Search

Despite the high global impacts of canine vector-borne diseases (CVBD) due to their wide distribution and zoonotic potential, the current epidemiological situation of CVBD in many tropical and subtropical regions remains unknown. This study examines the seroprevalence and molecular prevalence of Ehrlichia canis and other pathogens causing CVBDs (Leishmania infantum, Dirofilaria immitis, Babesia spp., Anaplasma spp. and Hepatozoon canis) in dogs living on the island of Boa Vista (Cape Verde Republic). Blood samples and infesting ticks were taken from 150 dogs across the island (stray, shelter, and pet dogs). Serum samples were tested using a rapid immunochromatographic test (Uranotest ® Quattro) that detects antibodies against E. canis, L. infantum, Anaplasma spp. and D. immitis antigen. Levels of serum antibodies against E. canis were measured using the immunofluorescence antibody test (IFAT). In addition, tick-borne pathogens in blood samples (Anaplasma spp., Babesia spp., Hepatozoon spp., and Ehrlichia canis) were detected by microscopy observation and/or PCR plus sequencing. The seroprevalence of E. canis was extremely high at 82% (123/150), as revealed by both immunochromatography and IFAT. Most dogs returning a seropositive test result (82.92%; 102/123) had antibody titres > 1:1280 but showed no clinical signs or notable laboratory abnormalities. Of the 123 animals testing seropositive for E. canis, 67 (54.47%) also presented antibodies against Anaplasma spp., and 13 (10.56%) showed the presence of Hepatozoon spp. gamonts in the blood smear. Ehrlichia canis infection was detected in 17.1% (25/146) of dogs tested by direct sequencing of polymerase chain reaction (PCR) products. Co-infections were detected in seven of these dogs: four dogs tested PCR-positive for both E. canis and A. platys, two dogs tested positive for E. canis and Hepatozoon spp., and one dog tested positive for E. canis, A. platys and Hepatozoon spp. Rhipicephalus sanguineus sensu lato was the only tick species found infesting the canine study population. The high prevalence of tick-borne pathogens detected in dogs from Boa Vista Island highlights a need for improved control measures designed to prevent the transmission of these pathogens., (© 2024. The Author(s).)

Purpose: Tick-transmitted parasites as Babesia gibsoni, Babesia vogeli, Ehrlichia canis, and Hepatozoon canis are major health concern for dogs. Owing to prevalence and infection severity, there is need of sensitive, specific, and affordable test for their simultaneous detection., Methods: Prevalence of B. gibsoni, B. vogeli, E. canis, and H. canis infections was assessed on 719 blood samples by microscopy and multiplex PCR assay targeting 18S rRNA (B. gibsoni & H. canis), ITS1 & 5.8S rRNA (B. vogeli) and VirB9 gene (E. canis). An internal control (canine-actin) was also included to increase the accuracy of assay and effect of associated risk factors with disease prevalence was also studied., Results: Microscopic prevalence of B. gibsoni, B. vogeli, E. canis and H. canis was 5.0%, 0.1%, 1.4% and 1.0%, respectively, whereas with multiplex PCR assay, the corresponding values were 8.9%, 1.1%, 2.6% and 5.1% besides concurrent infections of B. gibsoni & H. canis (0.4%), B. gibsoni & E. canis (0.4%), E. canis & H. canis (0.3%) and B. gibsoni & B. vogeli (0.1%). Analytical sensitivity of developed assay was 0.1pg (B. gibsoni & H. canis), 0.01pg (B. vogeli), and 1.0pg (E. canis). A ″fair″ (B. vogeli & H. canis) to ″substantial″ (B. gibsoni & E. canis) agreement between two tests was observed with data as statistically significant. Breed, sex and location were significantly associated with B. gibsoni infection., Conclusion: The developed multiplex PCR assay offers a potential solution to detect these pathogens simultaneously, aiding in timely diagnosis and effective disease management in suspected dogs., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Canine monocytic ehrlichiosis caused by Ehrlichia canis is an important rickettsial pathogen of dogs transmitted by Rhipicephalus sanguineus sensu lato ticks in India. Globally, molecular characterization of E. canis is done using different E. canis gene targets. This study aimed to characterize genetic diversity of uncultured Ehrlichia species from dogs by 16S rRNA and partial gp200 gene (termed as p43 region) sequences data. Phylogeny based on 16S rRNA gene did not reveal any region-specific lineages. The phylogeny based on 5' region of E. canis gp200 gene (termed as p43 region) revealed four major clusters (A, B, C and D) and the Indian isolates fall under clusters A and B. Cluster A is characterized by an insertion of unique 141 bp tandem repeat sequence. Similar tandem repeat sequence was found in one of the E canis isolates from east-Asia, suggesting a possible divergence within this species. The study shows evidence for divergence of a new lineage within E. canis. The location of this insertion at the 'ankyrin repeat domains' containing region is suggestive of its possible role in modulation of host responses., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bed bugs, common blood-feeding pests, have received limited attention regarding their potential involvement in emerging pathogen transmission. This study aimed to investigate the main vector-borne bacteria within bed bugs collected from Tunisian governorates and to genetically characterize the identified species. Molecular screening was conducted on field-collected bed bug samples, targeting zoonotic vector-borne bacteria from the Anaplasmataceae family, as well as the genera Rickettsia, Ehrlichia, Bartonella, and Borrelia. A total of 119 Cimex lectularius specimens were collected and grouped into 14 pools based on sampling Tunisian sites. Using genus-specific PCR assays, DNA of Rickettsia and Ehrlichia spp. was detected in a single pool. Sequencing and BLAST analysis of the obtained partial ompB and dsb sequences from positive samples revealed 100% similarity with those of Ehrlichia canis and Rickettsia felis available in GenBank. Obtained partial sequences showed phylogenetic similarity to R. felis and E. canis isolates found in dogs and ticks from American and European countries. To the best of our knowledge, this study is the first to investigate bed bugs in Tunisia and to report the worldwide identification of R. felis and E. canis DNA in the common bed bug, C. lectularius. These findings highlight the need for further research to explore the potential role of bed bugs in the epidemiology of these vector-borne bacteria., Competing Interests: Declaration of Competing Interest The authors declared that they have no potential conflicts of interest with respect to the research, authorship and publication of this article., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Ehrlichia canis has been the main hemopathogen affecting domestic dogs in Brazil. Even though tick-infested dogs may lead to household infestation and predispose human exposure and public health concern, no comprehensive study has surveyed humans, dogs, and environmental ticks altogether. Materials and Methods: Accordingly, the present study aimed to assess tick-infested households, identify tick species, perform serological (immunofluorescence assay) and molecular (PCR and q-PCR) detection of Ehrlichia in ticks, in the eighth biggest metropolitan area of Brazil. Results: Between 2007 and 2020, 233/5973 (3.9%) out of all complaints were from tick-infested households of 200 different addresses. Overall, 370/552 (67.0%) ticks were collected and identified as adult and 182/552 (33.0%) as immature forms of Rhipicephalus sanguineus s.l. complex; a single tick from one owner, a female tick of Amblyomma sculptum ; and 395 ticks from dogs, 319/395 (80.8%) adult and 72/395 (18.2%) immature forms of Rhipicephalus spp., and 4/395 (1.01%) female Amblyomma aureolatum . Overall, 2/135 (1.5%) owners and 13/136 (9.6%) dogs were seropositive for E. canis . The DNA of Anaplasmataceae family was molecularly detected in 16/50 (32.0%) R. sanguineus s.l. As expected, the number of monthly tick infestation complaints were directly associated, and mean ( p  = 0.01), maximum ( p  = 0.011), and minimum ( p  = 0.008) temperature were statistically significant and had a low positive correlation (0.24, 0.23, and 0.24, respectively). In addition, complaints were highly associated to all socioeconomic variables ( p  < 0.001), with the exception of the presence of vacant lots. Conclusions: Despite low samplings and human negative results, areas with low-income with adequate temperature and urban agglomerations have been shown to be associated risks for tick infestations, predisposing tick-borne diseases. In conclusion, monitoring should always be conducted in such areas, including One Health approach with serosurvey of owners and dogs, along with identification and molecular screening of ticks.

Anaplasma phagocytophilum, Anaplasma platys and Ehrlichia canis, responsible of diseases in dogs, are tick-borne pathogens with a proven or potential zoonotic role that have shown increasing prevalence worldwide. The aims of this retrospective study were to assess the frequency of Anaplasma spp. and Ehrlichia spp. exposure in dogs tested in a veterinary teaching hospital in Italy over a 9-year period, to compare the performance of the diagnostic tests used, to evaluate correlations with clinical data, and to genetically analyse the identified bacteria. During the study period, 1322 dogs tested by at least one of the rapid immunoenzymatic test, indirect immunofluorescent antibody test or end-point PCR assay for Anaplasmataceae detection were included. Dogs were tested if they had clinical signs or clinicopathological alteration or risk factors related to infection, and if they were potential blood-donor animals. Ninety-four of 1322 (7.1%) dogs tested positive for at least one pathogen: 53 (4.3%) for A. phagocytophilum, one (0.1%) for A. platys and 63 (4.6%) for E. canis. The number of dogs tested increased and the positivity rate progressively declined over the years. Comparison of tests showed a near-perfect agreement between serological tests and a poor agreement between PCR and indirect assays. A breed predisposition has been highlighted for A. phagocytophilum infection in hunting breed dogs and for E. canis infection in mixed breed dogs. Phylogeny confirmed potential zoonotic implications for A. phagocytophilum and showed no correlation of the identified bacteria with the geographical origin. Our study provides new insights into possible risk factors in dogs and evidenced discordant results between different tests, suggesting that a combination of serological and molecular assays is preferable for a correct diagnosis., (© 2024. The Author(s).)

The brown dog tick, Rhipicephalus sanguineus is a complex of tick species with an unsettled species concept. In Europe, R. sanguineus is considered mainly a Mediterranean tick with sporadic findings in central and northern Europe. R. sanguineus is known as a vector of a range of pathogens of medical and veterinary importance, most of which not yet reported as autochthonous in Hungary. A total of 1839 ticks collected by veterinarians from dogs and cats were obtained in Hungary. The study aims at precise determination of ticks identified as R. sanguineus and detection of pathogens in collected ticks. All ticks were morphologically determined and 169 individuals were identified as R. sanguineus. A subset of 15 ticks was selected for molecular analysis (16S rDNA, 12S rDNA, COI). Phylogenetic analyses invariably placed sequences of all three markers into a single haplotype identified as R. sanguineus sensu stricto. All 169 brown dog ticks were tested for the presence of A. platys, E. canis, R. conorii, B. vogeli and H. canis. None of the investigated ticks was positive for the screened pathogens, though A. phagocytophilum sequence was detected in a single tick., Competing Interests: Declaration of competing interest The authors have declared that they have no conflict of interest related to this study., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Anaplasmosis and ehrlichiosis are important tick-borne rickettsial diseases of medical and veterinary importance that cause economic losses in livestock. In this study, the prevalence of Anaplasma ovis, Ehrlichia canis and Ehrlichia chaffeensis was investigated in ticks collected from sheep in various farms in Van province, which is located in the Eastern Anatolian Region of Turkey. The ticks used in this study were collected by random sampling in 26 family farm business in 13 districts of Van province. A total of 688 ticks were collected from 88 sheep and 88 tick pools were created. All ticks identified morphologically as Rhipicephalus bursa. Phylogenetic analysis of Chaperonin and 16S rRNA gene sequences confirmed A. ovis, E. canis and E. chaffeensis in this study. Of the 88 tick pools tested, 28.41% (25/88) were positive for at least one pathogen. Anaplasma DNA was detected in five of the 88 pools (5.68%), E. canis DNA was detected in 19 of the 88 pools (21.59%), and E. chaffeensis DNA was detected in one of the 88 pools (1.14%) of R. bursa ticks. To our knowledge, this is the first report describing the presence of A. ovis, E. canis, and E. chaffeensis in R. bursa ticks collected from sheep in Turkey. Further studies are needed to investigate other co-infections in sheep in Turkey., (© 2024 The Authors. This is an open access article under the CC BY-NC-ND 4.0 license.)

Background: Ixodid ticks, particularly Rhipicephalus sanguineus s.l., are important vectors of various disease-causing agents in dogs and humans in Cuba. However, our understading of interactions among tick-borne pathogens (TBPs) in infected dogs or the vector R. sanguineus s.l. remains limited. This study integrates microfluidic-based high-throughput real-time PCR data, Yule's Q statistic, and network analysis to elucidate pathogen-pathogen interactions in dogs and ticks in tropical western Cuba., Methods: A cross-sectional study involving 46 client-owned dogs was conducted. Blood samples were collected from these dogs, and ticks infesting the same dogs were morphologically and molecularly identified. Nucleic acids were extracted from both canine blood and tick samples. Microfluidic-based high-throughput real-time PCR was employed to detect 25 bacterial species, 10 parasite species, 6 bacterial genera, and 4 parasite taxa, as well as to confirm the identity of the collected ticks. Validation was performed through end-point PCR assays and DNA sequencing analysis. Yule's Q statistic and network analysis were used to analyse the associations between different TBP species based on binary presence-absence data., Results: The study revealed a high prevalence of TBPs in both dogs and R. sanguineus s.l., the only tick species found on the dogs. Hepatozoon canis and Ehrlichia canis were among the most common pathogens detected. Co-infections were observed, notably between E. canis and H. canis. Significant correlations were found between the presence of Anaplasma platys and H. canis in both dogs and ticks. A complex co-occurrence network among haemoparasite species was identified, highlighting potential facilitative and inhibitory roles. Notably, H. canis was found as a highly interconnected node, exhibiting significant positive associations with various taxa, including A. platys, and E. canis, suggesting facilitative interactions among these pathogens. Phylogenetic analysis showed genetic diversity in the detected TBPs., Conclusions: Overall, this research enhances our understanding of TBPs in Cuba, providing insights into their prevalence, associations, and genetic diversity, with implications for disease surveillance and management., (© 2024. The Author(s).)

Background: Ehrlichia canis is transmitted by ticks causing Canine monocytic ehrlichiosis, which is considered one of the most critical tickborne pathogens. Materials and Methods: This study aimed to identify by PCR technique E. canis in ticks associated with dogs from urban and rural homes in Nuevo Leon, Mexico. The study was conducted at 13 localities in eight municipalities from 2012 to 2021. Results: A total of 1873 ticks of three species were captured: Amblyomma tenellum , Dermacentor variabilis , and Rhipicephalus sanguineus s . l . The overall infection rate of E. canis in ticks was 59.12% (149/252). Of the 15 sequences, three haplotypes were identified. Conclusion: The urban transmission cycle of canine ehrlichiosis is demonstrated, where the potential vector is the tick R. sanguineus s.l.

This study aimed to determine the prevalence and phylogenetic analysis of Ehrlichia spp. in horses and dogs in Iran. Blood samples were collected from 400 animals, including 200 horses and 200 dogs, from five different provinces in Iran. Polymerase chain reaction (PCR) was used to detect Ehrlichia spp. based on amplification of the 16S rRNA gene. The semi-nested PCR method was used to amplify the dsb, TRP36, and gltA genes. The results showed that 4.5 % of the samples (3 % horses and 6 % dogs) were positive for Ehrlichia sp. The highest prevalence was observed in Kerman and Khuzestan, while the lowest was found in West Azerbaijan, Golestan, and Mazandaran. The study suggests that the populations of dogs and horses in the country should be considered important factors in the epidemiology of ehrlichiosis. Phylogenetic analysis based on the dsb and TRP36 genes revealed that the prevalent species were E. canis and E. ruminantium., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Canine monocytic ehrlichiosis is cause by Ehrlichia canis resulting in hematologic disorders and severe clinical signs. The aim of this study was to scrutinize the molecular detection and genetic diversity of E. canis based on the trp36 gene in dogs from Thailand's northern and central regions. A total of 120 dogs blood samples were amplified for trp36 gene of E. canis using the polymerase chain reaction (PCR). Forty-seven out of 120 dog blood samples (39.16%, 47/120) were positive for E. canis the trp36 DNA with 790 bp of PCR amplicon size. The factor significantly associated with E. canis infection is animal housing status (p < 0.05). Sequence and phylogenetic analysis showed that E. canis trp36 gene of Thailand isolates was clustered into 1st clade with similarity ranging from 95.65 to 100% together with the US genogroup. The 14 haplotypes of the trp36 gene shown in TCS network exhibited that haplotype #1-4 was found in Thailand. The entropy analysis of the trp36 gene illustrated 751 polymorphic sites and 271 entropy peaks of nucleic and amino acid sequences, respectively. Hence, these findings are crucial for better understanding the epidemiology of Ehrlichia infection and could be helpful for implementing control measures in Thailand., (© 2023. The Author(s).)

Background: In Europe, feline vector-borne infections are gaining importance because of the changing climate, expanding habitats of potential vectors and expanding pathogen reservoirs. The main objective of this study was to assess the prevalence of vector-borne pathogens (VBPs) in stray cats in Zaragoza, Spain, and to investigate potential risk factors for infection, including feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV)., Methods: Blood samples from stray cats presented to the veterinary faculty in Zaragoza between February 2020 and 2022 were tested by polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum, Anaplasma platys, Bartonella henselae, Ehrlichia canis, Rickettsia spp., haemotropic Mycoplasma spp., Hepatozoon spp., Leishmania infantum, piroplasms and microfilariae at the LABOKLIN laboratory. The cats were also tested for FeLV and FIV by PCR., Results: Nearly half of the cats (158/332, 47.6%) were positive for at least one VBP. Hepatozoon spp. were detected in 25.6%, haemotropic Mycoplasma spp. in 22.9%, B. henselae in 9.3% and L. infantum in 2.1% of the cats. Male sex had a statistically significant association with test results for haemotropic Mycoplasma spp. (odds ratio 1.38 [1.21;1.57]); regionality with Hepatozoon spp., B. henseale and FIV; and seasonality with Hepatozoon spp., haemotropic Mycoplasma spp., L. infantum and FeLV (P ≤ 0.05 each). A strong positive correlation was reported for the amount of rainfall and the number of cats that tested positive for Hepatozoon spp. (ρ = 753, P = 0.05). None of the cats tested positive for A. phagocytophilum, A. platys, E. canis, Rickettsia spp., piroplasms, or microfilariae. Co-infections with multiple VBPs were detected in 56 out of 332 cats (16.9%). Thirty-one of the 332 cats included in the study (9.3%) tested positive for FeLV (6.9%) and for FIV (3.6%). In 20/31 cats (64.5%) that tested positive for FeLV/FIV, coinfections with VBP were detected (P = 0.048, OR 2.15 [0.99; 4.64])., Conclusions: VBPs were frequently detected in stray cats in Zaragoza. In particular, regionality and seasonality had a statistically significant association with PCR results for most VBPs included in the study., (© 2023. The Author(s).)

Animal infection models are crucial for studying various aspects of Ehrlichia canis infections. To understand the pathogenesis of the first Chinese isolate of E. canis and simulate the natural progression of canine ehrlichiosis, we developed a model with 18 Beagle dogs that consisted of E. canis initial infection (days 0-17), treatment with doxycycline or rifampicin (days 18-32), recovery (days 33-66), E. canis reinfection (days 67-91), and Babesia vogeli superinfection (days 92-116). We measured body weight and rectal temperature every other day, drew blood every 4 days for routine hematology and biochemistry tests, and for quantification of E. canis and B. vogeli by quantitative PCRs. In this study, the first isolate of E. canis from China was used to experimentally infect dogs, and the infected dogs exhibited clinical signs of acute severe ehrlichiosis, including high fever, loss of appetite, dehydration, and body weight loss, confirming the similar pathogenicity of E. canis in China as compared to isolates from other regions. Infection with E. canis and B. vogeli led to reduced body weight and fever in dogs. Doxycycline treatment led to absence of E. canis DNA in infected dogs, while rifampicin treatment lowered the blood E. canis copy number up to 1.5 folds. E. canis-free infected dogs after doxycycline treatment were successfully re-infected with E. canis, indicating dogs with antibodies are still at risk of re-infection. Super-infection with B. vogeli resulted in higher fever, more severe anemia, and a reduced number of platelets. Splenectomized dogs showed significantly higher E. canis numbers during recovery and re-infection than intact dogs. The histological changes were observed in brain, lung, kidney, liver and spleen of the infected dogs. The findings in this study provide insights into clinical and hematologic responses, as well as effective treatment options, for dogs infected with the first Chinese isolate of E. canis, and may contribute to our understanding of the diagnosis and prevention of tick-borne diseases in dogs, including canine monocytic ehrlichiosis., Competing Interests: Declaration of Competing Interest The authors in the manuscript below do not have conflict of interest to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Rickettsial pathogens including Ehrlichia canis and Anaplasma platys are bacteria that cause parasitic infections in dogs such as canine monocytic ehrlichiosis (CME) and canine cyclic thrombocytopenia (CCT), respectively affecting mortality and morbidity worldwide. An accurate, sensitive, and rapid method to diagnose these agents is essential for effective treatment. In this study, a recombinase polymerase amplification (RPA) coupled with CRISPR-Cas12a methods was established to detect E. canis and A. platys infection in dogs based on the 16S rRNA. The optimal condition for DNA amplification by RPA was 37 °C for 20 min, followed by CRISPR-Cas12a digestion at 37 °C for one hour. A combination of RPA and the cas12a detection method did not react with other pathogens and demonstrated strong sensitivity, detecting as low as 100 copies of both E. canis and A. platys. This simultaneous detection method was significantly more sensitive than conventional PCR. The RPA-assisted cas12a assay provides specific, sensitive, rapid, simple and appropriate detection of rickettsial agents in canine blood at the point-of-care for diagnostics, disease prevention and surveillance., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

The coinfections by some microorganisms have been related to severe diseases in humans and animals, where immunosuppressive agents favor opportunistic behavior of other pathogens. A 4-month-old, female mixed-breed dog with a two-week history of inappetence, prostration, emaciation, and respiratory distress was admitted at a veterinary hospital in Brazil. Tachycardia, pale mucous membranes, severe respiratory distress, and a large number of ticks (Rhipicephalus sanguineus s.l.) in different body regions were observed at clinical examination. Hematological examination of dog showed leukocytosis, neutrophilia, mild anemia, and thrombocytopenia, whereas unremarkable values in biochemical tests. Thoracic radiography revealed a pleural effusion image. Blood and the pleural fluid (purulent aspect) samples were subjected to qPCR (16S rRNA and dsb genes) and sequencing, which identified Ehrlichia canis and Anaplasma platys coinfection. An aggregate of coccoid-to-branching or long filamentous microorganisms, surrounded by pyogranulomatous inflammatory reaction was seen at the cytology of the pleural fluid. Bacteriological culture of pleural effusion showed colonies compatible with the genus Nocardia, which revealed gram-positive filamentous organisms with a tendency of fragmentation and were identified as Nocardia otitidiscaviarum in mass spectrometry (MALDI-TOF MS). Therapy of N. otitidiscaviarum isolate using levofloxacin (supported by a previous in vitro susceptibility testing) and doxycycline for E. canis and A. platys resulted in complete resolution of the clinical picture. Here, we report for the first time a triple coinfection by Nocardia otitidiscaviarum, A. platys, and E. canis in a dog with pleural effusion, where debilitating or immunosuppressive conditions induced by A. platys and E. canis coinfection probably contributed to the opportunistic behavior of N. otitidiscaviarum., (© 2023. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Background: There are few reports of tick-borne pathogens infecting dogs living in indigenous communities of Brazil. Herein, we aimed to molecularly detect vector-borne pathogens in dogs from two indigenous communities in the Brazilian Amazon. Materials and Methods: We surveyed 327 dogs raised in Amazon region at 2 distinct indigenous ethnicities for the molecular detection of tick-borne pathogens (114 from Tapirapé and 213 from Karajá indigenous ethnicity). Whole blood samples were subjected to PCR and sequencing for Ehrlichia , Babesia , and Hepatozoon . Univariate and multivariate analyses were used to investigate the factors affecting the pathogen infection patterns in dogs. Results: Among the 327 blood samples, 40 were positive for Ehrlichia canis (12.2%), 2 for Anaplasma platys (0.61%), and 204 were positive for Hepatozoon canis (66.5%). Binary Logistic Regression showed association between E. canis infection and ethnicity ( p  = 0.010) and tick attachment ( p  = 0.041). Karajá dogs were 3.4 times (95% CI 1.3-8.5) more likely to be positive for E. canis than Tapirapé dogs. Dogs with ticks were 2.5 times more likely (95% CI 1.0-7.6) to be positive for E. canis than dogs without ticks. Conclusions: Our survey expands the knowledge regarding the presence of vector-borne pathogens in dogs from indigenous communities in the Amazon region.

The Anaplasmataceae family includes obligate, arthropod-transmitted intracellular bacteria that can be zoonotic and potentially fatal. Studies focusing on the interaction between neotropical primates and the agents of this family are scarce. The present study aimed to identify agents of the Anaplasmataceae family in the whole blood of free-living and captive neotropical primates in the State of Mato Grosso, Central-West Brazil. Thirty-eight samples of six nonhuman primate (NHP) species were collected in seven municipalities and analysed through polymerase chain reaction (PCR), nucleotide sequencing, and phylogenetic analysis of the dsb, groEL, 16S rRNA, and gltA genes. DNA fragments similar to those of Ehrlichia canis were detected in Sapajus apella and Ehrlichia chaffeensis from Mico melanurus. The sequences generated in this study and homologous sequences retrieved from GenBank® were used for phylogenetic analyses to characterize the Ehrlichial agents detected in NHPs. The agents were then grouped into clades corresponding to different isolates from the NHP species. In addition, an Anaplasma sp. closely related to Anaplasma marginale was identified in two S. apella individuals. These findings shed light on the susceptibility of neotropical NHPs to Anaplasmataceae agents. These bacteria are known to be transmitted by ticks, which can also serve as possible sources of infection for other animals, including humans., Competing Interests: Declarations of Competing Interest None., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Background: Vector-/tick-borne pathogens (V/TBPs) pose a potential threat to human and animal health globally. Information regarding canine V/TBPs is scarce and no specific study has been conducted so far to explore the microbial diversity within ticks infesting dogs from Pakistan. Herein, this knowledge gap is addressed by assessing the genetic diversity and prevalence pattern of V/TBPs in ixodid ticks with special implications for public and canine health., Methods: A total of 1150 hard ticks were collected from 300 dogs across central Khyber Pakhtunkhwa (KP), Pakistan. After morpho-molecular identification, 120 tick samples were screened for the presence of V/TBPs by amplifying 16S rRNA/gltA (Rickettsia/Ehrlichia and Wolbachia sp.), 18S rRNA (Theileria sp.) and cox1 (Dirofilaria sp.) genes through PCR followed by sequencing and phylogenetic study., Results: In toto, 50 ixodid ticks (50/120, 41.7%) were found positive for V/TBPs DNA. The detected V/TBPs were categorized into five genera and eight species, viz. Ehrlichia (E. canis and Ehrlichia sp.), Rickettsia (R. massiliae, R. raoultii and Rickettsia sp.), Theileria (T. annulata), Dirofilaria (D. immitis) and Wolbachia (Wolbachia sp.). The pathogen prevalence patterns showed that R. massiliae was the most prevalent zoonotic V/TBP (19.5%), followed by E. canis (10.8%), Rickettsia sp. (7.5%), R. raoultii (6.7%), T. annulata (5.8%), D. immitis (5.8%), Wolbachia sp. (4.2%) and Ehrlichia sp. (3.3%), respectively. Among the screened tick species, most Rhipicephalus sanguineus sensu lato samples were found positive for V/TBP DNA (20/20,100%) followed by Rh. turanicus sensu stricto (13/20, 65%), Hyalomma dromedarii (8/20, 40%), Rh. haemaphysaloides (6/20, 30%), Hy. excavatum (2/20, 10%) and Rh. microplus (1/20, 5%). Co-occurrence of V/TBP was also detected in tick specimens (single V/TBP infection: 32 ticks; double and triple: 13 and 5 tick samples). The detected pathogens shared a phylogenetic relationship with similar isolates published in NCBI GenBank from Old and New World countries., Conclusion: Ixodid ticks infesting dogs harbor a diverse array of V/TBPs including zoonotic agents from Pakistan. Furthermore, the presence of D. immitis in ticks that infest dogs raises the possibility that this parasite has either attained its dead-end host (i.e. the tick) while feeding on dogs or has expanded its range of intermediate/paratenic hosts. Further research work is needed to investigate the epidemiology and confirm the vector competence of screened tick species for these pathogens from Pakistan., (© 2023. The Author(s).)

Ehrlichiosis is an infectious disease caused by Ehrlichia canis (E. canis) genus and arthropod vectors. It is considered endemic in many parts of the world among dogs. But due to lack of research on cats, there isn't enough information available. The limited reports available on feline Ehrlichiosis relied on the detection of morulae in leukocytes. The current study was designed to detect the molecular prevalence of E. canis in cats along with associated risk factors and hematological analysis. A total of 384 blood samples from cats were collected from various veterinary hospitals and shelter homes and tested by microscopy and Polymerase Chain Reaction (PCR) to identify E. canis. The prevalence of E. canis has been reported at 5/384 (1.30%) and (14/384) 3.65% in cats through microscopy and PCR respectively. DNA sequences revealed significant resemblance with each other and variable resemblance with other Ehrlichia spp. sequences of different species from various countries already deposited on NCBI. Moreover, hematobiochemical and risk factor analysis were also carried out revealing significant results. This study reports first molecular detection of E. canis in client-owned and sheltered cats located in District Lahore, Punjab, Pakistan. Further studies should be conducted to identify its occurrence in the feline population of Pakistan so that control and prevention strategies must be planned accordingly. Due to the zoonotic impact of this pathogen and in perspective of one health, endemic regions of the disease should be identified and possible control measures should be implemented in these regions to minimize the spread of disease to non-endemic regions of the world and from animals to humans., Competing Interests: Competing interest The authors declare no financial or non-financial interests to disclose., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Ehrlichia canis and Babesia vogeli are vector-borne pathogens that infect blood cells and produce the diseases Canine Monocytic Ehrlichiosis (CME) and Babesiosis in dogs. Considering the lack of studies on these pathogens in Colombia, this study aims to determine the molecular prevalence and genetic characterization of E. canis and Babesia spp., in dogs from the Metropolitan Area of Bucaramanga (MAB), Santander, a region with one of the greatest pet densities in Colombia. One hundred eighty-five dogs were surveyed and analyzed through molecular, clinical, and hematological approaches. The molecular detection of E. canis and Babesia spp., was performed by conventional PCR targeting the dsb and 18S rRNA genes, respectively. To identify genogroups, E. canis positive samples underwent a hemi-nested PCR of the trp36 gene, and the PCR products were subsequently sequenced. Molecular analyses showed a prevalence of 13% (24/185; CI 95%, 8.1 - 18.0%) and 1.09% (2/185; CI 95,% -0.43 - 2.6%) for E. canis and B. vogeli respectively, as well as the presence of the genogroups US (USA), BR (Brazil), and CR (Costa Rica), in 62.5, 16.6, and 16.6% of E. canis positive samples, respectively. Values of hematocrit, hemoglobin, platelets, erythrocytes, white blood cell (WBC) count, lymphocytes, and eosinophils showed significant differences between animals infected with the different genogroups of E. canis (p< 0.05). In contrast, hematocrit values, hemoglobin, platelets, red blood cells, and creatine kinase MB isoenzyme (CK-MB) were lower in B. vogeli positive animals. Statistical analysis indicated that E. canis infection was associated with specific socioeconomic sectors as well as with some household features (p< 0.05). In conclusion, our results present evidence of the circulation of multiple genogroups of E. canis in the MAB, which is associated with different geographical origins and clinical traits. Epidemiological analyses suggest a need to increase molecular surveillance and prevention campaigns especially in lower socioeconomic sectors., Competing Interests: Declaration of Competing Interest All the authors have participated in the study, and no conflicts of interest have been disclosed., (Copyright © 2022. Published by Elsevier GmbH.)

Ehrlichia canis is a common tick-borne intracellular pathogen causing canine monocytic ehrlichiosis (CME) in dogs worldwide. The aims of this study were to investigate the genetic diversity and antigenicity of E. canis based on the p28 and trp36 genes in dogs in Thailand. The E. canis p28 and trp36 genes were amplified by the polymerase chain reaction (PCR) and cloned for sequencing and bioinformatic analyses. 36% (44/120) of dog blood samples were positive for E. canis DNA consisting of p28 (31%, 14/44) and trp36 (69%, 30/44) genes with 792 and 882 bp of PCR products size, respectively. The E. canis TRP36 from all Thailand sequences exhibited encoded nine amino acids (TEDSVSAPA) with 11 copies of tandem repeats along the sequences. The phylogenetic trees of E. canis, using the p28 and trp36 genes, exhibited that the Thailand isolates fell into two clades and one clade with similarity ranging from 55.95 to 100% and 100%, respectively. The results of diversity analysis revealed 10 and 20 haplotypes of the p28 and trp 36 genes, respectively. The entropy analysis of the p28 and trp36 nucleic acid sequences showed 442 and 1321 high entropy peaks respectively, whereas those of the P28 and TRP36 amino acid sequences showed 477 and 388 high entropy peaks, respectively. For B-cell epitopes analysis, the conserved amino acid of P28 and TRP36 sequences has been also demonstrated. Therefore, the results could be utilized to improve the understanding of phylogenetic relationship, genetic diversity and antigenicity of E. canis Thailand isolates., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Vector-borne pathogens (VBPs) are primarily transmitted by arthropod vectors, but secondary ways of transmission have been described, including via venereal route. Nonetheless, there is still limited research on possible sexual transmission of VBPs in dogs. We molecularly investigated the presence of vector-borne pathogens in semen from dogs living in an area where these agents are endemic. Upon PCR testing, seven out of 22 (31.8%) semen samples tested positive for at least one VBP, whereas simultaneous positivity to two or more pathogens was detected in three (13.6%) dogs. Among pathogens detected in semen, Trypanosoma cruzi (n = 1) and Leishmania infantum (n = 3) were identified to species level by restriction fragment length polymorphism analysis. Attempts to sequence PCR products from other pathogens were unsuccessful, but coupled epidemiological and molecular data suggest the presence of Anaplasma platys (n = 5), Babesia vogeli (n = 1) and Ehrlichia canis (n = 1) in semen from dogs. Further experimental studies would be needed to confirm the sexual transmission hypothesis for these VBPs and also the possible implications of these findings for canine reproduction., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Epidemiology and distributions of canine tick-borne diseases as well as their veterinary and zoonotic significance are poorly understood in Algeria. The present study describes a molecular investigation of important tick-borne pathogens in Rhipicephalus sanguineus sensu stricto collected from domestic dogs in steppe and high plateau areas of central and eastern Algeria. In total, 1,043 ticks were collected from 147 dogs, including 756 ticks from 124 dogs in the steppe region of Djelfa and 287 ticks from 23 dogs in the high plateau area of Bordj Bou Arreridj. Ticks were divided into 384 pools (309 pools from Djelfa and 75 pools from Bordj Bou Arreridj) and tested for genomic materials of Crimean-Congo hemorrhagic fever virus (CCHFV) as well as DNA for Anaplasma phagocytophilum, Anaplasma platys, Ehrlichia canis, Rickettsia spp., Babesia spp., and Hepatozoon spp. using PCR, sequencing and phylogenetic analysis. Hepatozoon spp. was most prevalent, with 160 positive pools (41.7%), and 12 of these were sequenced and identified as Hepatozoon canis. Babesia spp. was detected in 50 samples (13.0%), of which 11 were sequenced and identified as Babesia vogeli. A. platys and E. canis were detected in 92 (24.0%) and 15 (3.9%) of tested samples, respectively. Rickettsia spp. were detected in 24 (6.3%) samples, including 11 samples identified as R. massiliae, 6 samples identified as R. conorii conorii, and 7 samples could not be identified to species level. All 384 pools tested negative for CCHFV and A. phagocytophilum. In addition to detection of R. conorii conorii, R. massiliae, and E. canis, the present study provides the first molecular data for occurrence of A. platys, B. vogeli, and H. canis in Rh. sanguineus s.s. infesting dogs in Algeria. Further large scale studies should be conducted to better understand the epidemiology, distributions, and importance of canine tick-borne pathogens in Algeria., (Copyright © 2022. Published by Elsevier B.V.)

Ehrlichia canis has gained importance over the years as a zoonotic bacterium, nevertheless in Mexico is unknown the extent of the problem in animals and public health. The country had a few studies carried out locally using serology and molecular tests as diagnostic methods. Ehrlichiosis is not considered endemic in the central valley of Mexico, because the climatic conditions in the region have not allowed the vector (Rhipicephalus sanguineus) to establish itself adequately, therefore, diagnosis is not used in clinical practice in this area. A nested PCR (nPCR) offers rapid results with high sensitivity and specificity regardless of cost. The use of a recombinant positive control provides the advantage of timely diagnosis, follow-up treatment and allows the clinician to decide. In this work, the nPCR reported by Wen et al. (J Clin Microbiol 35(7):1852-2185, 1997) was used for the diagnosis of E. canis by modifying the reaction conditions to improve the detection of the test. We constructed a recombinant positive control to nPCR as diagnostic technique for E. canis, also we modified the reaction conditions to improve detection of the test which allowed the diagnosis of E. canis in dogs in the Mexican Republic using 53 samples from dogs with positive serological diagnosis of Ehrlichiosis, some of them from the valley of Mexico. Currently, this nPCR is offered to public at the Faculty of Veterinary Medicine and Zootechnics of the National Autonomous University of Mexico at an accessible cost and allows to begin to generate epidemiological information to know distribution of the bacterium., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Canine monocytic ehrlichiosis (CME) is caused by the tick-borne pathogen Ehrlichia canis, an obligate intracellular Gram-negative bacterium of the family Anaplasmataceae with tropism for canine monocytes and macrophages. The trp36 gene, which encodes for the major immunoreactive protein TRP36 in E. canis, has been successfully used to characterize the genetic diversity of this pathogen in different regions of the world. Based on trp36 sequence analysis, four E. canis genogroups, United States (US), Taiwan (TWN), Brazil (BR) and Costa Rica (CR), have been identified. The aim of this study was to characterize the genetic diversity of E. canis in Cuba based on the trp36 gene., Methods: Whole blood samples (n = 8) were collected from dogs found to be infested with the tick vector Rhipicephalus sanguineus sensu lato (s.l.) and/or presenting clinical signs and symptoms of CME. Total DNA was extracted from the blood samples and trp36 fragments were amplified by PCR. Nucleotide and protein sequences were compared using alignments and phylogenetic analysis., Results: Four of the trp36 sequences obtained (n = 8) fall within the phylogenetic cluster grouping the US genogroup E. canis strains. The other E. canis trp36 sequences formed a separate and well-supported clade (94% bootstrap value) that is phylogenetically distant from the other major groups and thus represents a new genogroup, herein designated as the 'Cuba (CUB) genogroup'. Notably, dogs infected with the CUB genogroup presented frequent hemorrhagic lesions., Conclusions: The results of this study suggest that genetic diversification of E. canis in Cuba is associated with the emergence of E. canis strains with increased virulence., (© 2022. The Author(s).)

Recent surveys in Southeast Asia, including Cambodia, have identified canine vector-borne pathogens (VBPs), including those with zoonotic potential, as highly prevalent. The lack of veterinary care alongside the close association semidomesticated dogs have with humans in the region exacerbates these zoonotic risks. Nonetheless, the number of studies investigating such pathogens and the threats they pose to dog and human health is limited. Here, we utilize a next-generation sequencing (NGS)-based metabarcoding protocol to conduct an assumption-free characterization of the bacterial, apicomplexan, and kinetoplastid blood-borne pathogens of free-roaming dogs from across Cambodia. From 467 dogs at five field sites, 62% were infected with one of eight confirmed pathogens, comprising Anaplasma platys (32%), Ehrlichia canis (20%), Hepatozoon canis (18%), Babesia vogeli (14%), Mycoplasma haemocanis (13%), the zoonotic pathogen Bartonella clarridgeiae (3%), Candidatus Mycoplasma haematoparvum (0.2%), and Trypanosoma evansi (0.2%). Coinfections of between two and four VBPs were common with 28% of dogs found to have a mixed infection. Moreover, DNA from putatively infectious agents belonging to the bacterial family and genera Coxiella, Mycobacterium, Neisseria, Rickettsiaceae, Treponema, and two uncharacterized Mycoplasma species were identified, in addition to protozoan genera Colpodella, Parabodo, and Bodo. Using a multiple logistic regression model, the presence of ectoparasites, abnormal mucous membranes, anemia, and total protein were found as predictors of canine VBP exposure. This study represents the first time an NGS metabarcoding technique has been used to holistically detect the bacterial and protozoan hemoparasites communities of dogs through an in-depth survey, highlighting the power of such methods to unearth a wide spectrum of pathogenic organisms in an unbiased manner., (© 2021 Wiley-VCH GmbH.)

Canine monocytic ehrlichiosis caused by Ehrlichia canis infection is a life-threatening vector-borne disease in dogs worldwide. Routine blood smear has very low sensitivity and cannot accurately provide a quantitative result. Conventional PCR (cPCR) and real-time PCR (qPCR) are widely used as molecular methods for E. canis detection. qPCR is quantitative but relies on standard curves of known samples. To overcome this difficulty, this study developed a new E. canis quantitative detection method, using droplet digital polymerase chain reaction (ddPCR). ddPCR was evaluated against cPCR and blood smears. PCR amplicons and genomic DNA (gDNA) from 12 microscopic positive samples were used to identify the limits of detection (LODs) in ddPCR and cPCR. Our ddPCR was assessed in 92 field samples, it was compared with cPCR and blood smears. ddPCR showed LOD=1.6 copies/reaction, or 78 times more sensitive than cPCR (LOD=126 copies/reaction), using PCR amplicons as a template, whereas both ddPCR and cPCR had equal LODs at 0.02 ng gDNA/reaction. In addition, ddPCR had 100% sensitivity and 75% specificity for E. canis detection compared to cPCR and no cross-reaction with other blood pathogens was observed. ddPCR identified more positive samples than cPCR and blood smear. ddPCR improved the overall performance of E. canis detection, with a better LOD and comparable sensitivity and specificity to cPCR. The technique might be helpful for diagnosis of E. canis in light infection, evaluating the number of E. canis and follow-up after treatment.

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., (Copyright © 2022. Published by Elsevier GmbH.)

Stray dogs may be highly exposed to vector-borne pathogens (VBPs), including zoonotic agents, and therefore may pose a high risk of spreading infections to other animals and humans. Among the Anaplasmataceae, Anaplasma phagocytophilum, A. platys and Ehrlichia canis are commonly identified species in dogs in Europe; however, information on the occurrence of these pathogens in canine populations from Bosnia and Herzegovina (B&H) is still lacking. Thus, the aim of this study was to determine the seroprevalence of Anaplasma spp. and Ehrlichia spp. in stray dogs in the Sarajevo region of B&H and to identify A. phagocytophilum, A. platys, E. canis and E. ewingii by molecular techniques. A total of 903 blood samples of stray dogs were screened by SNAP 4Dx Plus Test for the presence of antibodies against A. phagocytophilum/A. platys and E. canis/E. ewingii. Real-time PCR assays were performed for the detection of Anaplasmataceae, A. phagocytophilum, A. platys, E. canis and E. ewingii in seropositive dogs. Antibodies to A. phagocytophilum/A. platys and/or E. canis/E. ewingii were detected in 187 (20.7%) samples. Seroprevalence was highest for A. phagocytophilum/A. platys (184/903, 20.4%). Two dogs had antibodies to E. canis/E. ewingii, while one dog was found to have antibodies to A. phagocytophilum/A. platys and to E. canis/E. ewingii. Forty-eight (25.7%) of the 187 seropositive dogs examined by Real-time PCR were positive for Anaplasmataceae. A. phagocytophilum was detected in 45 (24%) samples, while one sample was positive for A. phagocytophilum and A. platys. Two samples positive for Anaplasmataceae tested negative in the species-specific PCRs. E. canis or E. ewingii could not be detected in any of the Ehrlichia-seropositive dogs. These findings highlight the need for dog health monitoring, improving the health and welfare of stray dog population, and establishment of effective surveillance systems to combat VBDs., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Anaplasma platys and Ehrlichia canis are members of the Anaplasmataceae family that cause disease in dogs and are mainly transmitted by Rhipicephalus sanguineus species group ticks. We performed a cross-sectional study on these pathogens across six bioclimatic regions of Chile, including 719 free-ranging rural dogs, 132 Andean foxes (Lycalopex culpaeus), and 82 South American gray foxes (Lycalopex griseus). Dog and fox blood samples were first screened for DNA of Anaplasmataceae followed by two Ehrlichia-specific protocols. Antibodies against Anaplasma sp. and E. canis were assessed by immunofluorescence in dogs. Ectoparasites were collected and identified, with the determination of the lineages of the Rhipicephalus sanguineus species group by molecular and phylogenetic analyses. Finally, potential risk factors for infection were investigated across the different bioclimatic regions and host species. All DNA amplicons obtained from the screening protocol corresponded to Anaplasma platys. The occurrence of both A. platys DNA and antibodies was confirmed in all six bioclimatic regions, except for regions at high altitude and/or without either R. sanguineus species group lineage present. Dogs infested with R. sanguineus ticks were significantly more prone to be infected and exposed to Anaplasma spp. Prevalence of DNA was significantly higher in juvenile (19%) than in adult dogs (9%), whereas the opposite was found for seroprevalence (19% versus 35%, respectively). Overall prevalence of A. platys DNA was higher in dogs (11%) than in foxes (4%), probably owing to markedly lower tick infestations in the foxes. Ehrlichia canis DNA was not detected in any sample, and antibodies against this pathogen were detected only in four dogs, in areas with both R. sanguineus lineages present. Free-ranging dogs in Chile could be favoring the maintenance of A. platys in all areas suitable for its tick vector. Although apparently infrequent, spillovers from dogs to foxes may be taking place and should be considered in management plans in Chile., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Anaplasma platys is a tick-transmitted rickettsial pathogen, which is known to be the etiologic agent for cyclic thrombocytopenia in its primary canine host. Infections with this pathogen are also reported in cats, cattle and people. Similarly, Ehrlichia canis is another tick-borne rickettsial pathogen responsible for canine monocytic ehrlichiosis and is also reported to cause infections in people. We describe infections in dogs with these two pathogens on the Caribbean island of Grenada, West Indies by detection using molecular methods. We utilized a 16S rRNA gene-based PCR assay to detect both Ehrlichia and Anaplasma species by screening 155 canine blood samples from asymptomatic dogs. We found 18.7 % of the dogs to be positive for A. platys and 16.8 % for E. canis. Samples that tested positive for A. platys were further assessed by sequence analysis targeting 16S rRNA, 23S rRNA, citrate synthase (gltA) and heat shock protein (groEL) genes. Phylogenetic analysis revealed high correlation of A. platys 16S rRNA and gltA gene sequences with the geographic origins, while 23S rRNA and groEL gene sequences clustered independent of the geographic origins. This study represents an important step in defining the widespread distribution of active rickettsial infections in Caribbean dogs with no apparent clinical signs, thus posing a high risk for canine health and to a lesser extent to humans, as most dogs in the Caribbean are free-roaming., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Present-day diagnostic tools and technologies for canine diseases and other vector-borne parasitic diseases hardly meet the requirements of an efficient and rapid diagnostic tool, which can be suitable for use at the point-of-care in resource-limited settings. Loop-mediated isothermal amplification (LAMP) technique has been always a method of choice in the development and validation of quick, precise, and sensitive diagnostic assays for pathogen detection and to reorganize point-of-care (POC) molecular diagnostics. In this study, we have demonstrated an efficient detection system for parasitic vector-borne pathogens like Ehrlichia canis and Hepatozoon canis by linking the LAMP assay to a smartphone via a simple, inexpensive, and a portable "LAMP box," All the components of the LAMP box were connected to each other wirelessly. This LAMP box was made up of an isothermal heating pad mounted below an aluminum base which served as a platform for the reaction tubes and LAMP assay. The entire setup could be connected to a smartphone via an inbuilt Wi-Fi that allowed the user to establish the connection to control the LAMP box. A 5 V USB power source was used as a power supply. The sensitivity of the LAMP assay was estimated to be up to 10 -6 dilution limit using the amplified, purified, and quantified specific DNA templates. It can also serve as an efficient diagnostic platform for many other veterinary infectious or parasitic diseases of zoonotic origin majorly towards field-based diagnostics.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Ehrlichia canis is among the most prevalent tick-borne pathogens infecting dogs worldwide, being primarily vectored by brown dog ticks, Rhipicephalus sanguineus sensu lato (s.l.). The genetic variability of E. canis has been assessed by analysis of different genes (e.g., disulfide bond formation protein gene, glycoprotein 19, tandem repeat protein 36 - TRP36) in the Americas, Africa, Asia, and in a single dog sample from Europe (i.e., Spain). This study was aimed to assess the variations in the TRP36 gene of E. canis detected in naturally infected canids and R. sanguineus s.l. ticks from different countries in Asia and Europe. DNA samples from dogs (n = 644), foxes (n = 146), and R. sanguineus s.l. ticks (n = 658) from Austria, Italy, Iran, Pakistan, India, Indonesia, Malaysia, the Philippines, Singapore, Thailand, Vietnam, and Taiwan were included in this study. Ehrlichia canis 16S rRNA positive samples (n = 115 from the previous studies; n = 14 from Austria in this study) were selected for molecular examination by analyses of TRP36 gene. Out of 129 E. canis 16S rRNA positive samples from dogs (n = 88), foxes (n = 7), and R. sanguineus s.l. ticks (n = 34), the TRP36 gene was successfully amplified from 52. The phylogenetic analysis of the TRP36 pre-repeat, tandem repeat, and post repeat regions showed that most samples were genetically close to the United States genogroup, whereas two samples from Austria and one from Pakistan clustered within the Taiwan genogroup. TRP36 sequences from all samples presented a high conserved nucleotide sequence in the tandem repeat region (from 6 to 20 copies), encoding for nine amino acids (i.e., TEDSVSAPA). Our results confirm the US genogroup as the most frequent group in dogs and ticks tested herein, whereas the Taiwan genogroup was present in a lower frequency. Besides, this study described for the first time the US genogroup in red foxes, thus revealing that these canids share identical strains with domestic dogs and R. sanguineus s.l. ticks., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Ehrlichia canis infections have been reported in humans in Venezuela and Costa Rica. In this study, 506 healthy residents and 114 dogs from four municipalities (Cauca, Colombia) were surveyed and blood samples collected. Antibodies to E. canis in human and canine sera were evaluated using the Tandem repeat protein 19 (TRP19) peptide ELISA and indirect immunofluorescence assay (IFA). Ehrlichia canis TRP19 antibodies were detected in only 1/506 human sera, but the single positive sample was negative by IFA. The majority (75/114; 66%) of dogs surveyed had antibodies to the E. canis TRP19 peptide by ELISA, and eight randomly selected sera were further confirmed by E. canis IFA. Genomic DNA samples obtained from 73 E. canis TRP19 ELISA-positive dog blood samples were examined by PCR targeting the 16S ribosomal ribonucleic acid (rRNA) gene. Ehrlichia canis 16S rRNA was amplified in 30 (41%) of the dogs, and 16 amplicons were selected for DNA sequencing, which confirmed that all were E. canis. A second PCR was performed on the 16 confirmed E. canis 16S rRNA PCR-positive samples to determine the TRP36 genotype by amplifying the trp36 gene. TRP36 PCR amplicon sequencing identified nine dogs infected with the U.S. E. canis TRP36 genotype (56%), one dog with the Brazilian genotype (6%), and six dogs with the Costa Rican genotype (38%). Moreover, these molecular genotype signatures were consistent with serologic analysis using TRP36 genotype-specific peptides. Notably, there was no serologic evidence of E. canis infection in humans, suggesting that E. canis infection in dogs in Cauca is not associated with zoonotic human infection.

Canine monocytic ehrlichiosis (CME), caused by transmitted Ehrlichia canis infection, is a major disease in dogs with worldwide distribution. Herein, a nucleic acid assay was established for the identification of E. canis infection employing a fluorescently labeled conformationally constrained pyrrolidinyl PNA probe (Flu-acpcPNA) designed to sequence-specifically target the 16S rRNA gene. The sensing principle is based on the excellent quenching ability of graphene oxide (GO) of the free PNA probe, that was diminished upon binding to the DNA target. The addition of DNase I improved the performance of the detection system by eliminating the nonspecific quenching capability of long-chain dsDNA and thus enhancing the fluorescence signaling. The assay was coupled with a recombinase polymerase amplification (RPA) technique, which could be performed under isothermal conditions (37 °C) without DNA denaturation and purification steps. The established method is simple to set up and execute, proving a rapid, specific, and sensitive detection of 16S rRNA gene of E. canis with a limit of detection at least 11.1 pM. This technique shows good potential for the visual detection of double-stranded DNA targets without the need for PCR or complicated instruments, which shows great promise for practical usage in resource limited areas.

The tick vector Rhipicephalus sanguineus is established as a complex of closely related species with high veterinary-medical significance, in which the presence of different genetic, morphological, and biological traits has resulted in the recognition of different lineages within taxa. One of the most striking differences in the "temperate" and "tropical" lineages of  R. sanguineus (s.l.) is the vector competence to Ehrlichia canis , suggesting that these ticks tolerate and react differently to pathogen infection. The present study addresses the SG and MG proteome of the R. sanguineus tropical and temperate lineages and compares their proteomic profile during E. canis infection. Batches of nymphs from the two lineages were allowed to feed on naïve and experimentally E. canis infected dogs and after molting, adults were dissected, and salivary glands and midgut tissues separated. Samples were screened for the presence of E. canis before proteomic analyses. The representation of the proteins identified in infected and non-infected tissues of each lineage was compared and gene ontology used for protein classification. Results highlight important differences in those proteomic profiles that added to previous reported genetic, biological, behavioral, and morphological differences, strengthening the hypothesis of the existence of two different species. Comparing infected and non-infected tissues, the results show that, while in midgut tissues the response to  E. canis  infection is similar in the salivary glands, the two lineages show a different pattern of protein representation. Focusing on the proteins found only in the infected condition, the data suggests that the cement cone produced during tick feeding may be implicated in pathogen infection. This study adds useful information to the debate on the controversial R. sanguineus systematic status, to the discussion related with the different vectorial competence occurring between the two lineages and identifies potential targets for efficient tick and tick-borne disease control., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sanches, Villar, Couto, Ferrolho, Fernández de Mera, André, Barros-Battesti, Machado, Bechara, Mateos-Hernández, de la Fuente, Antunes and Domingos.)

Tick-borne pathogens (TBPs) in dogs have attracted much attention over the last decade since some are now known to be zoonotic and pose a threat to both animal and human health sectors. Despite the increase in the number of studies on canine TBPs worldwide, only a few studies have been conducted in resource-limited countries where research priority is given to food animals than companion animals. In the present study, the occurrence of TBPs of the genera Babesia, Hepatozoon, Anaplasma, and Ehrlichia was investigated in 209 owned and stray dogs in three major cities in Malawi through molecular techniques. Among the examined dogs, 93 (44.5%) were infected with at least one TBP. The detection rates were 23.1% for Babesia rossi, 2.9% for B. vogeli, 19.1% for Hepatozoon canis, 2.4% for Anaplasma platys, and 3.8% for Ehrlichia canis. This is the first molecular study that has provided evidence that dogs in Malawi are infected with TBPs. Sensitization is required for veterinary practitioners, dog handlers, and pet owners as the detected pathogens affect the animals' wellbeing. Further studies focusing on rural areas with limited or no access to veterinary care are required to ascertain the extent of the TBP infection in dogs.

A multiplex PCR assay was standardized and evaluated to simultaneously detect the DNA of Babesia vogeli, Ehrlichia canis and Hepatozoon canis in dogs of selected districts of Punjab state, India. Amplicons of 602 bp, 380 bp and 306 bp corresponding to B. vogeli (18S rRNA gene), E. canis (VirB9 gene), and H. canis (18S rRNA gene) were obtained, without any non-specific amplification. The results of multiplex PCR assay were further compared with the corresponding singleplex PCR assay. The diagnostic sensitivity and specificity of multiplex PCR assay with respect to singleplex PCR assay in the detection of B. vogeli, E. canis and H. canis varied from 50% to 100% and 92.08% to 98.79%, respectively revealing "moderate" to "very good" agreement by kappa value statistics. Blood samples from 322 dogs collected from selected districts of Punjab state, India, when screened by microscopy revealed the prevalence of B. vogeli, E. canis and H. canis as 0.31%, 0.93% and 1.86%, respectively whereas with multiplex PCR assay the values were 0.93%, 10.24% and 4.65%, respectively, with concurrent infection of E. canis & H. canis (1.86%) and B. vogeli & E. canis (0.31%). The diagnostic sensitivity and specificity of multiplex PCR assay with respect to microscopy in the detection of B. vogeli, E. canis and H. canis varied from 69.15% to 100% and 85.11% to 92.33%, respectively revealing "fair" agreement by kappa value statistics and the data was statistically significant. The analytical sensitivity of multiplex PCR assay in the detection of B. vogeli, E. canis and H. canis was 100 pg, 10 pg and 0.1 pg, respectively, whereas the values for the singleplex counterpart were 0.1 pg, 0.01 pg and 0.01 pg. Furthermore, various risk factors viz. age, breed, sex, season and districts were non-significantly associated with the prevalence of these haemoparasites except for E. canis that revealed a significant association with districts by multiplex PCR assay. Therefore the multiplex PCR assay developed may be useful in identification of the aetiological agents of these diseases during their early phase, which may in turn be useful in development of better health care and appropriate treatment of suspected dogs, particularly in endemic regions., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Nowadays, Ehrlichia canis receives increasing attention because of its great morbidity and mortality in animals. Dogs in the subclinical and chronic phases can be asymptomatic, and serological tests show cross-reactivity and fail to differentiate between current and past infections. Moreover, there could be low parasitaemia, and E. canis might be found only in target organs, hence causing results to be negative by polymerase chain reaction (PCR) on blood samples., Methods: We evaluated by PCR the prevalence of E. canis in blood, liver, spleen, lymph node and bone marrow samples of 59 recently euthanised dogs that had ticks but were clinically healthy., Results: In total, 52.55% of the blood PCRs for E. canis were negative, yet 61.30% yielded positive results from tissue biopsies and were as follows: 63.15% from bone marrow; 52.63% from liver; 47.36% from spleen; and 15.78% from lymph node. In addition, 33% had infection in three tissues (spleen, liver and bone marrow)., Conclusions: Our results show the prevalence of E. canis from tissues of dogs that were negative by blood PCR. Ehrlichia canis DNA in tissue was 30% lower in dogs that tested negative in PCR of blood samples compared to those that were positive. However, it must be taken into account that some dogs with negative results were positive for E. canis in other tissues.

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.

Folate pathways components were demonstrated to be present in RNA-sequencing data obtained from uninfected and pathogen-infected Rhipicephalus ticks. Here, PCR and qPCR allowed the identification of folate-related genes in Rhipicephalus spp. ticks and in the tick cell line IDE8. Genes coding for GTP cyclohydrolase I (gch-I), thymidylate synthase (ts) and 6-pyrovoyltetrahydropterin (ptps) were identified. Differential gene expression was evaluated by qPCR between uninfected and infected samples of four biological systems, showing significant upregulation and largest fold-change for the gch-I gene in the majority of the biological systems, supporting the selection for functional analysis by RNAi silencing. Efficient knockdown of the gch-I gene in uninfected and Ehrlichia canis-infected IDE8 cells showed no detectable impact on the capacity of the bacteria to invade or replicate in the tick cells. Overall, this work demonstrated an increase in the expression of some folate-related genes, though not always statistically significantly, in the presence of infection, suggesting gene expression modulation of these pathways, either as a tick response to an invader or manipulation of the tick cell machinery by the pathogens to their advantage. This discovery points to folate pathways as interesting targets for further studies., (© 2019 Blackwell Verlag GmbH.)

Climatic changes have influenced the temporal and spatial distribution of diseases. In livestock-grazing areas, rodents are reservoirs of zoonotic pathogens; therefore, they play an important role in the transmission of diseases affecting domestic animals and humans. The objective of this study was to investigate the presence of the zoonotic agents: Anaplasma phagocytophilum, Borrelia burgdorferi, Ehrlichia canis and Rickettsia rickettsii, as well as the presence of viral RNA from the Bunyaviridae, Togaviridae and Flaviviridae families, in wild rodents from animal production units in central Mexico. The samples were obtained from wild rodents that had access and contact with animal production units. A total of 92 rodents were captured, and samples of blood, serum and organs, such as spleen, kidney, heart and liver, were obtained. The serum was used to detect antibodies against Anaplasma phagocytophilum, Borrelia burgdorferi, Ehrlichia canis and Rickettsia rickettsii by an immunofluorescence antibody test (IFAT); the blood was used for PCR analysis; and the organs were used to obtain RNA (cDNA) to perform RT-PCR. By IFAT, all samples were positive to A. phagocytophilum and E. canis, and negative to B. burgdorferi and R. rickettsii. The samples that were positive to IFAT were used to confirm the presence of pathogen by PCR analysis. The results from the PCR were as follows: 34 samples were positive to A. phagocytophilum, and 59 to E. canis. There was no amplification of genetic material from the Bunyaviridae, Flaviviridae and Togaviridae virus families from the organs that were sampled, which suggests that the samples obtained did not contain RNA specific to these families. This is the first immuno-molecular prospecting study on vector-borne diseases in central Mexico demonstrating the presence of A. phagocytophilum and E. canis in wild rodents living in cattle grazing areas., (© 2020 Blackwell Verlag GmbH.)

Ehrlichia canis is the etiologic agent of a highly prevalent tick-borne disease, canine monocytic ehrlichiosis (CME). Four defined E. canis genotypes based on the trp36 gene sequences have been reported, three of them identified in North or South America. The diversity of E. canis has been investigated using genetic and serologic approaches based on distinct 36 kDa tandem repeat protein (trp36) gene sequences that have been reported. The main objectives of this study were to determine the prevalence of E. canis infection in dogs from Medellín, Colombia by PCR and determine the E. canis diversity using molecular and serologic approaches. Blood was collected from dogs (n = 300) with clinical signs of CME for PCR detection of E. canis 16S rRNA, dsb and trp36 DNA. Phylogenetic analysis of trp36 gene sequences was performed using MEGA. A serological evaluation was performed using immunofluorescence microscopy and ELISA with species-specific peptides from E. canis TRP19 and TRP36 (3 genotypes) and E. chaffeensis (TRP32). E. canis DNA (16S rRNA and/or dsb) was detected in 18 % (53/300) of dogs by PCR amplification. The trp36 gene was amplified and sequenced from 35/53 16S rRNA/dsb PCR positive samples revealing three genotypes: United States (US; n = 21), Costa Rica (CR; n = 11), and Brazil (BR; n = 3). Most dogs (33/35) with detectable trp36 DNA had anti-E. canis TRP19 and TRP36 peptide antibodies that corresponded to the genotype detected by PCR. Dogs that had antibodies to the TRP19 peptide (82/300; 38 %), also had antibodies to one or more genotype-specific TRP36 peptides. Based on TRP36 serology, the dogs exhibited highest frequency of infection with the US genogroup (US = 26), followed by the CR genogroup (CR = 19) and the BR genogroup (BR = 11). Notably, 26/53 trp36 PCR positive dogs had detectable antibodies to multiple E. canis genotypes (US/BR/CR = 8, BR/CR = 7, US/CR = 6 and US/BR = 5) suggesting coinfection or multiple sequential infections with different genotypes. Colombian dogs did not have antibodies to E. chaffeensis as determined by a TRP32 species-specific ELISA. Our results demonstrate the presence of three previously defined genotypes in North and South America in Colombian dogs (US, BR, CR). These results also demonstrate that TRP19 and TRP36 serology can provide valuable information regarding E. canis exposure and the potential genotype(s) involved in infection., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Canine vector-borne diseases (CVBDs) pose a major health problem in dogs globally, with the potential to cause zoonoses, in particular in developing countries where scientific knowledge on the topic is minimal. Blood samples and ticks were collected from stray dogs in Tamil Nadu, South India to assess the prevalence of CVBD-causing pathogens (Anaplasma spp., Babesia spp., Ehrlichia spp., Hepatozoon spp., filarioids and Leishmania spp.). Of the 230 dogs examined, 229 (99.6%) were infested by ticks (mean intensity, 5.65) with Rhipicephalus sanguineus sensu lato and Rhipicephalus haemaphysaloides being morphologically identified in the 98.3% and 1.7% of the infested dogs, respectively. Overall, the 67.8% (n = 156) of dogs was positive for at least one pathogen with Hepatozoon canis being the most prevalent (37.8%) followed by Anaplasma platys (22.6%), Ehrlichia canis (16.1%) Babesia vogeli (10%), Anaplasma phagocytophilum (0.4%) and Babesia gibsoni (0.4%). Two filarioids (Dirofilaria sp. "hongkongensis" and Brugia malayi, 0.4%) were diagnosed in sampled animals. Co-infection with H. canis and A. platys was the most prevalent (8.3%, P = 0.00001), whilst all animals scored negative for Leishmania spp.. Out of 295 ticks analysed, 215 R. sanguineus s.l. (75.4%) and 8 R. haemaphysaloides (88.9%) were positive for at least one pathogen with H. canis as the predominant species (42.5%), followed by A. platys (33.8%), E. canis (16.9%), B. vogeli (3.8%) and A. phagocytophilum (0.3%). Fifty-six dogs (35.9%) harboured the same pathogen as the respective tick specimens, while 29 dogs (18.6%) had a different pathogen. Thirteen sequence types (STs) were identified for H. canis, with ST2 (49.4%) as the most representative in dogs and ST1 (73.5%) in ticks. Similarly, seven STs were found for Anaplasma spp. (i.e., five for A. platys, one for A. phagocytophilum and one for Anaplasma sp.), with ST2 as the most representative in dogs (70.6%) and ST3 (52.5%) in ticks for A. platys. Only one ST was identified for B. vogeli, B. gibsoni, E. canis, D. sp. "hongkongensis" and B. malayi. Regular surveillance and adoption of adequate treatment and control measures are needed to reduce the risk of disease-causing pathogens in stray dogs and of pathogens with zoonotic potential., Competing Interests: Declaration of Competing Interest The authors declare no competing interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The aim of the present study was to detect Cercopithifilaria bainae and other tick-borne pathogens and to perform molecular characterization of the tick Rhipicephalus sanguineus s.l. collected from dogs. Ticks (n = 432, including 8 larvae, 59 nymphs, and 365 adults) were sampled from domiciled dogs (n = 73) living in Campo Grande, Mato Grosso do Sul (Midwest Brazil). All ticks were morphologically identified as R. sanguineus. Genomic DNA was extracted in pools (three to five ticks per animal) and was used for definition of R. sanguineus haplotypes (based on 16S rRNA analysis) and pathogen identification (Cercopithifilaria sp., Ehrlichia canis, Anaplasma platys, Hepatozoon canis, Babesia vogeli and Rickettsia spp.). Rhipicephal us sanguineus specimens were identified as haplotypes A and B. DNA of Cercopithifilaria bainae (43.83%; 32/73), Ehrlichia canis (24.65%; 18/73), Anaplasma platys (19.17%; 14/73), and Hepatozoon canis (5.47%; 4/73) was detected. The identity of pathogens was confirmed by DNA sequence analysis. The present study confirms the presence of haplotypes A and B of R. sanguineus in the state of Mato Grosso do Sul and its importance as a vector of several pathogens of veterinary concern. Finally, this is the first report to identify C. bainae in ticks in the Midwestern region of Brazil.

Ehrlichia canis is the common blood pathogen infected dogs in Thailand that significantly affect dog health and caused canine monocytic ehrlichiosis which leads to anaemia, high morbidity also mortality rates. This study was performed to analyse associated risk factors and evaluate the significance of haematological responses of dogs infected with E. canis in Phitsanulok province, the northern part of Thailand. Blood samples were collected from 94 dogs, 27 (28.7%) dogs have been confirmed E. canis infection by nested PCR method. Mostly of infected dogs had hypohemoglobinemia (<12.1 g/dl), leucocytosis (>15.5×10 3 /μl), neutrophilia (>10.6×10 3 /μl) and thrombocytopenia (<170×10 3 /μl). However, only thrombocytopenia was statistically different between E. canis infected and non-infected groups. Additionally, no significant statistical relationship between E. canis infection rate and sex, age or breed apparent. These data supported that infection with E. canis is endemic in dogs and thrombocytopenia may highlight during infection which reliability to use in the clinical diagnosis of E. canis infection.

Rickettsial infections in dogs of Mexico were investigated. A total of 246 dogs were blood sampled and initially screened to detect Ehrlichia canis, E. chaffeensis, E. ewingii, Anaplasma phagocytophilum and Rickettsia rickettsii by a quantitative real-time PCR (qPCR) assay. Sixty-five dogs were monitored and sampled twice 7-8 months apart. Using the qPCR, 72 positive dogs to E. canis were detected (prevalence of 29.26%). These dogs were also tested by nested PCR to detect the same pathogens. None of the studied dogs were positive to E. chaffeensis, E. ewingii, R. rickettsii nor A. phagocytophilum by both PCR assays. The cumulative incidence of E. canis infection was 38.46%. Sequencing analysis of the nested PCR products revealed 100% and 98.1% identity of E. canis and R. parkeri, respectively. We found a dog co-infected with E. canis and R. parkeri., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Ehrlichia canis and Anaplasma platys are intracellular tick-transmitted bacteria that infect dogs; there is evidence for limited zoonotic potential as well. The prevalence of E. canis in Colombia has been evaluated in different regions; however little is known about the prevalence or distribution of A. platys. Neither pathogen has been studied in the Magdalena region, thus the purpose of our study was to assess the prevalence of these pathogens in dogs attending veterinary clinics from the cities of Santa Marta and Ciénaga, and to assess possible associated risk factors for infection. A. platys and E. canis infections in blood were evaluated by Taqman PCR assays. E. canis was detected in 26/170 (15.3%, 95% CI 10.4%-21.8%) and A. platys in 34/168 (20.2%, 95% CI 14.6%-27.3%) of all dogs tested. Eleven dogs (6.5%, 95% CI 3.4-11.7%) were coinfected with both pathogens. Sequencing results showed low diversity within E. canis and within A. platys strains, however a strain of E. canis detected in our study area is genetically distinct from strains reported in another city of Colombia. Our results suggest that for A. platys, Santa Marta dogs were at greater risk than Ciénaga dogs, and that purebred dogs were at slightly lower risk in both areas. The confirmation of these pathogens in northern Colombia should cause concern for the possible co-transmission of these agents to humans or animals in the region., (Copyright © 2019 Elsevier B.V. All rights reserved.)