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.

Isothermal Detection of Canine Blood Parasite (Ehrlichia canis) Utilizing Recombinase Polymerase Amplification Coupled with Graphene Oxide Quenching-Based Pyrrolidinyl Peptide Nucleic Acid.

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.

An innovative and user-friendly smartphone-assisted molecular diagnostic approach for rapid detection of canine vector-borne diseases.

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.

Molecular detection and characterization of tick-borne hemoparasites and Anaplasmataceae in dogs in major cities of Malawi.

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.

Molecular epidemiology of parasitic protozoa and Ehrlichia canis in wildlife in Madrid (central Spain).

Wildlife species are involved in the transmission of diverse pathogens. This study aimed to monitor raccoons (Procyon lotor), American minks (Neovison vison), and red foxes (Vulpes vulpes) as potential reservoirs in central Spain. Specifically, 200 spleen and fecal samples (from 194 raccoons, 3 minks, and 3 foxes) were analyzed molecularly by PCR/qPCR and sequencing for the presence of piroplasmids, Hepatozoon spp., Toxoplasma gondii, and Ehrlichia canis infections in the Community of Madrid (Spain). Biological samples were obtained in the years 2014, 2015, and 2016. No pathogen DNA was found in fecal samples. In contrast, analysis of raccoon spleen samples revealed that Toxoplasma was the most prevalent pathogen (prevalence 3.6 ± 2.6%), followed by Hepatozoon canis and E. canis (each with a prevalence of 2.57 ± 2.2%). Hepatozoon canis was also diagnosed in all three of the analyzed foxes. Analysis of yearly prevalence showed that tick-borne pathogens were less frequent in raccoon in 2015, a dry and warm year compared both to 2014 and 2016. These data suggest that fecal PCR assays are unsuitable for detection of DNA of non-erythrocytic pathogens. Furthermore, they demonstrate that the raccoon (an invasive species often living in proximity to domestic areas) and the red fox are putative reservoirs for pathogenic organisms in the Community of Madrid.

Detection and molecular characterisation of Ehrlichia canis in naturally infected dogs in South West Nigeria.

Canine ehrlichiosis is an important tick-borne rickettsial disease mainly caused by Ehrlichia canis. This study aimed to detect and characterise E. canis in dogs in Abeokuta, Nigeria by microscopy and nested PCR. Blood samples were collected from 205 dogs, thin smears were made, field-stained, and DNA was extracted from the blood samples. A partial region of the 16S rRNA gene was amplified by polymerase chain reaction (PCR) and sequenced unidirectionally. Ehrlichial morulae were detected in three dogs (1.5%). The PCR test revealed that 47 dogs (22.9%) were positive for E. canis. The lengths of the sequences obtained range from 374 bp to 376 bp with an average G-C content of 37% and 98-99% homology with the reference sequences in GenBank. The aligned autochthonous sequences were less polymorphic. The phylogenetic analysis separated sequences reported previously in Nigeria from the autochthonous sequences. The present work shows that the strain of E. canis detected in the study area is genetically different from those reported in the northern part of Nigeria and more closely related to sequences from Brazil and India.

Molecular survey of Anaplasma platys and Ehrlichia canis in dogs from Campo Grande, Mato Grosso do Sul, Brazil.

This study investigated the frequency of infection by Anaplasma platys and Ehrlichia canis in dogs submitted to animal health centers in Campo Grande, state of Mato Grosso do Sul, Brazil. E. canis and A. platys showed infection frequencies of 55.75% and 16.96%, respectively. The identity of the two species was confirmed by DNA sequencing.

Detection of canine vector-borne diseases in eastern Poland by ELISA and PCR.

The aim of the study was to establish the prevalence of Ehrlichia canis, Anaplasma phagocytophilum and Borrelia burgdorferi in dogs in eastern Poland and to determine the factors associated with exposure (seroposity) or infection (PCR). Anti-A. phagocytophilum, anti-B. burgdorferi and anti-E. canis antibodies were determined in 400 dogs, using the SNAP 4Dx ® test (IDEXX Laboratories). In addition, PCRs were performed for the detection of E. canis, A. phagocytophilum and B. burgdorferi DNA. In reference to the risk factor analysis, a regression logistic model was determined for each aetiological agent. The overall seroprevalence was highest for B. burgdorferi (11.0 %), followed by A. phagocytophilum (8.0 %) and E. canis (1.5 %). Eleven healthy dogs were found to be infected with A. phagocytophilum, as determined by PCR, while the remainder were seronegative. For B. burgdorferi, the DNA of the spirochetes was detected in the blood of 20 dogs, while the presence of anti-B. burgdorferi IgG was detected in the sera of ten of these. For E. canis, none of the dogs tested positive by PCR. Tick control was included as a protective factor for A. phagocytophilum and B. burgdorferi, while the origin (rural) was included as a risk factor for B. burgdorferi and A. phagocytophilum infection. In addition, breed (pure) was a risk factor for B. burgdorferi infection, and sex (female) was a risk factor for E. canis.

Development and evaluation of a loop-mediated isothermal amplification assay for detection of Ehrlichia canis DNA in naturally infected dogs using the p30 gene.

Canine monocytic ehrlichiosis (CME) is a common tick-borne disease caused by the rickettsial bacterium Ehrlichia canis (Rickettsiales: Anaplasmataceae). In view of the different stages and variable clinical signs of CME, which can overlap with those of other infections, a conclusive diagnosis can more readily be obtained by combining clinical and hematological evaluations with molecular diagnostic methods. In this study, a loop-mediated isothermal amplification (LAMP) assay targeting the p30 gene of E. canis was developed. The assay was developed using DNA extracted from E. canis-infected cultures of the macrophage cell line DH82 and samples from dogs testing positive for E. canis DNA by PCR. The LAMP assay was compared to a p30-based PCR assay, using DNA extracted from EDTA-anticoagulated blood samples of 137 dogs from an endemic region in Brazil. The LAMP assay was sensitive enough to detect a single copy of the target gene, and identified 74 (54.0%) E. canis DNA-positive samples, while the p30 PCR assay detected 50 positive samples (36.5%) among the field samples. Agreement between the two assays was observed in 42 positive and 55 negative samples. However, 32 positive samples that were not detected by the PCR assay were identified by the LAMP assay, while eight samples identified as E. canis-positive by PCR showed negative results in LAMP. The developed E. canis LAMP assay showed the potential to maximize the use of nucleic acid tests in a veterinary clinical laboratory, and to improve the diagnosis of CME.

Colorimetric detection of Ehrlichia canis via nucleic acid hybridization in gold nano-colloids.

Canine monocytic ehrlichiosis (CME) is a major thick-bone disease of dog caused by Ehrlichia canis. Detection of this causal agent outside the laboratory using conventional methods is not effective enough. Thus an assay for E. canis detection based on the p30 outer membrane protein gene was developed. It was based on the p30 gene amplification using loop-mediated isothermal DNA amplification (LAMP). The primer set specific to six areas within the target gene were designed and tested for their sensitivity and specificity. Detection of DNA signals was based on modulation of gold nanoparticles' surface properties and performing DNA/DNA hybridization using an oligonucleotide probe. Presence of target DNA affected the gold colloid nanoparticles in terms of particle aggregation with a plasmonic color change of the gold colloids from ruby red to purple, visible by the naked eye. All the assay steps were completed within 90 min including DNA extraction without relying on standard laboratory facilities. This method was very specific to target bacteria. Its sensitivity with probe hybridization was sufficient to detect 50 copies of target DNA. This method should provide an alternative choice for point of care control and management of the disease.

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.

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, epidemiological, and hematological evaluation in Ehrlichia canis infected dogs from an endemic region in Egypt.

Background: Canine monocytic ehrlichiosis (CME) is considered a multisystemic, life-threatening, rickettsial, and tick-borne disease that affects canine species and is caused by Ehrlichia canis (E. canis). Clinical signs of CME vary from asymptomatic to severe illness with three clinical phases. E. canis has the potential to infect humans. Aim: This study aimed to provide recent information as there is limited data about the disease in Egypt. Therefore, this work was conducted to study the molecular prevalence of E. canis and evaluate the corresponding risk factors, hematology, biochemistry, and molecular characterization of the genus Ehrlichia and E. canis species among Egyptian dogs. Methods: One hundred eighty dogs of both sexes from 3 months to 8 years from different breeds: stray and foreign breeds were examined for clinical signs in all seasons in two delta governorates: El-Dakahlia and El-Gharbia. Blood samples were collected from dogs for microscopic and haemato-biochemical analysis, and then molecular characterization of the genus Ehrlichia and species-specific E. canis was performed, followed by sequencing and phylogenetic analysis. Results: Out of 180 samples examined by polymerase chain reaction (PCR) assay, 42 (23.33%) were positive for the genus of Ehrlichia and the species-specific E. canis. Only twenty-four dogs (13.33%) were positive for PCR, infested with ticks, and showed fever, anemia, loss of body weight, pale mucous membrane of gum and conjunctiva, blindness, paralysis, hemoglobinuria, and Melena. The univariate logistic regression revealed that all variables, including age, season, tick infestation, hemorrhage from natural orifices, and ectoparasitic treatments per year, showed statistical significance (p ≤ 0.05), except breed and sex, which also did not exhibit any relation between CME infection in multivariate logistic regression. The presence of morulae inside leukocytes in 66 dogs out of the total examined 180 (36.67%), only 39 (59.1%) were positive for morulae and PCR-positive for E. canis. Dogs positive for E. canis suffered from anemia, severe thrombocytopenia, the absolute value of WBCs and their fractions, alanine aminotransferas (ALT), AST, ALKP, γ-GT, total. P, T.BIL, urea, globulin, and creatinine were significantly increased in dogs infected with E. canis when compared to those with negative PCR results, while the levels of albumin and A: G ratios were significantly decreased. Conclusion: The current study proves the existence of E. canis in El-Dakahlia and El-Gharbia governorates, and this is the first large-scale study concerning the epidemiological, clinicopathological examination, molecular characterization, sequencing, and phylogenetic analysis of reported from the center of the Delta of the Nile in Egypt.

Molecular characterization of Ehrlichia canis from naturally infected dogs reveals a novel Asiatic-lineage and co-circulation of multiple lineages in India.

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.

First detection of Rickettsia felis and Ehrlichia canis in the common bed bug Cimex lectularius.

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.

One Health Approach on Ehrlichia canis: Serosurvey of Owners and Dogs, Molecular Detection in Ticks, and Associated Risk Factors in Tick-Infested Households of Southern Brazil.

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.

Detection of Anaplasma spp. and Ehrlichia spp. in dogs from a veterinary teaching hospital in Italy: a retrospective study 2012-2020.

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.

Rhipicephalus sanguineus from Hungarian dogs: Tick identification and detection of tick-borne pathogens.

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.

Molecular Survey of Tick-Borne Haemoparasites of Dogs by Multiplex Polymerase Chain Reaction from Punjab, India.

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.