracellular life cycle. The discovery of many E. chaffeensis proteins crucial for its continuous in vivo growth will serve as a major resource for investigations aimed at defining pathogenesis and developing novel therapeutics for this and related pathogens of the rickettsial family Anaplasmataceae.

Ehrlichiosis-Induced Atrial Flutter: An Unusual Cause of Atrial Flutter.

Tick-borne illness has been increasingly on the rise, since the first human case was reported in the late 1980s. Ehrlichia chaffeensis is one of the most common reported causes of tick-borne illness, particularly in the southern states of the United States. The clinical picture presents as a paradigm to the clinician, often missing the diagnosis without an appropriate history being taken and sometimes mistreated for other conditions. With the number of cases on the rise, new manifestations and clinical presentations due to E chaffeensis continue to be reported. Our case report is one such case in a 46-year-old male from Arkansas, with known exposure to multiple tick bites who presented with classical symptoms and laboratory values of tick-borne illness leading to atrial flutter. This unusual manifestation of atrial flutter due to tick-borne illness is rare and poorly understood. Further studies on tick-borne illness due to E chaffeensis may be needed to understand the systemic causes of the bacteria. In addition, in our case report, we bring to attention the standard presentation (symptoms, signs, and laboratory values) of tick-borne illness due to E chaffeensis along with the current standard for diagnosis and treatment.

An Entry-Triggering Protein of Ehrlichia Is a New Vaccine Candidate against Tick-Borne Human Monocytic Ehrlichiosis.

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes human monocytic ehrlichiosis, an emerging disease transmitted by the Lone Star tick, Amblyomma americanum. E. chaffeensis outer membrane protein entry triggering protein of Ehrlichia (EtpE) is necessary for bacterial entry into human cells. We investigated the role of EtpE in transmission of the bacteria from tick to human cells and whether or not vaccination with EtpE can prevent transmission of ehrlichiae from ticks to mammals. An antiserum against the recombinant C terminus of EtpE (rEtpE-C), which binds a mammalian cell-surface receptor and triggers bacterial entry, significantly inhibited E. chaffeensis transmission from infected tick cells to human monocytes in culture. Each of five specific-pathogen-free dogs were vaccinated with rEtpE-C along with an immunostimulating complex or were sham vaccinated with the complex alone. Dogs vaccinated with rEtpE-C developed high antibody titers against rEtpE-C and produced interferon-γ-secreting cells, as assessed with the ELISpot assay. All 10 dogs were challenged with A. americanum adult ticks infected as nymphs by syringe inoculation with E. chaffeensis Upon challenge, both the vaccinated and control dogs became infected by day 1 post-tick attachment, but the majority of rEtpE-C-vaccinated dogs rapidly cleared the infection from the bloodstream as soon as day 7, whereas most of sham-vaccinated dogs remained infected at day 35. Peripheral blood leukocytes from vaccinated dogs had significantly elevated interferon-γ mRNA levels and secreted significantly elevated interferon-γ soon after tick attachment. Thus, the EtpE-C vaccine represents the first ehrlichial protein vaccine demonstrated to reduce bacterial infection in mammals upon challenge with infected ticks.IMPORTANCE The incidence of tick-borne diseases has risen dramatically in the past two decades and continues to rise. Discovered in 1986 and designated a nationally notifiable disease in 1998 by the Centers for Disease Control and Prevention, human monocytic ehrlichiosis, which is caused by the bacterium Ehrlichia chaffeensis, is one of the most prevalent, life-threatening, emerging tick-borne zoonoses in the United States. We investigated the role of the E. chaffeensis protein EtpE in transmission of the bacterium from tick to human cells and in vaccinated dogs with EtpE to assess the efficacy of vaccination against E. chaffeensis-infected tick challenge. Our results help fill gaps in our understanding of E. chaffeensis-derived protective antigens that could be used in a candidate vaccine for immunization of humans to counter tick-transmitted ehrlichiosis.

Anaplasma phagocytophilum infection in cats: A literature review to raise clinical awareness.

PRACTICAL RELEVANCE: Granulocytic anaplasmosis is a disease in humans and animals caused by the Gram-negative bacterium Anaplasma phagocytophilum within the family Anaplasmataceae. The pathogen is transmitted by ticks of the Ixodes species. Infections with A phagocytophilum have often been described in dogs but reports on natural infections in cats are rare. An infection with A phagocytophilum should be considered as a differential diagnosis in cats if the history reveals tick infestation and/or outdoor access in combination with the relevant clinical signs. GLOBAL IMPORTANCE: A phagocytophilum is also important in human medicine because of its zoonotic potential. Due to the risk of vector-borne infections for both feline and public health, cats should be protected with ectoparasiticides, especially in endemic areas. AIM: The aim of this review is to give an overview of the published data and summarise the epidemiology, pathogenesis, diagnosis, clinical signs and therapy of feline granulocytic anaplasmosis. As clinical signs are vague and non-specific, this review aims to raise awareness of A phagocytophilum infection, both among clinicians, so that they consider testing potentially exposed cats, and scientists, in order to prompt further research. EVIDENCE BASE: Sixteen publications describing 55 cats have been reviewed. Thirty-four cats were well diagnosed based on guidelines of the European Advisory Board on Cat Diseases and blood analyses were performed to varying extents for these cats. Because of the limited number of studies and a lack of knowledge in cats, clinical signs and blood analyses are compared with available data in dogs.

Transovarial transmission of Borrelia spp., Rickettsia spp. and Anaplasma phagocytophilum in Ixodes ricinus under field conditions extrapolated from DNA detection in questing larvae.

BACKGROUND: Ixodes ricinus constitutes the main European vector tick for the Lyme borreliosis pathogen Borrelia burgdorferi (sensu lato), the relapsing fever borrelia Borrelia miyamotoi, as well as Anaplasma phagocytophilum and several Rickettsia species. Under laboratory conditions, a transovarial transmission to the next tick generation is described for Rickettsia spp. and Borrelia spp., especially regarding B. miyamotoi, whereas the efficiency of transovarial transfer under field conditions is largely unstudied. METHODS: In order to better estimate the potential infection risk by tick larvae for humans and animals, 1500 I. ricinus larvae from 50 collected "nests" (larvae adhering to the flag in a clumped manner) were individually examined for Borrelia, Rickettsia and A. phagocytophilum DNA using quantitative real-time PCR (qPCR). RESULTS: Thirty-nine of 50 nests each (78.0%, 95% CI: 64.0-88.5%) were positive for Borrelia spp. and Rickettsia spp. DNA, and in three nests (6.0%, 95% CI: 1.3-16.5%) A. phagocytophilum DNA was detected. Overall, DNA from at least one pathogen could be detected in 90.0% (45/50, 95% CI: 78.2-96.7%) of the nests. Of the 1500 larvae, 137 were positive for Borrelia spp. DNA (9.1%, 95% CI: 7.7-10.7%), 341 for Rickettsia spp. DNA (22.7%, 95% CI: 20.6-24.9%) and three for A. phagocytophilum DNA (0.2%, 95% CI: 0-0.6%). Quantity of Borrelia spp. and Anaplasma spp. DNA in positive larvae was low, with 2.7 × 100Borrelia 5S-23S gene copies and 2.4 × 101A. phagocytophilum msp2/p44 gene copies detected on average, while Rickettsia-positive samples contained on average 5.4 × 102gltA gene copies. Coinfections were found in 66.0% (33/50, 95% CI: 51.2-78.8%) of the nests and 8.6% (38/443, 95% CI: 6.1-11.6%) of positive larvae. In fact, larvae had a significantly higher probability of being infected with Borrelia spp. or Rickettsia spp. when both pathogens were present in the nest. CONCLUSIONS: This study provides evidence for transovarial transmission of Rickettsia spp. and Borrelia spp. in I. ricinus under field conditions, possibly facilitating pathogen persistence in the ecosystem and reducing the dependence on the presence of suitable reservoir hosts. Further studies are needed to prove transovarial transmission and to explain the surprisingly high proportion of nests containing Rickettsia and/or Borrelia DNA-positive larvae compared to infection rates in adult ticks commonly reported in other studies.

Seroprevalence of viral and vector-borne bacterial pathogens in domestic dogs (Canis familiaris) in northern Botswana.

BACKGROUND: Domestic dogs (Canis familiaris) have the potential to act as disease reservoirs for wildlife and are important sentinels for common circulating pathogens. Therefore, the infectious disease seroprevalence among domestic dogs in northern Botswana may be indicative of pathogen exposure of various wildlife species. The objective of this study was to assess the seroprevalence of Ehrlichia spp., Borrelia burgdorferi, Anaplasma spp., Dirofilaria immitis, canine adenovirus, canine parvovirus, and canine distemper virus in domestic dogs as proxies of disease prevalence in the local wildlife in the Okavango Delta region of Botswana. Statistical analysis assessed crude and factor-specific seroprevalence proportions in relation to age, sex, and geographical location as predictors of seropositivity. Logistic regression was used to identify adjusted predictors of seropositivity for each of the pathogens of interest. RESULTS: Samples from 233 dogs in a total of seven locations in Maun, Botswana, and surrounding villages were collected and serologically analyzed. No dogs were seropositive for B. burgdorferi, while low seroprevalence proportions were observed for Anaplasma spp. (2.2%) and D. immitis (0.9%). Higher seroprevalence proportions were observed for the tick-borne pathogen Ehrlichia spp. (21.0%), and 19.7% were seropositive for canine adenovirus (hepatitis). The highest seroprevalence proportions were for canine parvovirus (70.0%) and canine distemper virus (44.8%). The predictors of seropositivity revealed that adults were more likely to be seropositive for canine adenovirus, canine distemper virus, and canine parvovirus than juveniles, and location was a risk factor for canine adenovirus, canine distemper virus, canine parvovirus, and Ehrlichia spp. CONCLUSIONS: Results indicate that increasing tick control and vaccination campaigns for domestic dogs may improve the health of domestic animals, and potentially wildlife and humans in the Okavango Delta since viral and vector-borne bacterial pathogens can be transmitted between them.

Prevention of transmission of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum by Ixodes spp. ticks to dogs treated with the Seresto® collar (imidacloprid 10% + flumethrin 4.5%).

The capability of imidacloprid 10% + flumethrin 4.5% (Seresto®) collars to prevent transmission of Borrelia burgdorferi sensu lato (Bbsl) and Anaplasma phagocytophilum (Ap) by naturally infected ticks was evaluated in two studies with 44 dogs. In each study, one group served as non-treated control, whereas the other groups were treated with the Seresto® collar. All dogs were exposed to naturally Bbsl- and Ap-infected hard ticks (Ixodes ricinus, Ixodes scapularis). In study 1, tick infestation was performed on study day (SD) 63 (2 months post-treatment [p.t.]); in study 2, it was performed on SD 32 (one month p.t.) respectively SD 219 (seven months p.t.). In situ tick counts were performed 2 days after infestation. Tick counts and removals followed 6 (study 1) or 5 days (study 2) later. Blood sampling was performed for the detection of specific Bbsl and Ap antibodies and, in study 1, for the documentation of Ap DNA by PCR. Skin biopsies were examined for Bbsl by PCR and culture (only study 1). The efficacy against Ixodes spp. was 100% at all time points. In study 1, two of six non-treated dogs became infected with Bbsl, and four of six tested positive for Ap; none of the treated dogs tested positive for Bbsl or Ap. In study 2, ten of ten non-treated dogs became infected with Bbsl and Ap; none of the treated dogs tested positive for Bbsl or Ap; 100% acaricidal efficacy was shown in both studies. Transmission of Bbsl and Ap was successfully blocked for up to 7 months.

Phylogenetic profiling for zoonotic Ehrlichia spp. from ixodid ticks in the Eastern Cape, South Africa.

Ticks are obligate hematophagous parasite of vertebrate that transmit a range of pathogenic microorganisms that can cause diseases in livestock and humans. The range of tick-borne disease causative agents infecting domestic animals and humans has recently increased. Several significant zoonotic tick-borne diseases such as ehrlichiosis among others are on the increase worldwide. This study was designed to investigate the occurrence of zoonotic Ehrlichia spp. from samples collected from livestock in selected communities in the Eastern Cape Province, South Africa. Tick samples were manually collected from domesticated animals in selected homesteads. The ticks were morphologically identified to species and tested for Ehrlichia infection via polymerase chain reaction (PCR), using genus-specific disulphide bond formation protein (dsbA) gene primers. This was followed by sequence analysis of amplicons and phylogeny. Of the 1,200 ticks collected, Amblyomma hebraeum was most prevalent (n = 335; 27.9%), followed by Rhipicephalus appendiculatus (n = 274; 22.8%), Rhipicephalus decoloratus; (n = 224; 18.7%) and Rhipicephalus eversti eversti (n = 200, 16.7%). Ehrlichia DNA was detected in 19/1,200 (1.6%) of the screened DNA samples. A homology search of the generated sequences revealed a high percentage of identity between 95% and 98% with other homologous dsbA gene sequences of other Ehrlichia species in GenBank. Phylogenetic analysis showed that the obtained sequences clustered unambiguously with other Ehrlichia sequences from different geographical regions of the world. We concluded that Ehrlichial pathogens are vectored by the ticks collected from domesticated animals in the study areas, thus suggesting concern for public health, as some of the recovered pathogens are zoonotic in nature and could pose serious public health risk through human exposure to tick bites.

Occurrence of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Babesia microti in Ixodes ricinus ticks collected from selected areas of Opolskie Province in south-west Poland.

INTRODUCTION: Ticks (Acari: Ixodida) are vectors and/or reservoirs of many pathogens, i.e. Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Babesia microti. These pathogens are ethiological agents of such diseases as Lyme borreliosis, human granulocytic anaplasmosis and human babesiosis. OBJECTIVE: The aim of the study was to evaluate the role of the Ixodes ricinus in the transmission of Borrelia burgdorferi sensu lato, Borrelia afzelii, Borrelia garinii, Borrelia burgdorferi sensu stricto, Anaplasma phagocytophilum and Babesia microti in Opolskie Province in Poland. MATERIAL AND METHODS: DNA from 222 ticks was isolated by the ammonia method. The pair of primers specific to the flagelline gene was used to detect of B. burgdorferi s. l. To detect of genospecies of this spirochete, three pairs of internal primers were used. In turn, two pairs of primers specific to the 16S rDNA gene and the 18S rRNA were used, respectively, for the detection of A. phagocytophilum and B. microti. Borrelia burgdorferi s. l., A. phagocytophilum, and B. microti were detected in 4.5%, 2.7% and 5.4% of examined ticks, respectively. RESULTS AND CONCLUSIONS: Of the ten ticks infected with B. burgdorferi s. l., B. afzelii was found in seven, undefinied genospecies in two, and mixed infection with B. afzelii and B. burgdorferi s. s. in one. The study demonstrated the potential risk of exposure of humans and animals to infections of B. burgdorferi s. l., A. phagocytophilum and B. microti in the examined area of Poland.

Molecular Detection of Anaplasma phagocytophilum from Larvae of Haemaphysalis longicornis in Ibaraki, Japan.

We collected 1,084 ticks by flagging vegetation in Tsukuba and Moriya (Ibaraki, Japan), where several cases of canine granulocytic anaplasmosis were reported. The DNA of the collected ticks was molecularly examined for infection with the family Anaplasmataceae. Twenty-six positive samples of Anaplasmataceae-specific PCR of partial 16S rRNA gene were subjected to semi-nested PCR, covering the divergent regions, the gene, and sequence analysis. Anaplasma phagocytophilum was detected in 3 pools of Haemaphysalis longicornis larvae and A. bovis from a H. flava male. Sequences of both amplicons had high homologies to those from dogs in our previous studies in Ibaraki. These results suggest that Haemaphysalis ticks are candidate vectors of A. phagocytophilum and A. bovis in Ibaraki, Japan.

Human ehrlichiosis at a tertiary-care academic medical center: Clinical associations and outcomes of transplant patients and patients with hemophagocytic lymphohistiocytosis.

BACKGROUND: Ehrlichiosis is an acute febrile tick-borne disease which can rarely be a trigger for secondary hemophagocytic lymphohistiocytosis (HLH). METHODS: We reviewed our experience with Ehrlichia infections at a tertiary-care academic medical center. RESULTS: Over 10 years, 157 cases of ehrlichiosis were identified. Ten patients (6.4%) had infection with E. ewingii, 7(4.5%) of whom were transplant patients as compared to 3(1.9%) non-transplant patients (p = .035). Transplant patients were more likely to have leukopenia and elevated creatinine compared to immunocompetent patients; length of hospital stay and early mortality were not different between the two groups. Ten patients met the HLH-2004 diagnosis criteria, which could be an underestimation of HLH occurrence as most patients were not completely evaluated for these criteria. We calculated the H-Score to find the probability of HLH; 25 patients scored high making the occurrence rate of HLH at least 16%. Ehrlichia-induced HLH patients (N = 25) had more anemia, thrombocytopenia, elevated creatinine and AST. Moreover, they had a significantly longer hospital stay (median 9 days) compared to patients without HLH (median 4 days) (p = .006). CONCLUSIONS: Ehrlichia-induced HLH is a potential serious complication with relatively high occurrence rate; patients manifest severe disease with end-organ damage requiring longer hospital stay.

Transmission of Anaplasma phagocytophilum (Foggie, 1949) by Ixodes ricinus (Linnaeus, 1758) ticks feeding on dogs and artificial membranes.

BACKGROUND: The interplay of speed of activity of acaricidal products and tick-borne pathogen transmission time is the major driver for disease prevention. This study aimed to investigate the time required for transmission of Anaplasma phagocytophilum by adult Ixodes ricinus ticks in vivo on dogs, and to confirm the time required for transmission observed in vivo, in vitro. METHODS: Nymphs of I. ricinus were experimentally infected with an A. phagocytophilum strain of canine origin. Dogs were allocated to 6 groups of 3 dogs each. Groups 1-5 were infested with 50 A. phagocytophilum-infected female adult ticks on Day 0. Ticks were removed post-infestation at 3, 6, 12, 24 and 48 h. Dogs in Group 6 were infested with 60 A. phagocytophilum-infected female adult ticks (left on dogs until engorged). Dogs were observed daily for general health and clinically examined on Day 0, and weekly from Day 14. Blood was collected for qPCR and serological analysis on Day 0 (pre-challenge) and weekly thereafter. In the in vitro study each artificial feeding chamber was seeded with 10 adult ticks (5 male/5 female), attachment assessed, and blood pools sampled for qPCR at 6 h intervals up to 72 h after first tick attachment. RESULTS: Anaplasma phagocytophilum specific antibodies and DNA were detected in all 3 dogs in Group 6. No A. phagocytophilum-specific antibodies or DNA were detected in any dogs in Groups 1-5. All dogs remained healthy. Female tick attachment in 60 artificial feeding chambers over 72 h ranged between 20-60%. Anaplasma phagocytophilum DNA was detected in the blood collected from 5% of chambers sampled at 6 h, with the highest number of positive samples (16.3%) observed at 36 h. CONCLUSIONS: Transmission of A. phagocytophilum by I. ricinus ticks starts within a few hours after attachment but establishment of infections in dogs is apparently dependent on a minimum inoculation dose that was only observed when ticks attached for greater than 48 h. These findings highlight the need for acaricidal products to exert a repellent and/or rapid killing effect on ticks to forestall transmission and subsequent disease.

Role of mustelids in the life-cycle of ixodid ticks and transmission cycles of four tick-borne pathogens.

BACKGROUND: Elucidating which wildlife species significantly contribute to the maintenance of Ixodes ricinus populations and the enzootic cycles of the pathogens they transmit is imperative in understanding the driving forces behind the emergence of tick-borne diseases. Here, we aimed to quantify the relative contribution of four mustelid species in the life-cycles of I. ricinus and Borrelia burgdorferi (sensu lato) in forested areas and to investigate their role in the transmission of other tick-borne pathogens. Road-killed badgers, pine martens, stone martens and polecats were collected in Belgium and the Netherlands. Their organs and feeding ticks were tested for the presence of tick-borne pathogens. RESULTS: Ixodes hexagonus and I. ricinus were found on half of the screened animals (n = 637). Pine martens had the highest I. ricinus burden, whereas polecats had the highest I. hexagonus burden. We detected DNA from B. burgdorferi (s.l.) and Anaplasma phagocytophilum in organs of all four mustelid species (n = 789), and Neoehrlichia mikurensis DNA was detected in all species, except badgers. DNA from B. miyamotoi was not detected in any of the investigated mustelids. From the 15 larvae of I. ricinus feeding on pine martens (n = 44), only one was positive for B. miyamotoi DNA, and all tested negative for B. burgdorferi (s.l.), N. mikurensis and A. phagocytophilum. The two feeding larvae from the investigated polecats (n = 364) and stone martens (n = 39) were negative for all four pathogens. The infection rate of N. mikurensis was higher in feeding nymphs collected from mustelids compared to questing nymphs, but not for B. burgdorferi (s.l.), B. miyamotoi or A. phagocytophilum. CONCLUSIONS: Although all stages of I. ricinus can be found on badgers, polecats, pine and stone martens, their relative contribution to the life-cycle of I. ricinus in forested areas is less than 1%. Consequently, the relative contribution of mustelids to the enzootic cycles of I. ricinus-borne pathogens is negligible, despite the presence of these pathogens in organs and feeding ticks. Interestingly, all four mustelid species carried all stages of I. hexagonus, potentially maintaining enzootic cycles of this tick species apart from the cycle involving hedgehogs as main host species.

Seroprevalence and current infections of canine vector-borne diseases in Nicaragua.

BACKGROUND: Vector-borne diseases constitute a major problem for veterinary and public health, especially in tropical regions like Central America. Domestic dogs may be infected with several vector-borne pathogens of zoonotic relevance, which may also severely compromise canine health. METHODS: To assess the prevalence of canine vector-borne diseases in Nicaragua, 329 dogs from seven cities, which were presented to the veterinarian for various reasons, were included in this study. Dogs were examined clinically and diagnostic blood samples were taken for analysis of packed cell volume (PCV) and presence of microfilariae as well as antigen of Dirofilaria immitis and antibodies to Ehrlichia spp., Anaplasma spp. and Borrelia burgdorferi (sensu lato) by use of a commercially available rapid ELISA. To detect current infections, specific PCRs for the detection of E. canis, A. platys and A. phagocytophilum were carried out on blood samples of the respective seropositive dogs. Microfilaremic blood samples, as well as D. immitis antigen positive samples were further subjected to PCR and subsequent sequencing for filarial species identification. RESULTS: Antibodies against Ehrlichia spp. were present in 62.9% of dogs, while Anaplasma spp. seroprevalence was 28.6%. Antibodies against species of both genera were detected in 24.9% of dogs. Borrelia burgdorferi (s.l.) antibodies were not detected. Dirofilaria immitis antigen was present in six animals (1.8%), two of which also showed D. immitis microfilariae in buffy coat. In addition to D. immitis, Acanthocheilonema reconditum was identified by PCR and sequencing in two of four additional microfilaremic blood samples, which were tested negative for D. immitis antigen. Current E. canis infections as defined by DNA detection were present in 58.5% of Ehrlichia-seropositive dogs, while 5.3% of Anaplasma-seropositive dogs were PCR-positive for A. platys, 2.2% for A. phagocytophilum and 16.0% for both Anaplasma species. Current E. canis infection had a statistically significant negative impact on PCV, whereas no relationship between infection status and clinical signs of disease could be observed. CONCLUSIONS: These results indicate that canine vector-borne diseases are widespread in Nicaragua and that dogs may constitute a reservoir for human infection with E. canis, A. phagocytophilum and D. immitis. Thus, the use of repellents or acaricides to protect dogs from vector-borne diseases is strongly recommended.