Serological evidence of Ehrlichia minasensis infection in Brazilian dogs.

Ehrlichia spp. are important tick-borne pathogens of animals in Brazil, and Ehrlichia canis is the most prevalent species infecting dogs. Moreover, Ehrlichia minasensis has also recently been identified as a novel ehrlichial agent that infects cattle in Brazil. The objective of this study was to determine whether dogs could be infected by E. minasensis. To investigate this possibility, sera (n = 429) collected from dogs in the Pantanal region were retrospectively analyzed for the presence of antibodies against E. canis and E. minasensis. Canine sera were screened by two isolates of E. canis in indirect immunofluorescence assay (IFA) and the majority (n = 298; 69.4%) had antibodies with endpoint titers ranging from 80 to 327,680. In order to further confirm E. canis-specific antibodies, IFA positive sera were analyzed by ELISA using E. canis-specific peptides (i.e. TRP19 and TRP36 US/BR/CR), which detected E. canis antibodies in 80.2% (239/298) of the dog sera. Fifty-nine (13.7%) samples had detectable antibodies to E. canis by IFA but were negative by E. canis peptide ELISA. These sera were then tested by E. minasensis IFA (Cuiaba strain) as antigen and 67.8% (40/59) were positive (titers ranging from 80 to 20,480). Eleven sera had antibody titers against E. minasensis at least two-fold higher than observed for E. canis and suggests that these dogs were previously infected with E. minasensis. The results of the present study suggest that multiple ehrlichial agents infect dogs in Brazil, which highlights the need to consider different Ehrlichia spp. in Brazilian dogs, particularly in areas where dogs are frequently exposed to multiple tick species. This investigation is the first to provide serologic evidence of E. minasensis infection in dogs from Brazil.

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.

Diversity unearthed by the estimated molecular phylogeny and ecologically quantitative characteristics of uncultured Ehrlichia bacteria in Haemaphysalis ticks, Japan.

Ehrlichia species are obligatory intracellular bacteria transmitted by arthropods, and some of these species cause febrile diseases in humans and livestock. Genome sequencing has only been performed with cultured Ehrlichia species, and the taxonomic status of such ehrlichiae has been estimated by core genome-based phylogenetic analysis. However, many uncultured ehrlichiae exist in nature throughout the world, including Japan. This study aimed to conduct a molecular-based taxonomic and ecological characterization of uncultured Ehrlichia species or genotypes from ticks in Japan. We first surveyed 616 Haemaphysalis ticks by p28-PCR screening and analyzed five additional housekeeping genes (16S rRNA, groEL, gltA, ftsZ, and rpoB) from 11 p28-PCR-positive ticks. Phylogenetic analyses of the respective genes showed similar trees but with some differences. Furthermore, we found that V1 in the V1-V9 regions of Ehrlichia 16S rRNA exhibited the greatest variability. From an ecological viewpoint, the amounts of ehrlichiae in a single tick were found to equal approx. 6.3E+3 to 2.0E+6. Subsequently, core-partial-RGGFR-based phylogenetic analysis based on the concatenated sequences of the five housekeeping loci revealed six Ehrlichia genotypes, which included potentially new Ehrlichia species. Thus, our approach contributes to the taxonomic profiling and ecological quantitative analysis of uncultured or unidentified Ehrlichia species or genotypes worldwide.

The co-infection with Ehrlichia minasensis, Anaplasma marginale and Anaplasma platys is not associated with anemia in beef cattle in the Brazilian Pantanal.

The Anaplasmataceae family is composed of obligatory intracellular Gram-negative bacteria transmitted by arthropod vectors. In Brazil, with the exception of Anaplasma marginale, little is known about the occurrence of other Anaplasma and Ehrlichia species infecting cattle. The present study aimed at investigating the occurrence of Anaplasma spp. and Ehrlichia spp. in beef cattle (Bos indicus) sampled in the Brazilian Pantanal, an area prone to periodic flooding and endemic for bovine trypanosomiasis. Blood samples from 400 cattle were collected and screened by PCR assays based on rrs and dsb genes from Anaplasma spp. and Ehrlichia spp., respectively. Positive samples for Anaplasma spp. were subjected to qPCR assays based on the msp-2 gene and nPCR based on the groEL gene. As a result, 4.75% (19/400) and 48.12% (167/347) were positive for Anaplasma platys and Ehrlichia minasensis, respectively. Besides, positivity of 56.75% (227/400) for A. marginale and seropositivity of 90.75% (363/400) for Trypanosoma vivax were found. A high rate of co-infection was observed (67.25%), from which the co-infection by A. marginale and E. minasensis was more frequently found in calves than cows. Interestingly, none of the animals presenting co-infection showed anemia or other clinical signs. The present study showed, for the first time, the occurrence of A. platys and E. minasensis in beef cattle in the southern Pantanal, as well as a high rate of co-infection by A. marginale, E. minasensis and T. vivax in the sampled animals.

Effect of rodent density on tick and tick-borne pathogen populations: consequences for infectious disease risk.

BACKGROUND: Rodents are considered to contribute strongly to the risk of tick-borne diseases by feeding Ixodes ricinus larvae and by acting as amplifying hosts for pathogens. Here, we tested to what extent these two processes depend on rodent density, and for which pathogen species rodents synergistically contribute to the local disease risk, i.e. the density of infected nymphs (DIN). METHODS: In a natural woodland, we manipulated rodent densities in plots of 2500 m2 by either supplementing a critical food source (acorns) or by removing rodents during two years. Untreated plots were used as controls. Collected nymphs and rodent ear biopsies were tested for the presence of seven tick-borne microorganisms. Linear models were used to capture associations between rodents, nymphs, and pathogens. RESULTS: Investigation of data from all plots, irrespective of the treatment, revealed a strong positive association between rodent density and nymphal density, nymphal infection prevalence (NIP) with Borrelia afzelii and Neoehrlichia mikurensis, and hence DIN's of these pathogens in the following year. The NIP, but not the DIN, of the bird-associated Borrelia garinii, decreased with increasing rodent density. The NIPs of Borrelia miyamotoi and Rickettsia helvetica were independent of rodent density, and increasing rodent density moderately increased the DINs. In addition, NIPs of Babesia microti and Spiroplasma ixodetis decreased with increasing rodent density, which had a non-linear association with DINs of these microorganisms. CONCLUSIONS: A positive density dependence for all rodent- and tick-associated tick-borne pathogens was found, despite the observation that some of them decreased in prevalence. The effects on the DINs were variable among microorganisms, more than likely due to contrasts in their biology (including transmission modes, host specificity and transmission efficiency). The strongest associations were found in rodent-associated pathogens that most heavily rely on horizontal transmission. Our results draw attention to the importance of considering transmission mode of a pathogen while developing preventative measures to successfully reduce the burden of disease.

Molecular identification of Ehrlichia, Anaplasma, Babesia and Theileria in African elephants and their ticks.

Although historical records indicate the presence of Ehrlichia and Babesia in African elephants, not much is known about their prevalence and diversity in elephants and their ticks, Amblyomma thollonii and Rhipicephalus humeralis. We amplified and sequenced the hypervariable V4 region of the 18S rRNA gene of Babesia and Theileria and the heat shock protein gene (groEL) of Ehrlichia/Anaplasma in DNA extracted from elephant blood (n = 104) and from elephant ticks (n = 52). Our results showed that the African elephants were infected with a novel Babesia spp. while A. thollonii was infected with Theileria bicornis and Theileria cf. velifera. This is the first record of T. bicornis; a protozoan that is linked to fatal infection in rhinoceros in a tick. Elephants and their ticks were all infected with a species of Ehrlichia like that identified in Japanese deer. The prevalence of Babesia spp., Theileria spp. and Ehrlichia spp. in ticks was higher than that of their elephant hosts. About 13.5% of elephants were positive for Theileria or Babesia while 51% of A. thollonii ticks and 27% of R. humeralis ticks were positive for Theileria or Babesia. Moreover, 5.8% of elephants were positive for Ehrlichia or Anaplasma compared to 19.5% in A. thollonii and 18% in R. humeralis. There was no association between the positive result in ticks and that of their elephant hosts for either Babesia spp., Theileria spp. or Ehrlichia spp. Our study reveals that the African elephants are naturally infected with Babesia spp and Ehrlichia spp and opens up an opportunity for further studies to determine the role of elephant as reservoirs of tick-borne pathogens, and to investigate their potential in spreading these pathogens as they range extensively. The presence of T. bicornis in A. thollonii also suggests a need for experiments to confirm its vector competence.

A molecular survey of Anaplasma, Ehrlichia, Bartonella and Theileria in ticks collected from southeastern China.

To investigate the prevalence of Anaplasma, Ehrlichia, Bartonella and Theileria, we collected ticks from small mammals in six counties of Zhejiang Province in southeastern China. Polymerase chain reaction (PCR) amplification was performed to test Anaplasma, Ehrlichia, Bartonella and Theileria in tick samples. Positive PCR products were sequenced and then compared with previously published sequences deposited in GenBank using BLAST. About 292 adult ticks were captured and the dominant tick species were Ixodes sinensis and Haemaphysalis longicornis. Overall, 34 ticks (11.6%) were tested positive for at least one pathogen of Anaplasma, Ehrlichia, Bartonella and Theileria. Rates of PCR-positivity to Anaplasma, Ehrlichia, Bartonella and Theileria were 5.5, 1.7, 2.4 and 2.4%, respectively. Positive rates of Anaplasma, Bartonella and Theileria were significantly different among ticks of different species. Prevalence of Anaplasma and Theileria varied significantly among ticks of different counties. Anaplasma, Ehrlichia, Bartonella and Theileria were widely prevalent in ticks in Zhejiang Province suggesting other tick-borne pathogens should also be suspected if patients had history of tick bites.

Novel Ehrlichia sp. detected in Magellanic penguins (Sphenicus magellanicus) and in the seabird tick Ixodes uriae from Magdalena Island, southern Chile.

Ehrlichia spp. are obligatory intracellular microorganisms that infect hematopoietic, endothelial or blood cells of mammals. Ticks are the only vectors of these agents in nature. To date, the role of birds and their associated ticks as reservoirs of ehrlichiae remains almost unexplored. In this study, we performed a molecular screening for bacteria of Anaplasmataceae family in samples of spleen (n = 72) and lung (n = 17), recovered from 72 carcasses of Magellanic penguins (Spheniscus magellanicus) in Brazil and Chile. One apparently unengorged tick (Ixodes uriae) was also collected while wandering upon one of the carcasses and submitted to molecular analyses as well. Through conventional and nested PCR protocols three genes (16S rRNA, dsb and groEL) of a new Ehrlichia sp. were partially characterized upon organs of three penguins and in the tick coming from Magdalena Island (Chile). First matches after BLASTn comparisons showed that our sequences share 99.4% (16S rRNA), 94.6% (groEL) and 79.3% (dsb) of identity with "Candidatus Ehrlichia ornithorhynchi", Ehrlichia sp. NS101 and Ehrlichia canis CCZ, respectively. Matrixes of genetic distance including other representatives of the Ehrlichia genus point a 99.4%, 94.0%, and 80.0% of identity with 16S rRNA, groEL and dsb genes from Ehrlichia sp. It25, Ehrlichia sp. NS101, and Ehrlichia chaffeensis San Louis, respectively. A Bayesian phylogenetic analysis of Anaplasmataceae 16S rRNA gene places the detected Ehrlichia sp. into a group with Ehrlichia sp. BAT and Ehrlichia sp. Natal. Although depicting different topologies, Bayesian unrooted phylogenetic trees constructed for groEL and dsb genes position this Ehrlichia sp. into well-supported branches, which reinforces the finding of a new taxon. For the moment, any pathogenic effect of this new Ehrlichia sp. on penguins is still unknown. However, this fact becomes important to assess from a conservation point of view since populations of Magellanic penguins are currently threatened and in an ongoing decrease.

Ehrlichia Isolate from a Minnesota Tick: Characterization and Genetic Transformation.

Ehrlichia muris subsp. eauclairensis is recognized as the etiological agent of human ehrlichiosis in Minnesota and Wisconsin. We describe the culture isolation of this organism from a field-collected tick and detail its relationship to other species of Ehrlichia The isolate could be grown in a variety of cultured cell lines and was effectively transmitted between Ixodes scapularis ticks and rodents, with PCR and microscopy demonstrating a broad pattern of dissemination in arthropod and mammalian tissues. Conversely, Amblyomma americanum ticks were not susceptible to infection by the Ehrlichia Histologic sections further revealed that the wild-type isolate was highly virulent for mice and hamsters, causing severe systemic disease that was frequently lethal. A Himar1 transposase system was used to create mCherry- and mKate-expressing EmCRT mutants, which retained the ability to infect rodents and ticks.IMPORTANCE Ehrlichioses are zoonotic diseases caused by intracellular bacteria that are transmitted by ixodid ticks. Here we report the culture isolation of bacteria which are closely related to, or the same as the Ehrlichia muris subsp. eauclairensis, a recently recognized human pathogen. EmCRT, obtained from a tick removed from deer at Camp Ripley, MN, is the second isolate of this subspecies described and is distinctive in that it was cultured directly from a field-collected tick. The isolate's cellular tropism, pathogenic changes caused in rodent tissues, and tick transmission to and from rodents are detailed in this study. We also describe the genetic mutants created from the EmCRT isolate, which are valuable tools for the further study of this intracellular pathogen.

Bacterial and protozoal pathogens found in ticks collected from humans in Corum province of Turkey.

BACKGROUND: Tick-borne diseases are increasing all over the word, including Turkey. The aim of this study was to determine the bacterial and protozoan vector-borne pathogens in ticks infesting humans in the Corum province of Turkey. METHODOLOGY/PRINCIPAL FINDINGS: From March to November 2014 a total of 322 ticks were collected from patients who attended the local hospitals with tick bites. Ticks were screened by real time-PCR and PCR, and obtained amplicons were sequenced. The dedected tick was belonging to the genus Hyalomma, Haemaphysalis, Rhipicephalus, Dermacentor and Ixodes. A total of 17 microorganism species were identified in ticks. The most prevalent Rickettsia spp. were: R. aeschlimannii (19.5%), R. slovaca (4.5%), R. raoultii (2.2%), R. hoogstraalii (1.9%), R. sibirica subsp. mongolitimonae (1.2%), R. monacensis (0.31%), and Rickettsia spp. (1.2%). In addition, the following pathogens were identified: Borrelia afzelii (0.31%), Anaplasma spp. (0.31%), Ehrlichia spp. (0.93%), Babesia microti (0.93%), Babesia ovis (0.31%), Babesia occultans (3.4%), Theileria spp. (1.6%), Hepatozoon felis (0.31%), Hepatozoon canis (0.31%), and Hemolivia mauritanica (2.1%). All samples were negative for Francisella tularensis, Coxiella burnetii, Bartonella spp., Toxoplasma gondii and Leishmania spp. CONCLUSIONS/SIGNIFICANCE: Ticks in Corum carry a large variety of human and zoonotic pathogens that were detected not only in known vectors, but showed a wider vector diversity. There is an increase in the prevalence of ticks infected with the spotted fever group and lymphangitis-associated rickettsiosis, while Ehrlichia spp. and Anaplasma spp. were reported for the first time from this region. B. microti was detected for the first time in Hyalomma marginatum infesting humans. The detection of B. occultans, B. ovis, Hepatozoon spp., Theileria spp. and Hemolivia mauritanica indicate the importance of these ticks as vectors of pathogens of veterinary importance, therefore patients with a tick infestation should be followed for a variety of pathogens with medical importance.

Anaplasma, Ehrlichia and Rickettsia species infections in cats: European guidelines from the ABCD on prevention and management

OVERVIEW: Anaplasma species, Ehrlichia species and Rickettsia species are vector-borne pathogens infecting a wide variety of mammals, but causing disease in very few of them. Infection in cats: Anaplasma phagocytophilum is the most important feline pathogen among these rickettsial organisms, and coinfections are possible. Little information is available on the pathogenesis of these agents in cats. Clinical signs are usually reported soon after tick infestation. They are mostly non-specific, consisting of fever, anorexia and lethargy. Joint pain may occur. Infection in humans: Some rickettsial species ( A phagocytophilum, Ehrlichia chaffeensis, Ehrlichia ewingii, Rickettsia conorii, Rickettsia rickettsii, Rickettsia felis, Rickettsia typhi and Candidatus Neoehrlichia mikurensis) are of zoonotic concern. Direct contact with cat saliva should be avoided because of potential contamination by R felis. Infected cats are 'sentinels' of the presence of rickettsial pathogens in ticks and fleas in a given geographical area, and they signal a risk for people exposed to vectors.

Experimental evaluation of Peromyscus leucopus as a reservoir host of the Ehrlichia muris-like agent.

BACKGROUND: The Ehrlichia muris-like agent (EMLA) is a newly recognized human pathogen in the North Central United States. Although blacklegged ticks (Ixodes scapularis) have been identified as capable vectors, wild reservoirs have not yet been established for EMLA. As key hosts for I. scapularis, white-footed mice (Peromyscus leucopus) are important reservoirs for various tick-borne pathogens, and potentially, for EMLA. The objective of this study was to evaluate reservoir competence in P. leucopus using a natural vector. RESULTS: Mice acquired EMLA infection from feeding ticks and were able to transmit infection to naïve ticks. Transmission between simultaneously feeding tick life stages was also demonstrated. Infections in mice were acute and severe, with systemic dissemination. Limited host survival and clearance of infection among survivors resulted in a narrow interval where EMLA could be acquired by feeding ticks. CONCLUSIONS: Peromyscus leucopus is a competent reservoir of EMLA and likely to play a role in its enzootic transmission cycle. The duration and severity of EMLA infection in these hosts suggests that tick phenology is a critical factor determining the geographic distribution of EMLA in North America.

Evaluation of Gulf Coast Ticks (Acari: Ixodidae) for Ehrlichia and Anaplasma Species.

Amblyomma maculatum Koch (the Gulf Coast tick) is an aggressive, human-biting ixodid tick distributed throughout much of the southeastern United States and is the primary vector for Rickettsia parkeri, an emerging human pathogen. Amblyomma maculatum has diverse host preferences that include white-tailed deer, a known reservoir for Ehrlichia and Anaplasma species, including the human pathogens E. ewingii and E. chaffeensis. To examine more closely the potential role of A. maculatum in the maintenance of various pathogenic Ehrlichia and Anaplasma species, we screened DNA samples from 493 questing adult A. maculatum collected from six U.S. states using broad-range Anaplasmataceae and Ehrlichia genus-specific PCR assays. Of the samples tested, four (0.8%) were positive for DNA of Ehrlichia ewingii, one (0.2%) was positive for Anaplasma platys, and one (0.2%) was positive for a previously unreported Ehrlichia species closely related to Ehrlichia muris and an uncultivated Ehrlichia species from Haemaphysalis longicornis ticks in Japan. No ticks contained DNA of Ehrlichia chaffeensis, Ehrlichia canis, the Panola Mountain Ehrlichia, or Anaplasma phagocytophilum. This is the first identification of E. ewingii, A. platys, and the novel Ehrlichia in questing Gulf Coast ticks; nonetheless the low prevalence of these agents suggests that A. maculatum is not likely an important vector of these zoonotic pathogens.

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.

Tick-borne pathogens of zoonotic and veterinary importance in Nigerian cattle.

BACKGROUND: Ticks and tick-borne diseases undermine cattle fitness and productivity in the whole of sub-Saharan Africa, including Nigeria. In this West African country, cattle are challenged by numerous tick species, especially during the wet season. Consequently, several TBDs are known to be endemic in Nigerian cattle, including anaplasmosis, babesiosis, cowdriosis and theilerioris (by Theileria mutans and Theileria velifera). To date, all investigations on cattle TBDs in Nigeria have been based on cytological examinations and/or on serological methods. This study aimed to ascertain the occurrence of tick-borne pathogens of veterinary and zoonotic importance in cattle in Nigeria using molecular approaches. METHODS: In October 2008, 704 whole blood samples were collected from indigenous cattle in the Plateau State, Nigeria. Analysis for tick-borne pathogens was conducted by means of PCR-based reverse line blotting (RLB) and sequencing targeting a panel of five genera of microorganisms (i.e. Babesia, Theileria, Anaplasma, Ehrlichia and Rickettsia spp.). RESULTS: In total, 561/704 (82.6%) animals were found infected, with 465 (69.6%) of them being infected by two or more microorganisms, with up to 77 possible combinations of pathogens detected. Theileria mutans was the most prevalent microorganism (66.3%), followed by Theileria velifera (52.4%), Theileria taurotragi (39.5%), Anaplasma marginale (39.1%), Anaplasma sp. (Omatjenne) (34.7%), Babesia bigemina (7.9%), Anaplasma centrale (6.3%), Anaplasma platys (3.9%), Rickettsia massiliae (3.5%), Babesia bovis (2.0%) and Ehrlichia ruminantium (1.1%). Calves were found significantly less infected than juvenile and adult cattle. CONCLUSIONS: This study provides updated, molecular-based information on cattle TBDs in Nigeria. The molecular approach employed allowed the diagnosis of numerous positive cases including carrier statuses, multiple infections and novel pathogen detections within the indigenous cattle population. Moreover, the RLB method here described enabled the detection of veterinary agents not only pertaining to bovine health, including also those of zoonotic importance. The high prevalence recorded for T. mutans, T. velifera, A. marginale, T. taurotragi and Anaplasma sp. (Omatjenne), suggests they may be endemically established in Nigeria, whereas the lower prevalence recorded for other microorganisms (i.e. A. centrale and B. bovis) highlights a less stable epidemiological scenario, requiring further investigations.

Co-Feeding Transmission of the Ehrlichia muris-Like Agent to Mice (Mus musculus).

The Ehrlichia muris-like agent (EMLA) is a newly recognized human pathogen found in Wisconsin and Minnesota. Ecological investigations have implicated both the blacklegged tick, Ixodes scapularis, and the white-footed mouse, Peromyscus leucopus, as playing roles in the maintenance of EMLA in nature. The work presented here shows that I. scapularis is an efficient vector of EMLA in a laboratory mouse model, but that Dermacentor variabilis, another frequent human biting tick found in EMLA endemic areas, is not. Additionally, I. scapularis larvae are able to acquire EMLA through co-feeding with infected nymphs. As EMLA only persists in mouse blood for a relatively short period of time, co-feeding transmission may play an important role in the maintenance of EMLA in ticks, and subsequently may play a role in limiting the geographic distribution of this pathogen in areas where co-feeding of larvae and nymphs is less common.

Tick-borne rickettsial pathogens in questing ticks, removed from humans and animals in Mexico.

Tick-borne rickettsial diseases (TBRD) are commonly encountered in medical and veterinary clinical settings. The control of these diseases is difficult, requiring disruption of a complex transmission chain involving a vertebrate host and ticks. The geographical distribution of the diseases is related to distribution of the vector, which is an indicator of risk for the population. A total of 1107 were collected by tick drag from forests, ecotourism parks and hosts at 101 sites in 22 of the 32 states of Mexico. Collected ticks were placed in 1.5 mL cryovials containing 70% ethanol and were identified to species. Ticks were pooled according to location/host of collection, date of collection, sex, and stage of development. A total of 51 ticks were assayed by polymerase chain reaction (PCR) to confirm species identification using morphological methods. A total of 477 pools of ticks were assayed using PCR techniques for selected tick-borne pathogens. Anaplasma phagocytophilum was the most commonly detected pathogen (45 pools), followed by, Ehrlichia (E.) canis (42), Rickettsia (R.) rickettsii (11), E. chaffeensis (8), and R. amblyommii (1). Rhipicephalus sanguineus was the tick most frequently positive for selected pathogens. Overall, our results indicate that potential tick vectors positive for rickettsial pathogens are distributed throughout the area surveyed in Mexico.

Note on Ehrlichia chaffeensis, Ehrlichia ewingii, and "Borrelia lonestari" infection in lone star ticks (Acari: Ixodidae), Nebraska, USA.

The lone star tick, Amblyomma americanum (L.) (Acari: Ixodidae), is established in southeastern Nebraska yet the prevalence of tick-associated microorganisms is not known. An initial PCR-based analysis for Ehrlichia chaffeensis, Ehrlichia ewingii, and Borrelia infection in host-seeking adult ticks collected in southeast Nebraska was conducted. A total of 251 adult ticks collected in six sites in southeast Nebraska were tested. E. chaffeensis, E. ewingii, and Borrelia spp. were present, and the prevalence of each was approximately 1.6%. This study demonstrates that Ehrlichia spp. are present in Nebraska lone star tick populations.

Bacterial Profiling Reveals Novel "Ca. Neoehrlichia", Ehrlichia, and Anaplasma Species in Australian Human-Biting Ticks.

In Australia, a conclusive aetiology of Lyme disease-like illness in human patients remains elusive, despite growing numbers of people presenting with symptoms attributed to tick bites. In the present study, we surveyed the microbial communities harboured by human-biting ticks from across Australia to identify bacteria that may contribute to this syndrome. Universal PCR primers were used to amplify the V1-2 hyper-variable region of bacterial 16S rRNA genes in DNA samples from individual Ixodes holocyclus (n = 279), Amblyomma triguttatum (n = 167), Haemaphysalis bancrofti (n = 7), and H. longicornis (n = 7) ticks. The 16S amplicons were sequenced on the Illumina MiSeq platform and analysed in USEARCH, QIIME, and BLAST to assign genus and species-level taxonomies. Nested PCR and Sanger sequencing were used to confirm the NGS data and further analyse novel findings. All 460 ticks were negative for Borrelia spp. by both NGS and nested PCR analysis. Two novel "Candidatus Neoehrlichia" spp. were identified in 12.9% of I. holocyclus ticks. A novel Anaplasma sp. was identified in 1.8% of A. triguttatum ticks, and a novel Ehrlichia sp. was identified in both A. triguttatum (1.2%) ticks and a single I. holocyclus (0.6%) tick. Further phylogenetic analysis of novel "Ca. Neoehrlichia", Anaplasma and Ehrlichia based on 1,265 bp 16S rRNA gene sequences suggests that these are new species. Determining whether these newly discovered organisms cause disease in humans and animals, like closely related bacteria do abroad, is of public health importance and requires further investigation.

A Tick Vector Transmission Model of Monocytotropic Ehrlichiosis.

BACKGROUND: Ehrlichioses are emerging, tick-borne diseases distributed worldwide. Previously established animal models use needle inoculation as a mode of infection; however, there is limited representation of natural transmission in artificially inoculated models compared with transmission by the tick vector. The objective of this study was to develop a tick vector transmission animal model of ehrlichial infection using a human pathogen, Ehrlichia muris-like agent (EMLA). METHODS: Ixodes scapularis larvae were fed on EMLA-infected mice, and after molting, infected nymphs were used to infest naive animals. RESULTS: Ehrlichiae were acquired by 90%-100% of feeding larvae. The majority of animals fed upon by infected nymphs developed sublethal infection with 27% lethality. Bacteria disseminated to all tissues tested with greatest bacterial loads in lungs, but also spleen, lymph nodes, liver, kidneys, brain, and bone marrow. Numerous foci of cellular infiltration, mitoses, and hepatocellular death were observed in liver. Mice infected by tick transmission developed higher antiehrlichial antibody levels than needle-inoculated animals. Tick-feeding-site reactions were observed, but there was no observed difference between animals infested with infected or uninfected ticks. CONCLUSIONS: For the first time we were able to develop a tick transmission model with an Ehrlichia that is pathogenic for humans.