First detection of Ehrlichia chaffeensis, Ehrlichia canis, and Anaplasma ovis in Rhipicephalus bursa ticks collected from sheep, Turkey.

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.

Microfluidic PCR and network analysis reveals complex tick-borne pathogen interactions in the tropics.

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.

Molecular Evidence of Ehrlichia canis (Rickettsiales: Anaplasmataceae) in Ticks (Ixodida: Ixodidae) Associated with Dogs (Carnivora: Canidae) from Nuevo Leon, Mexico.

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.

Phylogenetic analysis of Ehrlichia species isolated from horses and dogs in Iran.

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.

Molecular survey on vector-borne pathogens in clinically healthy stray cats in Zaragoza (Spain).

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.

Molecular occurrence and genetic diversity of Ehrlichia canis in naturally infected dogs from Thailand.

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.

Molecular Detection of Tick-Borne Pathogens in Dogs from Indigenous Communities, Amazon, Brazil.

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.

Genetic diversity of vector-borne pathogens in ixodid ticks infesting dogs from Pakistan with notes on Ehrlichia canis, Rickettsia raoultii and Dirofilaria immitis detection.

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.

Pleural effusion-related Nocardia otitidiscaviarum, Anaplasma platys and Ehrlichia canis coinfection in a dog.

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.

RPA/CRISPR-cas12a as a specific, sensitive and rapid method for diagnosing Ehrlichia canis and Anaplasma platys in dogs in Thailand.

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.

Molecular detection and genetic characterization of Ehrlichia canis and Ehrlichia sp. in neotropical primates from Brazil.

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.

Molecular evidence of Ehrlichia canis, associated risk factors and hematobiochemical analysis in client owned and shelter cats of Pakistan.

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.

Ehrlichia canis: Molecular characterization and genetic diversity based on the p28 and trp36 genes.

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.

Molecular characterization of Ehrlichia canis and Babesia vogeli reveals multiple genogroups associated with clinical traits in dogs from urban areas of Colombia.

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.

Molecular detection of vector-borne pathogens in semen from dogs in southeastern Brazil.

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.

Use of a recombinant positive control in the diagnostic of canine Ehrlichiosis from 16sRNA gen of Ehrlichia canis in Mexico City.

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.

Novel Ehrlichia canis genogroup in dogs with canine ehrlichiosis in Cuba.

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.

Molecular detection of tick-borne pathogens in Rhipicephalus sanguineus sensu stricto collected from dogs in the steppe and high plateau regions of Algeria.

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.

A newly developed droplet digital PCR for Ehrlichia canis detection: comparisons to conventional PCR and blood smear techniques.

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.

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.