Emerging Tick-Borne Diseases.

Increases in tick-borne disease prevalence and transmission are important public health issues. Efforts to control these emerging diseases are frustrated by the struggle to control tick populations and to detect and treat infections caused by the pathogens that they transmit. This review covers tick-borne infectious diseases of nonrickettsial bacterial, parasitic, and viral origins. While tick surveillance and tracking inform our understanding of the importance of the spread and ecology of ticks and help identify areas of risk for disease transmission, the vectors are not the focus of this document. Here, we emphasize the most significant pathogens that infect humans as well as the epidemiology, clinical features, diagnosis, and treatment of diseases that they cause. Although detection via molecular or immunological methods has improved, tick-borne diseases continue to remain underdiagnosed, making the scope of the problem difficult to assess. Our current understanding of the incidence of tick-borne diseases is discussed in this review. An awareness of the diseases that can be transmitted by ticks in specific locations is key to detection and selection of appropriate treatment. As tick-transmitted pathogens are discovered and emerge in new geographic regions, our ability to detect, describe, and understand the growing public health threat must also grow to meet the challenge.

Early diagnosis of Ehrlichia ewingii infection in a lung transplant recipient by peripheral blood smear.

Ehrlichiosis in lung transplant (LT) recipients is associated with severe outcomes. Ehrlichia ewingii is a less frequent cause of symptomatic ehrlichiosis, characterized by cytoplasmic inclusions (morulae) within circulating neutrophils. We report a case of E. ewingii infection in an LT recipient diagnosed promptly by blood smear exam and confirmed with molecular studies.

Expanded Geographic Distribution and Clinical Characteristics of Ehrlichia ewingii Infections, United States.

Ehrlichiosis is a bacterial zoonosis, spread through the bites of infected ticks, that is most commonly caused in the United States by infection with the bacterium Ehrlichia chaffeensis. We retrospectively reviewed samples from an 18-month study of ehrlichiosis in the United States and found that E. ewingii was present in 10 (9.2%) of 109 case-patients with ehrlichiosis, a higher rate of infection with this species than had previously been reported. Two patients resided in New Jersey and Indiana, where cases have not been reported. All patients with available case histories recovered. Our study suggests a higher prevalence and wider geographic distribution of E. ewingii in the United States than previous reports have indicated.

Ehrlichiosis in Dogs: A Comprehensive Review about the Pathogen and Its Vectors with Emphasis on South and East Asian Countries.

Ehrlichiosis in dogs is an emerging vector borne rickettsial zoonotic disease of worldwide distribution. In general, three Ehrlichial species (Ehrlichia canis, E. ewingii, and E. chaffeensis) are involved in infecting dogs. Among them, E. canis is the well-known etiological pathogen affecting platelets, monocytes, and granulocytes. Dogs act as a reservoir, while the main vector responsible for disease transmission is Rhipicephalus sanguineus. However, in east Asian countries, Haemaphysalis longicornis is considered the principal vector for disease transmission. This disease affects multiple organs and systems and has three clinical manifestations, including acute, subclinical, and chronic. Definitive diagnosis involves visualization of morulae on cytology, detection of antibodies through an indirect immunofluorescence test (IFAT), and DNA amplification by polymerase chain reaction (PCR). In canine ehrlichiosis, no predilection of age or sex is observed; however, Siberian Huskies and German Shepherds are more likely to develop severe clinical manifestations. Doxycycline, rifampicin, and minocycline are proven to be effective drugs against canine ehrlichiosis. This review is intended to describe a brief overview of Ehrlichia infection in dogs, its reported prevalence in east and south Asian countries, and the latest knowledge regarding chemotherapy and associated vectors responsible for the disease transmission. This manuscript also identifies the prevailing knowledge gaps which merit further attention by the scientific community.

Haematological Alterations Associated with Selected Vector-Borne Infections and Exposure in Dogs from Pereira, Risaralda, Colombia.

Infections due to Ehrlichia, Anaplasma, Dirofilaria, Mycoplasma, Babesia and Hepatozoon continue to be highly prevalent in dogs, especially in tropical and subtropical areas, where vectors of many of them are present. However, many clinical aspects of dogs have not been characterized in detail, including assessing the haematological alterations associated with them, particularly in Colombia and Latin America. A group of 100 dogs with Ehrlichia, Anaplasma, Dirofilaria, Mycoplasma, Babesia and Hepatozoon infections/exposure were assessed by blood smear serology (SNAP4DX) and PCR in Pereira, Colombia. We performed blood counts to evaluate anaemia, leukopenia/leukocytosis, neutropenia, neutrophilia, lymphopenia/lymphocytosis, monocytosis, eosinophilia, and thrombocytopenia, among other alterations. Bivariate analyses were performed on Stata®14, with significant p < 0.05. From the total, 85% presented ≥1 infection (past or present), 66% with coinfections (≥2 pathogens) (Ehrlichia 75%), and 89% presented clinical alterations. A total of 100% showed anaemia, 70% thrombocytopenia, 61% monocytosis, and 47% neutropenia, among other alterations. Additionally, 11% presented pancytopenia and 59% bicytopenia. The median platelet count was lower in infected dogs (126,000 cells/µL) versus non-infected (221,000 cells/µL) (p = 0.003). Thrombocytopenia was higher among infected dogs (75%) versus non-infected (40%) (p = 0.006), with a 91% positive predictive value for infection. Median neutrophil count was lower in infected dogs (6591 cells/µL) versus non-infected (8804 cells/µL) (p = 0.013). Lymphocytosis occurred only among those infected (27%) (p = 0.022). Leukopenia was only observed among infected dogs (13%). Pancytopenia was only observed among infected dogs. Ehrlichiosis and other hematic infections have led to a significant burden of haematological alterations on infected dogs, including pancytopenia in a tenth of them, most with thrombocytopenia and all anemic.

Seroprevalence canine survey for selected vector-borne pathogens and its relationship with poverty in metropolitan Pereira, Colombia, 2020.

Background: Tick-borne diseases (TBD) and dirofilariosis are currently not under surveillance in most Latin American countries. In addition, there is a significant lack of studies describing the current situation in most endemic areas, including Colombia. Therefore, seroprevalence studies are crucial for understanding the epidemiology of these vector-borne diseases. Methods: A serosurvey for TBD and dirofilariosis among 100 dogs was carried out in the municipality of Pereira, located in the Coffee-Triangle region, Colombia. Samples were tested using a rapid assay test system (SNAP® 4Dx®); based on an enzyme immunoassay technique' screening for antibodies to Anaplasma phagocytophilum/platys (sensitivity 99.1%)' Borrelia burgdorferi s.l. (98.8%), and Ehrlichia canis/ewingii (96.2%) by using specific antigens and checking for Dirofilaria immitis antigen based on specific antibodies (99.2%). Bivariate analyses were performed on Stata®14, significant p < 0.05. Findings: Global seroprevalence to the selected vector-borne pathogens was 74% (95%CI 65-83%). The highest seroprevalence was found for E. canis/ewingii (74%), followed by A. phagocytophilum/platys (16%). Seropositivity for Borrelia spp. and Dirofilaria spp. was 0%. All Anaplasma spp. seropositive dogs showed co-detection of Ehrlichia spp. (16%). Seroprevalence was significantly higher among dogs from families of lower socioeconomic status/level (I, 86%), followed by level II (74%), and III (36%) (p = 0.001). All dogs exhibiting anorexia (12%) were invariably seropositive (100%) (p = 0.029). Seroprevalence was higher among those showing mucocutaneous paleness (95%) compared to those without paleness (68%) (p = 0.013) (OR = 9.3; 95%CI 1.18-72.9). There was high variability in seroprevalence through the studied areas, ranging from 0% (La Libertad Park) up to Combia, Cesar Nader, Las Brisas and Saturno localities (100%) (p = 0.033). Interpretation: Given the high seroprevalence obtained in an area with documented ticks, there is a potential risk of zoonotic transmission to humans. Further seroprevalence studies in humans are needed to assess the prevalence of infections. Poverty is highly associated with these tick-borne pathogens in Pereira, as shown in the present study.

Molecular detection of zoonotic blood pathogens in ticks from illegally imported turtles in Italy.

International trade of animals may represent a gateway for the spreading of zoonotic pathogens and their vectors. Amongst animals, reptiles are commonly illegally imported worldwide, being Italy in the fifth position of importation of these animals. Thus, the current study analysed the pathogens associated with Hyalomma aegyptium ticks, which were collected from illegally imported tortoises from North Africa to Italy. All tick DNA samples were tested by conventional PCR for the presence of Anaplasma spp., Babesia spp., Borrelia spp., Coxiella burnetti, Ehrlichia spp., Hepatozoon spp., Rickettsia spp. and microfilariae of filarioids. Out of 22% (n=161) of ticks screened, 78.9% (n=127) were males and 21.1% (n=34) females. Among them, three male specimens collected from two different turtles (1.9%; 95% CI; 0.5-5.5) scored positive for Anaplasma spp./Ehrlichia spp., whereas all females were negative. BLAST analysis of the sequences obtained from positive samples revealed 99-99.3% nucleotide identity with the sequence of Ehrlichia ewingii available in GenBank. The finding of E. ewingii in ticks from imported reptiles warrants the need for imposing strict rules in the international trade of reptiles to effectively reduce the introduction of exotic pathogens and their vectors in new geographic areas.

Detection of Multiple Intracellular Bacterial Pathogens in Haemaphysalis flava Ticks Collected from Hedgehogs in Central China.

Tickborne intracellular bacterial pathogens including Anaplasma, Coxiella burnetti, Ehrlichia, and Rickettsia cause emerging infectious diseases worldwide. PCR was used to amplify the genes of these pathogens in Haemaphysalis flava ticks collected from hedgehogs in Central China. Among 125 samples including 20 egg batches, 24 engorged females, and 81 molted male and female adult ticks, the DNA sequences and phylogenetic analysis showed that the minimum infection rate of the ticks was 4% (5/125) for A. bovis, 3.2% (4/125) for C. burnetti, 9.6%, (12/125) for E. ewingii, and 5.6% for Rickettsia including R.japonica (3.2%, 4/125) and R. raoultii (2.4%, 3/125), respectively. The prevalence of these pathogens was significantly higher in dead engorged females (83.3%, 20/24) than in eggs (5%, 1/20) and molted ticks (8.6%, 7/81). Our study indicated that H. flava ticks could be infected with multiple species of tickborne pathogens including Anaplasma, C. burnetti, Ehrlichia, and Rickettsia in Central China, and the prevalence of these pathogens was reduced during transovarial and transstadial transmission in ticks, suggesting that ticks may not be real reservoirs but only vectors for these tickborne pathogens.

Effective control of the motile stages of Amblyomma americanum and reduced Ehrlichia spp. prevalence in adults via permethrin treatment of white-tailed deer in coastal Connecticut, USA.

The lone star tick, Amblyomma americanum, is a common human-biting species whose range has been largely restricted to the southeastern United States, until recent detections of established populations on Long Island, New York and throughout coastal southern New England. We evaluated the effectiveness of topical treatment of 10 % permethrin delivered via 4-poster devices to white-tailed deer, Odocoileus virginianus, in the management of a newly discovered A. americanum population in Norwalk, Connecticut. Using a high-density deployment of one 4-poster device/12.7 ha, we were successful in significantly reducing densities of host-seeking adults (93 % reduction), nymphs (92 %), and larvae (96 %) from 2018 to 2020. We also documented a significant reduction (87 %) in parasitizing adults and nymphs on white-tailed deer from 2018 to 2019. The prevalence of Ehrlichia chaffeensis and Ehrlichia ewingii combined in host-seeking adults declined significantly from 47 % at the time the A. americanum population was discovered in 2017 to 7% in 2020. However, the prevalence in nymphs remained static (∼9%) throughout the study period. These data demonstrate that, when properly deployed in a density-dependent manner in terms of deer abundance, 4-poster devices can effectively manage parasitizing and host-seeking A. americanum populations and reduce the prevalence of two ehrlichial species of public health importance.

Evaluation of a commercial microbial enrichment kit used prior DNA extraction to improve the molecular detection of vector-borne pathogens from naturally infected dogs.

Accurate detection of vector-borne pathogens (VBPs) is extremely important as the number of reported cases in humans and animals continues to rise in the US and abroad. Validated PCR assays are currently the cornerstone of molecular diagnostics and can achieve excellent analytical sensitivity and specificity. However, the detection of pathogens at low parasitemia still presents a challenge for VBP diagnosis, especially given the very low volume of specimens tested by molecular methods. The objective of this study is to determine if a commercially available microbial enrichment kit, used prior DNA extraction, is capable of expanding the overall microbial community and increasing detectable levels of VBPs in canine blood samples through host DNA depletion. This study used EDTA-whole blood samples from dogs naturally infected with varying parasitemia levels of either Anaplasma phagocytophilum, Babesia gibsoni, or Ehrlichia ewingii. For two VBPs, EDTA-blood samples were diluted to determine the effect of microbial concentration at low parasitemia. Paired EDTA-blood samples from each dog were subjected to traditional, automated DNA extraction with or without the microbial concentrating kit (MolYsis®) prior DNA extraction. Relative amounts of pathogen DNA in paired samples were determined by real-time PCR and Next-Generation Sequencing targeting conserved regions of 16S rRNA (for bacteria) and 18S rRNA (for protozoa). Results from the three molecular methods suggest that the microbial concentrating kit did not improve the detection of VBPs, although significantly reduced the presence of host DNA. Alternative methods for VBP enrichment in clinical samples prior to molecular testing should continue to be investigated, as it may significantly improve clinical sensitivity and reduce the number of false-negative results.

Uncommon Ehrlichia canis infection associated with morulae in neutrophils from naturally infected dogs in Brazil.

Ehrlichia and Anaplasma species are the most common tick-borne disease (TBD) pathogens in dogs worldwide. Ehrlichia canis, the aetiological agent of the Canine Monocytic Ehrlichiosis (CME), is known to replicate within the cytoplasm of mononuclear cells into clusters of organisms called morulae. However, detection of morulae in neutrophils is commonly observed in dogs infected by Ehrlichia ewingii or Anaplasma phagocytophilum. We report uncommon clinical cases of canine ehrlichiosis presenting morulae compatible with E. ewingii and A. phagocytophilum in dogs from two distinct regions of Brazil. Eight dogs were admitted to two veterinary teaching hospitals from Brazil, showing clinical or haematological signs suggestive of TBD. Blood or peritoneal fluid was withdrawn for haematological and cytologic analysis. All samples were evaluated by PCR assays for Ehrlichia and Anaplasma using genus-specific primers for dsb, 16S rRNA and groEL genes, followed by sequencing. Samples were also evaluated by nested PCR assays for the 16S rRNA gene of E. ewingii and groEL gene of A. phagocytophilum and Anaplasma platys. Seven dogs revealed thrombocytopenia, six dogs had monocytosis and five presented lymphopenia and anaemia. All dogs showed morulae structures compatible with Ehrlichia spp. in neutrophils and were PCR-positive for the dsb and 16S rRNA gene fragments of Ehrlichia, with sequences showing 100% identity with multiple E. canis sequences deposited in the GenBank™. Sequencing of 16S rRNA and groEL gene fragments from one PCR-positive dog showed 100% identity with A. platys. Overall, our data suggest that in endemic regions for E. canis, that is Brazil, the presence of morulae in neutrophils may indicate infection by this bacterium. Herein, morulae were also found in neutrophils present in the peritoneal fluid of a dog. Also, this is the first report of E. canis and Hepatozoon canis co-infection in neutrophils from naturally infected dogs confirmed by DNA sequencing.

Local and regional temporal trends (2013-2019) of canine Ehrlichia spp. seroprevalence in the USA.

BACKGROUND: In the USA, there are several Ehrlichia spp. of concern including Ehrlichia canis, Ehrlichia ewingii, Ehrlichia chaffeensis, Ehrlichia muris eauclarensis, and "Panola Mountain Ehrlichia". Of these, E. canis is considered the most clinically relevant for domestic dogs, with infection capable of causing acute, subclinical, and chronic stages of disease. Changes in climate, land use, habitats, and wildlife reservoir populations, and increasing contact between both human and dog populations with natural areas have resulted in the increased risk of vector-borne disease throughout the world. METHODS: A Bayesian spatio-temporal binomial regression model was applied to serological test results collected from veterinarians throughout the contiguous USA between January 2013 and November 2019. The model was used to quantify both regional and local temporal trends of canine Ehrlichia spp. seroprevalence and identify areas that experienced significant increases in seroprevalence. RESULTS: Regionally, increasing seroprevalence occurred within several states throughout the central and southeastern states, including Missouri, Arkansas, Mississippi, Alabama, Virginia, North Carolina, Georgia and Texas. The underlying local trends revealed increasing seroprevalence at a finer scale. Clusters of locally increasing seroprevalence were seen from the western Appalachian region into the southern Midwest, along the Atlantic coast in New England, parts of Florida, Illinois, Wisconsin and Minnesota, and in a couple areas of the Mountain region. Clusters of locally decreasing seroprevalence were seen throughout the USA including New York and the mid-Atlantic states, Texas, the Midwest, and California. CONCLUSIONS: Canine Ehrlichia spp. seroprevalence is increasing in both endemic and non-endemic areas of the USA. The findings from this study indicate that dogs across a wide area of the USA are at risk of exposure and these results should provide veterinarians and pet owners with the information they need to make informed decisions about prevention of tick exposure.

Isolation and Short-Term Persistence of Ehrlichia ewingii in Cell Culture.

Ehrlichia ewingii is the causative agent of human and canine granulocytic ehrlichiosis. Since its discovery in 1970, little work has been done to characterize the pathogen or study the transmission dynamics due to the inability to grow the agent in vitro. The aim of this study was to assess the suitability of multiple cell lines and media formulations for propagation of E. ewingii in cell culture. In this study, we present an overview of attempts to isolate E. ewingii from the buffy coat of goats naturally infected by Amblyomma americanum ticks, as well as a methodology for maintaining the pathogen for up to 16 weeks in culture. The most promising results were seen with HL-60 cells differentiated by the addition of 1.5% DMSO to the media and supplemented with 8 mM l-glutamine. Cultures were passaged multiple times, and fluorescence and morulae were observed by indirect fluorescent antibody test and Diff-Quik staining. It is our hope that this information will provide a foundation for future attempts to propagate and maintain E. ewingii in cell culture.

Ehrlichioses: An Important One Health Opportunity.

Ehrlichioses are caused by obligately intracellular bacteria that are maintained subclinically in a persistently infected vertebrate host and a tick vector. The most severe life-threatening illnesses, such as human monocytotropic ehrlichiosis and heartwater, occur in incidental hosts. Ehrlichia have a developmental cycle involving an infectious, nonreplicating, dense core cell and a noninfectious, replicating reticulate cell. Ehrlichiae secrete proteins that bind to host cytoplasmic proteins and nuclear chromatin, manipulating the host cell environment to their advantage. Severe disease in immunocompetent hosts is mediated in large part by immunologic and inflammatory mechanisms, including overproduction of tumor necrosis factor α (TNF-α), which is produced by CD8 T lymphocytes, and interleukin-10 (IL-10). Immune components that contribute to control of ehrlichial infection include CD4 and CD8 T cells, natural killer (NK) cells, interferon-γ (IFN-γ), IL-12, and antibodies. Some immune components, such as TNF-α, perforin, and CD8 T cells, play both pathogenic and protective roles. In contrast with the immunocompetent host, which may die with few detectable organisms owing to the overly strong immune response, immunodeficient hosts die with overwhelming infection and large quantities of organisms in the tissues. Vaccine development is challenging because of antigenic diversity of E. ruminantium, the necessity of avoiding an immunopathologic response, and incomplete knowledge of the protective antigens.

Development of antibodies to and PCR detection of Ehrlichia spp. in dogs following natural tick exposure.

Dogs exposed to ticks in the southern US may become infected with multiple species of Ehrlichia. To better define infection risk, blood samples collected from 10 dogs infested with ticks via a natural infestation model were evaluated by blood smear examination, PCR, patient-side ELISAs (SNAP® 4Dx® and SNAP® 4Dx® Plus), IFA, and peptide based ELISA for evidence of infection with Ehrlichia canis, E. chaffeensis, and/or E. ewingii. Although morulae were rarely identified in blood smears, every dog (10/10) became infected with Ehrlichia spp. as evidenced by nested PCR detection of E. chaffeensis (7/10) and E. ewingii DNA (10/10); real-time PCR detection of E. chaffeensis (0/10) and E. ewingii (9/10); seroconversion on two different patient-side ELISAs (4/10 or 10/10); seroconversion on IFA to E. canis (10/10, maximum inverse titer=128-4096, GMTMAX=548.7) and E. chaffeensis (10/10, maximum inverse titer=1024-32,768, GMTMAX=4096); and seroconversion on peptide specific ELISA to E. chaffeensis VLPT (7/10) and E. ewingii p28 (9/10). Rickettsemia with E. chaffeensis and E. ewingii, as determined by nested PCR, persisted in dogs for an average of 3.2 or 30.5 days, respectively. Ehrlichia canis was not detected in any dog by any method, and no dogs developed signs of clinical disease. Our data suggest that in areas where ticks are common, dogs are at high risk of infection with Ehrlichia spp., particularly E. ewingii and E. chaffeensis, and can serve as a sentinel for monitoring for the presence of these zoonotic pathogens.

Prevalence of Ehrlichia chaffeensis and Ehrlichia ewingii in Amblyomma americanum and Dermacentor variabilis collected from southeastern Virginia, 2010-2011.

Amblyomma americanum is the most commonly encountered tick species in southeastern Virginia, representing approximately 95% of the human-biting tick population in this area. Here we investigated the prevalence of Ehrlichia chaffeensis and Ehrlichia ewingii in questing Amblyomma americanum and Dermacentor variabilis ticks collected from multiple sites in southeastern Virginia from 2010 to 2011. Although both Ehrlichia species were detected in Amblyomma americanum, no evidence of either pathogen was found in Dermacentor variabilis. Prevalence of E. chaffeensis varied by location, ranging from 0 to 5.08% among Amblyomma americanum populations. Ehrlichia ewingii prevalence was slightly higher, ranging from 0 to 8.20% among A. americanum populations. We conclude that both pathogens are established in southeastern Virginia A. americanum populations, and that although there are no apparent temporal trends in Ehrlichia prevalence, there is variation among locations, suggesting the potential for disease hotspots.