Agent Of Ehrlichiosis Research Articles

Immuno- and expression analysis of Ehrlichia canis immunoreactive proteins

Ehrlichia canis is the primary etiologic agent of canine monocytic ehrlichiosis, a serious and sometimes fatal hemorrhagic disease of dogs. Diagnosis of E. canis infection is often retrospectively confirmed by serologic detection of antibodies by immunofluorescent microscopy. Our laboratory previously identified numerous major immunoreactive proteins with species-specific linear antibody epitopes that are useful for immunodiagnosis of CME. More recently, we have defined the entire antibody-reactive immunome of E. canis, substantially increasing the number of major immunoreactive proteins known to exist. In this study, we analyzed and compared seven recently identified antibody reactive E. canis proteins with established diagnostic antigens including tandem repeat proteins TRP19, TRP36 and TRP140 and observed comparable immunoreactivity. Many of these proteins were conserved in different E. canis strains. Multiple linear antibody epitopes were mapped in a highly conserved TRP (Ecaj_0126), including within the tandem repeat domain. Temporal antibody responses were examined, and multiple proteins reacted with antibodies in sera as early as 21 days post experimental infection. Host-specific expression of the proteins was examined which revealed that some proteins exhibited higher expression in mammalian cells, while others in tick cells. This study has identified new immunodiagnostic candidates that exhibit different host expression patterns, information which may be useful for developing ultrasensitive immunodiagnostics and effective vaccines for CME.

RipE expression correlates with high ATP levels in Ehrlichia, which confers resistance during the extracellular stage to facilitate a new cycle of infection.

Ehrlichiosis is a potentially life-threatening disease caused by infection with the obligatory intracellular bacteria Ehrlichia species. Ehrlichia japonica infection of mice provides an animal model of ehrlichiosis as it recapitulates full-spectrum and lethal ehrlichiosis in humans. The E. japonica transposon mutant of EHF0962, which encodes a previously uncharacterized hypothetical protein, is attenuated in both infection and virulence in mice. EHF0962 was hence named here as resistance-inducing protein of Ehrlichia (RipE). Using this ΔripE mutant, we studied how RipE protein contributes to Ehrlichia pathogenesis. Ehrlichia species have an intracellular developmental cycle and a brief extracellular stage to initiate a new cycle of infection. Majority of RipE proteins were expressed on the surface of the smaller infectious dense-core stage of bacteria. Extracellular ΔripE E. japonica contained significantly less adenosine triphosphate (ATP) and lost infectivity more rapidly in culture compared with wild-type (WT) E. japonica. Genetic complementation in the ΔripE mutant or overexpression of ripE in WT E. japonica significantly increased bacterial ATP levels, and RipE-overexpressing E. japonica was more virulent in mice than WT E. japonica. RipE is conserved among Ehrlichia species. Immunization of mice with recombinant RipE induced an in vitro infection-neutralizing antibody, significantly prolonged survival time after a lethal dose of E. japonica challenge, and cross-protected mice from infection by Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis. Our findings shed light on the extracellular stage of Ehrlichia, highlighting the importance of RipE and ATP levels in Ehrlichia for extracellular resistance and the next cycle of infection. Thus, RipE is a critical Ehrlichia protein for infection as such can be a potential vaccine target for ehrlichiosis.

The geographic limits and life history of the tropical brown dog tick, Rhipicephalus linnaei (Audouin, 1826), in Australia with notes on the spread of Ehrlichia canis

The tropical brown dog tick, Rhipicephalus linnaei, is a tick of much medical, veterinary, and zoonotic importance. This tick has a nearly world-wide distribution due to its ability to survive and propagate in kennels and houses. Rhipicephalus linnaei is the vector of Ehrlichia canis, the causative agent of canine monocytic ehrlichiosis, an often debilitating disease of canids and, occasionally, humans. To prevent incursion of E. canis into Australia, dogs entering Australia have been required to have a negative immunofluorescence antibody test for E. canis. In May 2020 however, E. canis was detected in Western Australia. The detection of E. canis in Australia prompted disease investigation and concerted surveillance for R. linnaei and E. canis in regions across Australia. These investigations revealed that R. linnaei was established far beyond the previously recognised geographic range limits of this tick. In the present paper, using records from various collections, published data, and data from our network of veterinarian collaborators and colleagues, we update the current geographic range of R. linnaei in Australia. Our analyses revealed that the geographic range of R. linnaei in Australia is much wider than was previously supposed, particularly in Western Australia, and in South Australia. We also map, for the first time, where E. canis has been detected in Australia. Last, we discuss the possible routes of incursion and subsequently the factors which may have aided the spread of E. canis in Australia which led to the establishment of this pathogen in Australia.

Correction for Zhi et al., "Cloning and Expression of the 44-Kilodalton Major Outer Membrane Protein Gene of the Human Granulocytic Ehrlichiosis Agent and Application of the Recombinant Protein to Serodiagnosis".

Correction for Zhi et al., "Cloning and Expression of the 44-Kilodalton Major Outer Membrane Protein Gene of the Human Granulocytic Ehrlichiosis Agent and Application of the Recombinant Protein to Serodiagnosis".

Multiple Ehrlichia chaffeensis genes critical for persistent infection in a vertebrate host are identified as nonessential for its growth in the tick vector; Amblyomma americanum.

Ehrlichia chaffeensis is a tick-transmitted monocytic ehrlichiosis agent primarily causing the disease in people and dogs. We recently described the development and characterization of 55 random mutations in E. chaffeensis, which aided in defining the critical nature of many bacterial genes for its growth in a physiologically relevant canine infection model. In the current study, we tested 45 of the mutants for their infectivity ability to the pathogen's tick vector; Amblyomma americanum. Four mutations resulted in the pathogen's replication deficiency in the tick, similar to the vertebrate host. Mutations causing growth defects in both vertebrate and tick hosts included in genes coding for a predicted alpha/beta hydrolase, a putative dicarboxylate amino acid:cation symporter, a T4SS protein, and predicted membrane-bound proteins. Three mutations caused the bacterial defective growth only in the tick vector, which represented putative membrane proteins. Ten mutations causing no growth defect in the canine host similarly grew well in the tick vector. Mutations in 28 genes/genomic locations causing E. chaffeensis growth attenuation in the canine host were recognized as non-essential for its growth in the tick vector. The tick non-essential genes included genes coding for many metabolic pathway- and outer membrane-associated proteins. This study documents novel vector- and host-specific differences in E. chaffeensis for its functional gene requirements.

CtrA activates the expression of glutathione S-transferase conferring oxidative stress resistance to Ehrlichia chaffeensis.

Ehrlichia chaffeensis, the causative agent of human monocytic ehrlichiosis (HME), is a Gram-negative obligatory intracellular bacterium, which infects and multiplies in human monocytes and macrophages. Host immune cells produce reactive oxygen species (ROS) to eliminate E. chaffeensis upon infection. E. chaffeensis global transcriptional regulator CtrA activates the expression of GshA and GshB to synthesize glutathione (GSH), the most potent natural antioxidant, upon oxidative stress to combat ROS damage. However, the mechanisms exploited by E. chaffeensis to utilize GSH are still unknown. Here, we found that in E. chaffeensis CtrA activated the expression of glutathione S-transferase (GST) upon oxidative stress, and E. chaffeensis GST utilizes GSH to eliminate ROS and confers the oxidative stress resistance to E. chaffeensis. We found that CtrA bound to the promoter regions of 211 genes, including gst, in E. chaffeensis using chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq). Recombinant E. chaffeensis CtrA directly bound to the gst promoter region determined with electrophoretic mobility shift assay (EMSA), and activated the gst expression determined with reporter assay. Recombinant GST showed GSH conjugation activity towards its typical substrate 2,4-dinitrochlorobenzene (CDNB) in vitro and peptide nucleic acid (PNA) transfection of E. chaffeensis, which can knock down the gst transcription level, reduced bacterial survival upon oxidative stress. Our results demonstrate that E. chaffeensis CtrA regulates GSH utilization, which plays a critical role in resistance to oxidative stress, and aid in the development of new therapeutics for HME.

Seroplrevalence of <i>Anaplasma phagocytophilum</i> and <i>Ehrlichia</i> sp. among people affected by tick bites

Background. In spring and summer, the population of the Baikal region regularly comes into contact with the pathogens transmitted through the bites of ixodid ticks. In the Center for Diagnosis and Prevention of Tick-Borne Infections (Irkutsk, Russian Federation), we annually detect anaplasmas of the Anaplasma phagocytophilum species, as well as Ehrlichia chaffeensis/E. muris in both ixodid ticks and blood samples from people who have been bitten by ticks. At the same time, there are no data in open sources on the incidence of human granulocytic anaplasmosis and human monocytic ehrlichiosis in the Baikal region. Currently, there is very little information on the studies of intensity of the immune response to anaplasmas and ehrlichia in people living in the surveyed area, although this information is critical for assessing the frequency of contacts and the risk of infection of people in a territory endemic for tick-borne infections. The aim. To update information on the presence and prevalence of specific immunoglobulins M and G to A. phagocytophilum and Ehrlichia sp. among the population of the Irkutsk Region affected by tick bites. Materials and methods. In total, 204 samples of blood serum from the residents of the Irkutsk Region who were registered to be bitten by ticks were analyzed for the presence of IgM and IgG to human monocytic ehrlichiosis and human granulocytic anaplasmosis agents. Results. IgG to A. phagocytophilum were found in 9 samples, IgG to E. chaffeensis/E. muris – in 1 sample; no IgM to both pathogens were found in any sample. Conclusions. The results obtained indicate regular infection of the population with anaplasmas and ehrlichia which is a testifies to the existence of active natural foci of human monocytic ehrlichiosis and human granulocytic anaplasmosis in the Baikal region. To clarify the real epidemic role of these infections, a detailed study of the immune status is required both among healthy individuals and among patients with symptoms of an infectious disease.

Serologic Evidence of Human Exposure to Ehrlichiosis Agents in Japan.

In retrospective analyses, we report 3 febrile patients in Japan who had seroconversion to antibodies against Ehrlichia chaffeensis antigens detected by using an immunofluorescence and Western blot. Our results provide evidence of autochthonous human ehrlichiosis cases and indicate ehrlichiosis should be considered a potential cause of febrile illness in Japan.

Molecular Investigation and Phylogenetic Analysis of Ehrlichia canis in Dogs in Siirt, Turkey

Ehrlichia canis is the primary etiologic agent of canine monocytic ehrlichiosis, a tick-transmitted disease of dogs. The aim of this study is to molecularly investigate the presence of E. canis and to reveal its prevalence in dogs in Siirt province. The animal material of the study is consisted of a total of 82 dogs. A region of the 16S ribosomal RNA gene of E. canis was targeted for PCR amplification. As a result of the conducted Nested-PCR, positivity was detected at the rate of 10.53% (4/38) in male dogs and 13.64% (6/44) in females, and Ehrlichia canis specific bands of size 389 bp were obtained in 10 (12.20%) dogs in total. The phylogenetic tree was constructed with the Maximum Likelihood (MCL) method, The nucleotide sequence was registered in the NCBI GenBank database with access numbers OK331365.1-OK331366. Early detection of the disease by means of hematological, serological, or molecular tests is very important in terms of prognosis. More studies should be performed to determine vector-disease relationships in this region about ticks that vector the disease.

Evaluating EcxR for Its Possible Role in Ehrlichia chaffeensis Gene Regulation.

Ehrlichia chaffeensis, a tick-transmitted intraphagosomal bacterium, is the causative agent of human monocytic ehrlichiosis. The pathogen also infects several other vertebrate hosts. E. chaffeensis has a biphasic developmental cycle during its growth in vertebrate monocytes/macrophages and invertebrate tick cells. Host- and vector-specific differences in the gene expression from many genes of E. chaffeensis are well documented. It is unclear how the organism regulates gene expression during its developmental cycle and for its adaptation to vertebrate and tick host cell environments. We previously mapped promoters of several E. chaffeensis genes which are recognized by its only two sigma factors: σ32 and σ70. In the current study, we investigated in assessing five predicted E. chaffeensis transcription regulators; EcxR, CtrA, MerR, HU and Tr1 for their possible roles in regulating the pathogen gene expression. Promoter segments of three genes each transcribed with the RNA polymerase containing σ70 (HU, P28-Omp14 and P28-Omp19) and σ32 (ClpB, DnaK and GroES/L) were evaluated by employing multiple independent molecular methods. We report that EcxR binds to all six promoters tested. Promoter-specific binding of EcxR to several gene promoters results in varying levels of gene expression enhancement. This is the first detailed molecular characterization of transcription regulators where we identified EcxR as a gene regulator having multiple promoter-specific interactions.

Genetic characterization of a novel Ehrlichia chaffeensis genotype from an Amblyomma tenellum tick from South Texas, USA

Ehrlichia chaffeensis is the causative agent of human monocytotropic ehrlichiosis (HME), a disease that ranges in severity from mild to fatal infection. Ehrlichia chaffeensis is maintained in a zoonotic cycle involving white-tailed deer (Odocoileus virginianus) as the main vertebrate reservoir and lone star ticks (Amblyomma americanum) as its principal vector. Through complete genomic analysis from human ehrlichial isolates and DNA sequences obtained from deer and tick specimens, nine strains of E. chaffeensis have been characterized. Few studies have examined the genetic diversity of E. chaffeensis in ticks, and some of these investigations have identified that the genetic sequences coincide with the circulating strains reported so far. Here, we report the first evidence of E. chaffeensis DNA from an unfed Amblyomma tenellum (formerly Amblyomma imitator) collected in South Texas. We characterized the genetic variation of this E. chaffeensis genotype using conserved gene markers such as rRNA, dsb, and groEL. We also used gene targets useful to distinguish genotypes, such as the variable length PCR target gene (VLPT) and 120-kDa gene, encoding the tandem-repeat proteins TRP32 and TRP120, respectively. Our results suggest a novel E. chaffeensis genotype that exhibited greater variability than other genotypes of E. chaffeensis and highlights the role for A. tenellum as a potential vector of E. chaffeensis.

Comparative genomic analysis of the first Ehrlichia canis detections in Australia

Ehrlichia canis (Rickettsiales; Anaplasmataceae) is one of the most prevalent tick-borne pathogens of dogs globally. The bacterium infects monocytes and is the aetiological agent of canine monocytic ehrlichiosis. For many decades Australia was thought to be free of the pathogen, but this abruptly changed in May 2020 when E. canis was detected in several dogs from Kununurra, Western Australia. Subsequent surveillance activities found unexpectedly large scale spread of E. canis throughout much of northern Australia. To gain insight into the genetic relationships of the Australian strain and its potential origin, we undertook a genomic analysis of E. canis positive domestic dog and tick (Rhipicephalus linnaei) samples from the north of Western Australia, the far north of South Australia and the Northern Territory, covering thousands of square kilometres. We obtained complete E. canis genomes from each of the three states, plus an additional 16 partial genomes, substantially increasing publicly available E. canis genetic resources. The Australian E. canis genomes were highly conserved across large geographic distances. Outside of Australia, the genomes were most similar to E. canis YZ-1 from China, although few reference sequences were available. We analysed the variable trp36 gene to obtain greater phylogenetic signal, which demonstrated that the Australian E. canis belonged to the Taiwan genotype, comprised of samples from Taiwan, China, Thailand and Turkey. Taken together, our findings suggest that E. canis in Australia may have originated from Asia or the Middle East and spread throughout northern and central Australia following its introduction.

Detection of Ehrlichia spp. in ticks collected from stray dogs in Central and Southeastern Iran

Ehrlichia is an etiologic agent of ehrlichiosis in humans and some animals. Rhipicephalus sanguineus is the main vector of the Ehrlichia canis and dogs, red foxes and yellow jackals are reservoirs of the bacterium. This tick has a worldwide distribution and is regarded as one of the commonest species of ticks in Iran. This research aimed to detect Ehrlichia spp. in R. sanguineus isolated from stray dogs in Central and Southeast Iran (Isfahan and Zabol), by using polymerase chain reaction (PCR) and to evaluate the prevalence of the microorganism in these two areas. Tick samples were collected from stray dogs in Isfahan and Zabol between April and June of 2018. The DNA extraction was performed with commercial kits. PCR was done to determine the 336 bp fragment related to Ehrlichia spp. Overall, 15.21% of pools in both areas were positive for Ehrlichia, 21.42% and 10% of pools were from Isfahan and Zabol respectively. The results confirmed the presence of Ehrlichia spp. in R. sanguineus in stray dogs revealing that dogs and their ticks may have a significant role in the epidemiology of the disease.

Development of a SONIX Vaccine to Protect Against Ehrlichiosis.

The obligately Gram-negative intracellular bacterium Ehrlichia that resides in mononuclear phagocytes is the etiologic agent of human monocytotropic ehrlichiosis (HME). HME is an emerging and often life-threatening, tick-transmitted infectious disease in the USA. Currently, three different Ehrlichia species can cause ehrlichiosis in humans in the USA-Ehrlichia chaffeensis, Ehrlichia ewingii, and Ehrlichia muris subspecies eauclairensis. Ehrlichia also causes diseases in companion animals and domesticated ruminants. Ehrlichia are vector-borne diseases and transmitted by tick bites. As yet there are no commercially available vaccines to protect against these pathogens. Previously we developed structure-based vaccines and subunit vaccines to protect against ehrlichiosis in animal models. Though the vaccines are efficient in inducing protection, there is a delay in clearing the pathogens in challenge studies. In this chapter we demonstrate the development of a SONIX vaccine that is more potent than conventional vaccines. The vaccination strategy may be useful in Emergency Use Authorization (EUA) scenarios during public health emergencies.

Ehrlichia canis morulae in peripheral blood lymphocytes of two naturally-infected puppies in Israel.

Ehrlichia canis is the major causative agent of canine monocytic ehrlichiosis (CME). Its morulae might be detected during the acute disease phase, usually within peripheral blood monocytes, but were uncommonly described within peripheral blood lymphocytes. This report describes two unrelated puppies, naturally infected with E. canis. In both, examination of stained peripheral blood smears revealed one to several cytoplasmic inclusions, characteristic of typical E. canis morulae, exclusively within lymphocytes. Ehrlichia canis infection was confirmed in both cases by blood sample real-time PCR. Both dogs were young and had comorbidities. One dog, based on whole blood PCR, was co-infected with Anaplasma platys and Babesia vogeli. The other had no other concurrent tick-borne infection based on PCR, but had bacterial cholangiohepatitis. These comorbidities, and the dogs' young age possibly contributed to the uncommon presence of E. canis morulae within peripheral blood lymphocytes rather than their typical presence in monocytes.

Analysis on the prokaryotic microbiome in females and embryonic cell cultures of Rhipicephalus sanguineus tropical and temperate lineages from two specific localities in Brazil.

Two lineages of Rhipicephalus sanguineus are known in Brazil: the temperate or southern and the tropical or northern populations. The distribution patterns of both lineages of R. sanguineus have epidemiological implications that can affect vectorial competence concerning Ehrlichia canis, the agent of canine monocytic ehrlichiosis. Intending to identify the microbiomes of both lineages and compare microorganisms in R. sanguineus, we used the 16S rRNA (V4-V5 region) gene-based metataxonomic approach, through NGS sequencing on the MiSeq Illumina platform. We selected specimens of females from the environment and samples of primary embryonic cell cultures, from both lineages, and this was the first study to investigate the prokaryotic microbiome in tick cell cultures. The results showed that many bacterial taxa detected in the samples were typical members of the host environment. A significant diversity of microorganisms in R. sanguineus females and in embryonic cell cultures from both lineages was found, with emphasis on the presence of Coxiella in all samples, albeit in different proportions. The Coxiella species present in the two lineages of ticks may be different and may have co-evolved with them, thus driving different patterns of interactions between ticks and the pathogens that they can harbor or transmit to vertebrate hosts.

<p class="Body"><strong>Detection of a <em>Rickettsia </em>sp. and an <em>Ehrlichia chaffeensis</em>-like organism in ticks parasitizing the endangered Florida Grasshopper Sparrow (<em>Ammodramus savannarum floridanus</em>)</strong></p>

Between 2013 and 2015, 163 resident endangered Florida Grasshopper Sparrows (Ammodramus savannarum floridanus) and four migratory Eastern Grasshopper Sparrows (A. savannarum pratensis) were examined for the presence of ticks in peninsular Florida. Thirteen Amblyomma maculatum and seven Haemaphysalis chordeilis ticks were removed from 13 Florida Grasshopper Sparrows. Two A. maculatum were discovered on two Eastern Grasshopper Sparrows. Polymerase chain reaction (PCR) and sequencing of resultant amplicons of some of the tick specimens were performed to determine if ticks were infected with pathogenic bacteria. Salivary gland and midgut contents of five of six (83%) of the H. chordeilis tested positive for a novel Rickettsia closely related to, but distinct from, Rickettsia aeschlimannii (causative agent of Mediterranean spotted fever-like illness), an infectious zoonotic bacterium that has not been previously reported in the United States. Four of 14 (29%) of the A. maculatum tested positive for an agent most closely related to an uncultured Ehrlichia previously isolated from Oriental house rats (Rattus tanezumi; 97.5% identity to GenBank KM817187), which is genetically similar to Ehrlichia chaffeensis (causative agent of human monocytic ehrlichiosis), another infectious zoonotic bacterium. Blood from 16 Florida Grasshopper Sparrows and one Eastern Grasshopper Sparrow tested negative for spotted fever group rickettsiae, Anaplasma spp. and Ehrlichia spp. We recommend that additional collections and screening of ticks and blood from Florida Grasshopper Sparrows be undertaken to determine the rates of infection with rickettsiae and ehrlichiae in these imperiled songbirds.

ДІАГНОСТИКА ТА ЛІКУВАННЯ ДЕЯКИХ ТРАНСМІСИВНИХ ХВОРОБ ДОМАШНІХ ТВАРИН

Протягом останніх років у всьому світі спостерігається збільшення кількості захворювань, що переносяться кліщами, особливо бореліозу, рикетсіозів (анаплазмоз, ерліхіоз), кліщового енцефаліту та інших. Кліматичні та екологічні зміни, міграція (переміщення) домашніх тварин призводять до змін епізоотологічної ситуації щодо трансмісивних захворювань. Проведено аналіз епізоотологічних, клінічних, лабораторних досліджень. Узагальнено дані наукових досліджень щодо трансмісивних захворювань, а саме бореліозу, ерліхіозу, вірусного кліщового енцефаліту, вірусного енцефаломієліту овець. Протягом останніх років інфекційні та інвазійні захворювання тварин, викликані вірусами, бактеріями і найпростішими, що передаються кліщами, є новою проблемою охорони здоров’я і ветеринарної практики. Багато таких захворювань є зоонозами і призводять до інвалідності та смертності людей і тварин. Іксодові кліщів часто нападають на тварин і людей та широко поширені по всій території Європи, а також беруть участь у передачі великої кількості трансмісивних захворювань. В даний час однією з найбільших загроз є збудники комплексу Borrelia burgdorferi s. l., які відносяться до спірохет і вражають різноманітні види ссавців та птахів та передаються кліщами (Ixodes ricinus, Ixodes hexagonus та Ixodes persulcatus). Захворювання має велике епідеміологічне значення для здоров’я людей. Діагностика та лікування недостатньо розроблені. Ehrlichia spp. – це грамнегативні, облігатні внутрішньоклітинні бактерії з родини Anaplasmataceae. В Європі Ehrlichia canis є етіологічним збудником моноцитарного ерліхіозу собак. Основним господарем E. canis є собака (інші собачі можуть служити резервуарними господарями); переносник – кліщ Rhipicephalus sanguineus. Кліщовий енцефаліт, а також вірусний енцефаломієліт овець – захворювання, що передаються іксодовими кліщами і становлять небезпеку для собак, котів та інших тварин, а також людей у Європі. В даний час усі ці захворювання набувають важливого епізоотологічного значення, оскільки діагностика та лікування є комплексними і ускладненими. Основним заходом профілактики захворювань серед собак є ефективний захист тварин від нападу кліщів. Трансмісивні хвороби – це різновид інфекційних та інвазійних хвороб тварин і людей, збудники яких поширюються від одного до іншого сприйнятливого суб'єкту за участі кровосисних членистоногих. З таких хвороб найбільш поширеними і клінічно значимими є: бореліоз, ерліхіоз, кліщовий енцефаліт тощо. В Україні системні дослідження зоонозних захворювань не проводились. Системний моніторинг збудників та ефективний контроль трансмісивних хвороб тварин являються основою покращення епідеміологічної ситуації серед населення.

Canine vector-borne disease in domestic dogs on Isla Santa Cruz, Galápagos.

Vector-borne diseases result in significant morbidity and mortality in domestic dogs in tropical and subtropical regions and also pose a potential threat to wildlife species and humans. Ehrlichia canis, the causative agent of canine monocytic ehrlichiosis (CME), has a high reported seroprevalence in dogs on Santa Cruz in the Galápagos Islands, Ecuador. Veterinary diagnostic and treatment resources are often scarce and clinical follow-up is lacking in the Galápagos. This study evaluated 58 dogs presenting to the Darwin Animal Doctors clinic in the city of Puerto Ayora on Santa Cruz Island during August of 2018. The seroprevalence of E. canis/Ehrlichia ewingii (48.3%), Anaplasma phagocytophilum/Anaplasma platys (12.1%), and Borrelia burgdorferi (0%), as well as the proportion of dogs actively infected with E. canis (12.1%) and E. ewingii (0%), are reported. Active infection was defined as the identification of antigen by PCR. Dogs with a packed cell volume (PCV)≤30% had a 10-fold risk of active infection with E. canis compared to dogs with a PCV≥31% (p=.0124). A PCV cutoff of 30% may be a useful screening tool for active E. canis infection in regions with high Ehrlichia seroprevalence, in the absence of other apparent causes of anemia. Dirofilaria immitis antigen was present in 6.9% of examined dogs, with the highest prevalence in the barrio Las Ninfas. PCR and Sanger sequencing were used to provide the first molecular identification of D. immitis in the Galápagos. This study updates the seropositivity and prevalence data of these canine vector-borne pathogens and highlights the need for continued surveillance in the region.

Expression and antigenic analysis of the recombinant TRP36 protein from Ehrlichia canis São Paulo strain for serologic tests.

Ehrlichia canis is the main etiological agent of canine monocytic ehrlichiosis (CME), a globally canine infectious disease. In Brazil, CME is considered to be endemic, and its prevalence can reach 65% in some states. The diagnosis of ehrlichiosis is important for treatment and epidemiological purposes. The E. canis TRP36 (Tandem Repeat Protein) protein elicits the earliest acute-phase antibody response observed during the course of the disease. This study aimed to generate the recombinant TRP36 protein from E. canis São Paulo strain and to evaluate its potential as a tool for the serologic diagnosis of CME. The E. canis São Paulo isolate was cultivated in DH82 lineage cells, and its genomic DNA was obtained. The bacterial DNA fragment encoding the entire ORF of TRP36 was cloned into the pBAD/Thio-TOPO vector and transformed into Escherichia coli DH10B competent cells with the trp36-bearing plasmid for protein expression. To evaluate the protein antigenicity, 16 canine serum samples were previously tested (by PCR and the commercial SNAP®4Dx® serological test). The results were in accordance with the SNAP®4Dx® test. Experiments using this recombinant protein as an antigen, targeting the development of a serologic test based on ELISA methodology, are the next step to produce a reliable, affordable and useful diagnostic tool for CME in Brazil.