Canine Monocytic Ehrlichiosis Research Articles

A newly developed droplet digital PCR for <i>Ehrlichia canis</i> 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.

Tumor venéreo transmissível canino com remissão espontânea: estudo de caso com ênfase aos exames clínico e citopatológico para monitoramento da evolução tumoral

Abstract The canine transmissible venereal tumor (TVTC) is a neoplasm transmitted mainly through copulation and with a high incidence in stray dogs in Brazil. In the process of tumor evolution of TVTC, the progression, stationary and regression phases are recognized. The host immunity is related to the disease’s biological behavior, however, spontaneous regression observation in cases of naturally occurring TVTC is uncommon. A canine patient was attended, after beeing rescued from the street, due to an ulcerated mass in the external genitália and tick infestation. Cytopathological examination, which diagnosed TVTC, and laboratory tests that showed mild anemia and severe thrombocytopenia were performed. In view of the impossibility of carrying out other exams, it was made the presumptive diagnosis of canine monocytic ehrlichiosis (CME), and treatment was instituted. During follow-up it was observed quick improvement in clinical signs and laboratory changes, as well as a reduction in tumor mass. A new cytopathological evaluation was carried out, and was verified increase in mature lymphocytes and plasmocytes in the midst of the tumor cells, finding compatible with the stationary phase of the disease. From that moment on, it was decided to perform only clinical and cytopathological follow-up. In the following evaluations, continuous clinical remission and cytopathological findings compatible with those described in the regression phase were observed, until its complete remission. It is considered that the improvement in the general health of the patient after the treatment of CME is related to the spontaneous regression of TVTC, and that simultaneous performance of serial clinical and cytopathological exams may be feasible and useful for monitoring the stages of evolution of TVTC.

Clinical and haemato-biochemical observations in dogs naturally infected with Canine monocytic ehrlichiosis

Clinical and haemato-biochemical observations in dogs naturally infected with Canine monocytic ehrlichiosis

Alteration in Serum Concentration of Canine C-Reactive Protein (CRP) Associated with Canine Monocytic Ehrlichiosis (CME) and its Amelioration by Conventional Treatment

Alteration in Serum Concentration of Canine C-Reactive Protein (CRP) Associated with Canine Monocytic Ehrlichiosis (CME) and its Amelioration by Conventional Treatment

Alterações hematológicas e histopatológicas na aplasia medulardecorrente da infecção por Ehrlichia canis em cão da raça Border Collie

The Canine Monocytic Ehrlichiosis (CME) is an infectious disease that commonly affects dogs of all breeds and ages. It is caused by the bacterium Ehrlichia canis and is transmitted by the tick Rhipicephalus sanguineus. The disease may pre-sent itself in the acute, subclinical, and chronic forms. The present study reports the case of a 2-year-old male Border Collie with advanced stage CME, attended at the Pet Clinic of the Veterinary Hospital of the University Federal de Jataí, which resul-ted in medullary aplasia. The diagnosis of marrow aplasia was based on the necroscopic and histopathological examinations. At necropsy, the diaphyses of the long bones were filled with diffuse, strongly whitish and pasty tissue, typical of the adipose tissue, also found in the femoral epiphyses. The histopathology showed unilocular adipose tissue as the major constituent of the bone marrow and rare islands of marrow cells. These findings were compatible with severe hypoplasia of the red bone mar-row and hyperplasia of the white bone marrow, affecting hematopoiesis, resulting in the laboratory alterations observed in the hematocrit, WBC, and plateletogram.

Alteration in Serum Concentration of Canine C-Reactive Protein (CRP) Associated with Canine Monocytic Ehrlichiosis (CME) and its Amelioration by Conventional Treatment

Alteration in Serum Concentration of Canine C-Reactive Protein (CRP) Associated with Canine Monocytic Ehrlichiosis (CME) and its Amelioration by Conventional Treatment

A Case Study of Assessment of Haemato-Biochemical Profile, Clinical Diagnosis and Therapeutic Management of Canine Monocytic Ehrlichiosis (Tropical Canine Pancytopenia) In A Male Doberman DOG

Ehrlichia canis is an obligate intracellular rickettsial agent that is transmitted by a brown dog tick called as Rhipicephalus sanguineus which is considered as the principal vector of this disease. A 3-year-old male Doberman dog weighing 25 kg is presented with the history of inappetence, fever, weakness, anemia, scanty feces, hemoglobinuria, shrunken eye ball with mild corneal opacity and reluctant to walk due to pain in the joints. Clinical examination revealed elevated rectal temperature of 104.30F, tachycardia, increased respiratory rate and pale mucous membrane. Blood and serum samples were collected for hematology and serum biochemistry respectively. On blood smear examination with Giemsa staining, Ehrlichia morulae were noticed in monocytes suggestive of Canine Monocytic Ehrlichiosis / Tropical Canine Pancytopenia.

Molecular detection and characterization of Anaplasma platys and Ehrlichia canis in dogs from the Caribbean

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.

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

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

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.

Magnetic resonance findings of meningoencephalitis in a dog seroreactive to Ehrlichia canis in United Kingdom

AbstractWe describe a novel clinical presentation and imaging findings in a dog seropositive to Ehrlichia canis. A dog presented with an acute non‐ambulatory tetraparesis and severe obtundation. MRI findings showed multifocal intra‐axial changes centered in the grey matter. The lesions caused mild mass effect and leptomeningeal contrast enhancement. The dog responded to treatment with the progressive reduction of antibody titers detected on the indirect fluorescent antibody test and complete resolution of the clinical signs. Canine monocytic ehrlichiosis should be considered a differential in dogs with thrombocytopenia and meningoencephalitis.

Molecular Prevalence and Identification of Ehrlichia canis and Anaplasma platys from Dogs in Nay Pyi Taw Area, Myanmar.

Ticks are vectors of different types of viruses, protozoans, and other microorganisms, which include Gram-negative prokaryotes of the genera Rickettsiales, Ehrlichia, Anaplasma, and Borrelia. Canine monocytic ehrlichiosis caused by Ehrlichia canis and canine cyclic thrombocytopenia caused by Anaplasma platys are of veterinary importance worldwide. In Myanmar, there is limited information concerning tick-borne pathogens, Ehrlichia and Anaplasma spp., as well as genetic characterization of these species. We performed nested PCR for the gltA gene of the genus Ehrlichia spp. and the 16S rRNA gene of the genus Anaplasma spp. with blood samples from 400 apparently healthy dogs in Nay Pyi Taw area. These amplicon sequences were compared with other sequences from GenBank. Among the 400 blood samples from dogs, 3 (0.75%) were positive for E. canis and 1 (0.25%) was positive for A. platys. The partial sequences of the E. canis gltA and A. platys 16SrRNA genes obtained were highly similar to E. canis and A. platys isolated from different other countries.

Epidemiological link between canine monocytic ehrlichiosis caused by Ehrlichia canis and the presence of Rhipicephalus sanguineus sensu stricto in Argentina.

In this work, we analyze data that support an epidemiological link between cases of canine monocytic ehrlichiosis (CME) by Ehrlichia canis and the presence of Rhipicephalus sanguineus sensu stricto as vector in an endemic area for this tick in Argentina. In a blood sample of a 1-year-old toy poodle with CME compatible clinical signs, which showed CME typical morulae in monocytes in Giemsa-stained blood smear, DNA of E. canis was detected by PCR. Further, DNA of E. canis was also detected in a female of R. sanguineus s.s. collected on the infected dog. Rhipicephalus sanguineus s.s. is the only member of the R. sanguineus group that prevails in the study area. The results of this study suggest that R. sanguineus s.s. may play a more important role in the transmission of E. canis than it was assumed so far. The epidemiological link between CME cases and R. sanguineus s.s. as vector in temperate areas of Argentina described in this work contrast previous studies which found that R. sanguineus sensu lato "tropical lineage" (which is absent in the study area) is competent to transmit E. canis but not R. sanguineus s.s.

Epistaksis ve Köpeklerdeki Klinik Yansımaları: 2007-2017 Arasında Kaydedilmiş 34 Vaka

Objective: Epistaxis is a relatively remarkable clinical symptom in dogs that should be included in the differential diagnoses of diseases. Material and Methods: The present author's interest in this subject arose following receipt of several cases within the last 10 years. The purpose of the present article was to evaluate 34 dogs with epistaxis retrospectively. Results: There was bilateral (n=13) or unilateral (n=21) epistaxis, in which chronicity was evident in 13 dogs. Etiology deemed infectious (n=28), non-infectious (n=5) and unknown origin (n=1) causes. The infectious causes involved 13 cases with canine visceral leishmaniasis and other 9 with canine monocytic ehrlichiosis, followed by 6 co-infected dogs. Activated partial thromboplastin time was significantly (p<0.001) prolonged in coinfected groups. Regarding mean prothrombin time, a statistically important prolongation (p<0.001) was evident among leishmaniasis within other groups. Mean FIB values deemed elevated among all infected groups in contrast to healthy ones (p<0.001). Mean platelet values were decreased in E. canis mono and co-infected groups (p<0.001). Non-infectious diseases consisted of firearm injury (n=2), nasal malignant melanoma (n=1), lymphoma (n=1) and autoimmune hemolytic anemia (n=1). Conclusion: Taking into account review of this case series, it might be suggested that epistaxis should be on the list of clinical signs for infectious and non-infectious causes, which should be promptly treated based on probable tests and relevant findings.

Development and pharmacokinetic evaluation of chewable doxycycline tablets in Beagle dogs: comparison with a commercial formulation and evaluation of co-administration with vitamin supplement on the bioavailability

Doxycycline (DOX) is the antibiotic of choice for the treatment of canine monocytic ehrlichiosis (CME), one of the main infectious diseases affecting dogs. Despite the therapeutic simplicity, the need for long-term use of DOX tablets (28 days) and gastrointestinal effects may pose problems. The aim of the study was to develop a DOX chewable tablet, evaluate its palatability and pharmacokinetic profile in comparison with a commercial formulation, Doxifin®, and evaluate the influence of co-administration with vitamin supplement on the bioavailability of doxycycline. The AUC0-t values found for DOX chewable tablets (13.85 ± 3.81 μg.h/mL) and Doxifin® (15.88 ± 4.38 μg.h/mL) did not differ significantly (p<0.05). The co-administration of the vitamin supplement Hemolitan® influenced the pharmacokinetic profile of DOX, leading to a decrease in bioavailability for chewable DOX tablets and Doxifin®, with relative bioavailability values (F) of 64% and 62%, respectively. Palatability evaluations confirmed the palatability of the chewable tablets. Therefore, a DOX chewable tablet is an important alternative that can overcome some of the challenges associated with the characteristic bitter taste of doxycycline, with considerable reduction animal stress, and with plasma doxycycline concentrations equivalent in practical terms to a conventional product.

New sensitive real-time PCR targeting p28 gene for detection of Ehrlichia canis in blood samples from dogs

ABSTRACT: This study aims to describe a new detection method of a quantitative real-time polymerase chain reaction (qPCR) targeting the 28 kDa outer membrane protein gene (p28) as well as to compare this method with a conventional PCR (cPCR), which targets the same gene, in order to evaluate the performance of the technique designed in this study in detecting Ehrlichia canis (E. canis). Optimum oligonucleotides concentrations were reached, and the analytical sensitivity and specificity of the qPCR were performed. A total of 218 dogs’ whole blood samples were conventionally collected for this study. The DNA was extracted from each sample. Subsequently, the samples were tested by an established cPCR and the new qPCR to compare each technique’s performances. This new qPCR method for the molecular detection of E. canis presented a detection limit of ten copies of the fragment and was considered specific for E. canis according to analytical specificity analyses performed in vitro and in silico. The standard curve revealed 100% efficiency and a coefficient of determination (R2) equivalent to 99.8%. Among the samples examined by qPCR, 24.31% were considered positive, significantly greater than those detected by cPCR (15.13%). The qPCR technique reached a higher sensitivity than the cPCR when targeting the p28 gene in detecting E. canis. The qPCR standardized in this study is an efficient method for confirming canine monocytic ehrlichiosis (CME) diagnosis and might provide the parasitemia monitoring during the disease treatment.

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

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

Canonical correlative analyses among an enzyme-linked immunosorbent assay using synthetic peptides, an indirect fluorescent antibody test, and hematologic measurements in dogs infected with Ehrlichia canis.

Immunoreactive tandem repeat proteins (TRPs) in amino acid sequences were identified and employed in the serologic diagnosis of canine monocytic ehrlichiosis (CME). This study evaluated using TRP19 and TRP36 synthetic protein antigens with enzyme-linked immunosorbent assays (ELISAs) and compared the results with an indirect fluorescent antibody test (IFAT) to diagnose CME in the serum of dogs with suspected CME. The sera of 243 dogs that exhibited clinical and hematologic signs suggestive of CME had IFATs performed. An ELISA with synthetic TRP19 and TRP36 antigens from two Ecanis genotypes (USTRP36 and BrTRP36) that circulate in Brazil were also performed. Canonical correlations and chi-square tests were evaluated on the results of these tests. Among the 243 dogs, 179 (73.6%) were reactive at an IFAT≥40, 167 (68.72%) reacted at the cut-off ≥160, and 149 (61.31%) reacted at ≥640. The ELISA revealed that 172 (70.8%) dogs reacted to TRP19 peptide, 163 (67.1%) reacted to USTRP36, and 114 (46.9%) reacted to BrTRP36. The canonical correlation analyses showed that the TRP19 ELISA was the most effective diagnostic method. Serum total protein levels showed strong positive correlations with the USTRP36 peptide. Qualitative analyses revealed an association between the TRP19 peptide and the presence of anemia, thrombocytopenia, and hyperproteinemia. The IFAT results at titers of ≥160 and ≥640 were associated with thrombocytopenia. The results of this study indicated that the TRP19 peptide was an excellent antigen and that the IFAT was diagnostically important in confirming the diagnosis of CME in serum samples.

Development of a Loop-Mediated Isothermal Amplification (LAMP) Assay Targeting the Citrate Synthase Gene for Detection of Ehrlichia canis in Dogs

Canine monocytic ehrlichiosis caused by Ehrlichia canis is one of the leading tick-borne diseases of dogs, particularly in tropical countries. A highly sensitive and specific diagnostic method is essential for early detection to facilitate treatment. This study was conducted to develop E. canis loop-mediated isothermal amplification (LAMP) assay, a highly sensitive yet simple molecular technique, targeting the citrate synthase (gltA) gene of E. canis. Canine blood samples were subjected to conventional PCR targeting E. canis gltA. After analysis of the sequences of PCR amplicons, LAMP primers were generated. The optimum temperature and time for the LAMP assay were determined using eight samples—after which, the effectiveness and reproducibility of LAMP were verified by testing 40 samples, which included PCR-positive and negative samples. The detection limit was also established. The optimal condition for the assay was 61 °C for 60 min. Compared to PCR, the LAMP assay had a relative sensitivity and specificity of 92.5 and 100%, respectively. Statistical analysis using McNemar’s test showed that the E. canis LAMP assay has no significant difference with PCR. Therefore, the LAMP assay developed in this study may be used as an alternative to PCR in the detection of E. canis.

Acute Canine Monocytic Ehrlichiosis and Sertoli Cell Tumour in an Eleven-‎Year-Old Dachshund Cross-Bred Dog

An eleven-year-old Dachshund cross-bred dog was presented to the Small Animal Clinic of Ahmadu Bello University Veterinary Teaching Hospital, Zaria, with complaints of reduced appetite noticed 2 days earlier, and an enlarged testis was noticed three months prior to presentation. Clinical diagnosis was based on history of reduced appetite and enlarged testis; physical findings such as pyrexia, tick infestation, congested ocular mucous membranes, generalized enlarged superficial lymph nodes, enlargement of the mammary glands, alopecia, atrophy of the right testis and discoloration of the scrotum with large, firm palpable mass; complete blood count revealed slight anaemia (PCV 34 %, neutrophilia (12.88 x 109/L) and presence of band cells (0.32 x 109/L); buffy coat smear showed the presence of intracytoplasmic morulae in just one monocyte in the peripheral blood smear; Ehrlichia canis antibodies was detected in serum using Ehrlichia test kit (immunochromatography); serum oestradiol-17β (42.05 pg/mL) and anti- Müllerian hormone (AMH; 25.50 μg/mL) were employed as serum tumour markers; transverse and longitudinal scrotal sonographs revealed diffuse non-homogenous tissues with increase in the echogenicity of the testicles; radiographic findings of thoraco-abdomino-pelvic regions revealed normal organ opacity indicating absence of metastases. Tissue samples were taken from the tumor following surgical excision and submitted for histopathology. The findings were large cytoplasmic vacuoles with slightly irregular nucleoli. It was confirmed to be sertoli cell tumor. The conditions were managed medically, and by surgical excision of the tumour and scrotal ablation. Outcome was successful, and patient started feeding 24 hours after the surgery.