Ehrlichia canis in dogs experimentally infected, treated, and then immune suppressed during the acute or subclinical phases.

BACKGROUND: Concerns for recrudescence of Ehrlichia canis infection arise when immunosuppressive drugs are used to treat immune-mediated diseases in dogs previously infected with E. canis. OBJECTIVES: Determine whether administration of prednisolone and cyclosporine would reactivate E. canis infection in dogs previously treated with doxycycline during the acute or subclinical phases. ANIMALS: Seven beagles previously experimentally infected with E. canis and administered doxycycline for 4 weeks were included. Three of the 7 dogs were incidentally concurrently infected with Anaplasma platys and Babesia vogeli and were administered 2 doses of imidocarb 2 weeks apart before enrollment in the current study. METHODS: Experimental study. Each dog was administered prednisolone and cyclosporine for 6 weeks. Clinical signs, complete blood cell count (CBC), polymerase chain reaction (PCR) assays for E. canis, A. platys, and B. vogeli DNA in blood, E. canis indirect fluorescent antibodies (IFA) titers, and flow cytometry for antiplatelet antibodies were monitored. RESULTS: All dogs completed the immunosuppressive protocol. No evidence for recrudescence of E. canis, A. platys, or B. vogeli were detected based on clinical signs or results of CBC, PCR, IFA, and flow cytometry for antiplatelet antibodies. E. canis IFA titers were negative in 5/7 dogs at the end of immunosuppressive protocol and were negative 6 months after the protocol in 5/5 dogs available for testing. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs administered with a 4-week course of doxycycline with or without imidocarb failed to show evidence of activation of E. canis infection after administration of a commonly used immune suppressive protocol.

Efficacy of Minocycline in Naturally Occurring Nonacute Ehrlichia canis Infection in Dogs.

BACKGROUND: Minocycline has been used in the treatment of Ehrlichia canis infection in dogs as an alternative to doxycycline, the recommended treatment. However, efficacy of this alternative therapy is unknown. OBJECTIVE: To assess the efficacy of minocycline in the treatment of natural occurring E. canis infection in dogs. ANIMALS: Ten privately owned dogs of mixed breed positive for E. canis by blood PCR. METHODS: Prospective, randomized clinical study. Dogs positive for E. canis by PCR were housed in a kennel environment and randomly allocated to receive doxycycline 10 mg/kg bodyweight PO once daily ("gold standard" control group) or minocycline (extralabel) 10 mg/kg bodyweight PO twice daily (treatment test group) for 28 days. Blood, analyzed by PCR to determine the presence or absence of E. canisDNA, was collected weekly during treatment starting on the first day of treatment and including through day 35, 7 days after the last treatment. RESULTS: In both groups, one dog tested negative after 7 days of treatment. For the doxycycline group, the latest time to a negative PCR test was after 3 weeks of treatment. For the minocycline group, the latest time was on day 28 of treatment. All dogs tested negative 7 days after the end of treatment. CONCLUSION AND CLINICAL IMPORTANCE: Minocycline can be an effective alternative to doxycycline for clearing E. canis from the blood in nonacute infections.

Comparative efficacy of oral administrated afoxolaner (NexGard™) and fluralaner (Bravecto™) with topically applied permethrin/imidacloprid (Advantix(®)) against transmission of Ehrlichia canis by infected Rhipicephalus sanguineus ticks to dogs.

BACKGROUND: The ability of the topical spot-on Advantix(®) (50 % permethrin/10 % imidacloprid) to prevent transmission of Ehrlichia canis by infected Rhipicephalus sanguineus ticks to dogs has previously been reported. The recent market introduction of chewable tablets containing the novel compounds, afoxolaner (NexGard™) and fluralaner (Bravecto™) enabled us to conduct a comparative efficacy study with respect to the ability of these three products to block transmission of E. canis by ticks to dogs. The speed of kill, immediate drop-off rate and anti-attachment efficacy of the respective products were also studied. METHODS: The study was a blinded parallel group design, wherein 32 dogs were randomised into four different groups of eight dogs. Group 1 served as negative placebo control, group 2 and 3 were treated on Days 0, 28 and 56 with NexGard™ and Advantix(®), respectively. Group 4 was dosed once on Day 0 with Bravecto™. For tick efficacy assessments 50 non-infected ticks were placed onto the dogs on Days 30, 35, 42, 49, 56, 63, 70, 77 and 84 and on animal tick counts were performed at 3 h, 6 h and 12 h after infestation. To evaluate the ability to block transmission of E. canis, each dog was challenged by releasing 80 adult E. canis-infected R. sanguineus ticks into their sleeping kennels on Days 31, 38, 45 and 52. The animals were monitored for clinical signs of monocytic ehrlichiosis (pyrexia and thrombocytopenia) and were tested for E. canis DNA by PCR and for specific antibodies using IFA. A dog was considered infected with E. canis if both PCR and IFA yielded positive test results up to Day 84. RESULTS: Mean arithmetic tick counts on dogs treated with the Advantix(®) spot-on were significantly (P < 0.0005) lower throughout the study as compared with the negative controls and was, with respect to the speed of kill and resulting onset of acaricidal efficacy, superior over NexGard™ and Bravecto™ at all time points in the 12 h period observed (3 h, 6 h and 12 h). None of the dogs treated with the Advantix(®) spot-on became infected with E. canis, whereas six out of eight untreated control dogs acquired the infection. Furthermore, E. canis infection was diagnosed in four out of eight dogs treated with NexGard™ and in two out of eight dogs treated with Bravecto™. CONCLUSIONS: The speed of kill of the two recently registered systemic compounds against R. sanguineus was not sufficiently fast to prevent transmission of E. canis and resulted in only low partial blocking and protection capacity while Advantix(®) effectively blocked transmission of E. canis to dogs in the challenge period and thus provided adequate protection for dogs against monocytic ehrlichiosis.

The efficacy of a generic doxycycline tablet in the treatment of canine monocytic ehrlichiosis.

The objective of the present study was to evaluate the therapeutic efficacy of a generic doxycycline tablet (DoxyVet) against Ehrlichia canis infection in dogs. Canine monocytic ehrlichiosis is caused by the bacterium E. canis and transmitted by the brown kennel tick (Rhipicephalus sanguineus). Six disease-free and tick-free dogs were infested with E. canis infected ticks. Once diagnosed (with polymerase chain reaction [PCR] analysis and platelet counts) as positive for infection, doxycycline tablets were administered orally once a day for 20 consecutive days, at a target dose level of 10 mg/kg. The actual dose administered was calculated as ranging between 10 mg/kg and 11.7 mg/kg. The PCR analysis, 28 days after the first administration of the tablets, failed to detect E. canis in any of the dogs. On Day 56 of the study, four of the dogs were diagnosed with E. canis for the second time and a fifth dog was diagnosed on Day 70. The platelet counts of the sixth dog remained within normal levels and it was discharged from the study on Day 84. Doxycycline tablets were then administered to the remaining five infected dogs for 28 consecutive days. Four of these dogs had no positive PCR results during the following 3 months. The fifth dog was diagnosed with E. canis for the third time 58 days after the last tablets of the second treatment had been administered, after which it was rescue treated (doxycycline for a further 28 days). The results indicate that doxycycline administered in tablet form (DoxyVet) at 10 mg/kg - 11.7 mg/kg body mass once daily for 28 consecutive days clears most dogs of infection. The importance of a concomitant tick-control programme is therefore stressed.

The spreading process of Ehrlichia canis in macrophages is dependent on actin cytoskeleton, calcium and iron influx and lysosomal evasion.

Ehrlichia canis is an obligate intracellular microorganism and the etiologic agent of canine monocytic ehrlichiosis. The invasion process has already been described for some bacteria in this genus, such as E. muris and E. chaffeensis, and consists of four stages: adhesion, internalisation, intracellular proliferation and intercellular spreading. However, little is known about the spreading process of E. canis. The aim of this study was to analyse the role of the actin cytoskeleton, calcium, iron and lysosomes from the host cell in the spreading of E. canis in dog macrophages in vitro. Different inhibitory drugs were used: cytochalasin D (actin polymerisation inhibitor), verapamil (calcium channel blocker) and deferoxamine (iron chelator). Our results showed a decrease in the number of bacteria in infected cells treated with all drugs when compared to controls. Lysosomes in infected cells were cytochemically labelled with acid phosphatase to allow the visualisation of phagosome-lysosome fusion and were further analysed by transmission electron microscopy. Phagosome-lysosome fusion was rarely observed in vacuoles containing viable E. canis. These data suggest that the spreading process of E. canis in vitro is dependent on cellular components analysed and lysosomal evasion.

The efficacy of Advantix® to prevent transmission of Ehrlichia canis to dogs by Rhipicephalus sanguineus ticks.

The capacity of a topical combination of imidacloprid and permethrin (Advantix(®)) to prevent transmission of Ehrlichia canis was studied in two groups of six dogs. One group served as controls, whereas the other group was treated. All dogs were exposed to E. canis-infected Rhipicephalus sanguineus ticks on Days 7, 14, 21 and Day 28 post acaricidal treatment. The adult R. sanguineus ticks were released into the individual kennels of the dogs to simulate natural tick exposure. In situ tick counts were conducted on Day 9, 16 and 23 and any remaining ticks were counted and removed on Day 30. The efficacy of the acaricidal treatment against R. sanguineus ranged between 96.1% and 98.9% at 48 h post-application and lasted up to 4 weeks. Four out of six control dogs became infected with E. canis, as demonstrated by the presence of specific E. canis antibodies and the detection by PCR of E. canis DNA in blood samples. These dogs became thrombocytopenic and displayed fever and were consecutively rescue-treated by doxycycline. None of the six treated dogs became infected with E. canis, as confirmed by the lack of specific antibodies and absence of E. canis DNA in blood samples. Advantix(®) prevented transmission of E. canis and provided protection against monocytic ehrlichiosis for 4 weeks post acaricidal treatment.

In silico characterization of three two-component systems of Ehrlichia canis and evaluation of a natural plant-derived inhibitor.

Two-component signal transduction systems (TCS) are important elements in the interaction of endobacteria with host cells. They are basically composed of two proteins, an environmental signal sensor and a response regulator, which activate genes involved in a wide range of bacterial responses to their environment. We analyzed three sets of genes corresponding to TCS of Ehrlichia canis, a common tick-borne canine pathogen and the etiologic agent of canine monocytic ehrlichiosis, in order to identify the characteristic domains of the sensor and response regulator components. Analysis of sequence alignments of the corresponding proteins indicated a high degree of similarity to other members of the Anaplasmataceae TCS proteins, demonstrating that they could be useful as universal targets for development of new drugs against these bacteria. We also evaluated by quantitative PCR inhibition of E. canis by (2H)-1,4-benzoxazin-3(4H)-one (BOA), the core compound of the plant phenolic compound DIMBOA, which shows inhibitory action against TCS of the phytopathogen Agrobacterium tumefasciens. This bacterium exerts its pathogenicity by transferring oncogenic DNA (T-DNA) into plant cells; this transfer is mediated through a type-IV secretion system, which is regulated by the VirA/VirG TCS. The process of infection and pathogenesis of E. canis is associated with the secretion of effector proteins into the host cell cytoplasm through a T4SS system, which blocks the cell defense response. We suggest that BOA, and possibly other plant phenolic compounds that are TCS inhibitors, can be exploited in the search for new antiehrlichial drugs to be used alone or as complements in the treatment of canine monocytic ehrlichiosis.

Failure of imidocarb dipropionate to clear experimentally induced Ehrlichia canis infection in dogs.

The recommended treatment for canine ehrlichiosis is tetracycline or its analog doxycycline, although recent reports have documented ineffective clearing of Erchlichia canis after doxycycline administration. Imidocarb dipropionate is used as an alternative treatment to tetracycline or is used in conjunction with doxycycline. The effectiveness of imidocarb dipropionate in clearing Ehrlichia species from the blood and tissues of dogs with E. canis infection has not been thoroughly evaluated. Fifteen dogs were experimentally infected with E. canis. Ten dogs were treated with imidocarb dipropionate (6.6 mg/kg, IM, 2 injections given 2 weeks apart). Five infected control dogs were not treated. Blood samples from all 15 dogs were E. canis DNA positive by PCR assay by 3 weeks after inoculation (PI), and E. canis antibodies were detected by IFA assay by 1 week PI. Blood platelet counts in all dogs were below the reference interval by 4 weeks PI. E. canis DNA was detected in bone marrow and splenic aspirates by PCR assay 4 weeks PI but not before infection. Bone marrow aspirates were E. canis DNA positive by PCR assay in 14/15 dogs, and splenic aspirates were E. canis DNA positive by PCR assay in 13/15 dogs. Blood samples from all treated and control dogs remained positive for E. canis DNA by PCR assay, and platelet counts remained below preinoculation values 13 weeks PI (6 weeks after 2nd treatment). As administered in this study, imidocarb dipropionate did not clear experimental E. canis infection in dogs.

Bone marrow mastocytosis in dogs with myelosuppressive monocytic ehrlichiosis (Ehrlichia canis): a retrospective study.

BACKGROUND: Bone marrow mastocytosis has been reported rarely in naturally occurring canine monocytic ehrlichiosis (CME). OBJECTIVES: The aims of the present study were to estimate the prevalence and magnitude of bone marrow mastocytosis in a case series of dogs with natural CME and to assess the association, if any, between mastocytosis and the clinical severity of the disease. METHODS: Seventy-six dogs with confirmed CME (Ehrlichia canis) were included in the study. Affected dogs were allocated into group A (n = 51) without bone marrow hypoplasia and group B (n = 25) with bone marrow hypoplasia. Twenty clinically healthy Beagles not previously exposed to E canis served as controls (group C). The main inclusion criteria for group A were documentation of normocellular to hypercellular bone marrow and complete clinical cure following a 4-week treatment with doxycycline, while those for group B were bone marrow hypoplasia and lack of response to doxycycline. Bone marrow aspirate smears from all 96 dogs were Giemsa-stained and examined for the presence of mast cells, which were calculated as a percentage of 1,000 nucleated cells (NCs). The prevalence of mastocytosis was compared among the 3 groups by the Pearson's chi-square test. RESULTS: Bone marrow mastocytosis (>0.1% of NCs) was found in 5 (20%) dogs in group B (range, 0.5-2.5% of NCs; median, 1% of NCs). One dog in each of groups A and C had 0.1% mast cells in the marrow. The prevalence of bone marrow mastocytosis in dogs in group B was significantly higher (P = .004) than in groups A and C. CONCLUSION: Bone marrow mastocytosis can be seen in a substantial number of dogs with E canis-induced myelosuppression.

Acquired amegakaryocytic thrombocytopenia--four cases and a literature review.

Acquired amegakaryocytic thrombocytopenla was diagnosed in four dogs. Initial platelet counts in all four dogs were less than 50,000 x 10(9)/litre and initial bone marrow examinations revealed megakaryocytic hypoplasia with minimal changes in the erythroid and myeloid cell lines. Two dogs had evidence of idiopathic immune-mediated disease and two dogs had evidence of associated infectious disease. One dog had a positive antibody titre to Borrella burgdorferi, and one dog had positive titres to both Ehrlichia canis and B. burgdorferi. Treatment consisted of prednisone and cyclophosphamide for the dogs with presumptive immune-mediated disease, and prednisone and tetracycline for the dogs with positive antibody titres to the Infectious organisms. Both dogs with evidence of associated infectious disease responded to treatment. A postmortem examination did not reveal the underlying aetiology in the two dogs with presumptive idiopathic immune-mediated disease.

Evaluation of antibiotic susceptibilities of Ehrlichia canis, Ehrlichia chaffeensis, and Anaplasma phagocytophilum by real-time PCR.

We determined MICs of antibiotics against Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia canis by real-time quantitative PCR. The doubling times of the organisms were established: 19 h for E. chaffeensis, 26 h for A. phagocytophilum, and 28 h for E. canis. In comparison to the reference method for determining sensitivities, which uses Diff-Quick staining, our PCR assay was very sensitive and specific. We confirmed that doxycycline and rifampin are highly active against these bacteria and found variable susceptibilities to fluoroquinolones; A. phagocytophilum was susceptible, but E. canis and E. chaffeensis were only partly susceptible. Beta-lactam compounds, cotrimoxazole, macrolide compounds, and telithromycin showed no activity against any of the three organisms. Thiamphenicol was found to be more active than chloramphenicol. For the first time, we showed that these three species have numerous point mutations in their 23S RNA genes, with those at positions 754, 2057, 2058, 2059, and 2611 (Escherichia coli numbering) known to confer resistance to macrolide compounds in other bacteria. The role of each of these mutations in resistance to these drugs should be investigated in the future. Our study confirms previous reports that quantitative PCR is a reliable method for determining antibiotic susceptibility; therefore, it might be useful for screening new drugs.

Assay of fipronil efficacy to prevent canine monocytic ehrlichiosis in endemic areas.

Our objective was to evaluate the efficacy of fipronil for the prevention of Ehrlichia canis transmission to dogs by Rhipicephalus sanguineus in two endemic areas situated in Africa (Dakar and Djibouti). We carried out controlled trials in kennels for 1 year on 248 dogs, mainly police dogs and military working dogs. Eight groups were studied in a multi-centre study. Fifty five fipronil treated dogs were located in two separated kennels (G3, 37 dogs in Djibouti and G8, 18 dogs in Dakar). G1 (66 dogs) and G2 (60 dogs) were untreated control groups located in Djibouti, whereas G4 (32 dogs), G5 (13 dogs), G6 (18 dogs) and G7 (4 dogs) were the control groups located in Dakar. The epidemiological status of each group is known. G1 and G2 dogs were not kept in kennels, whereas G3, G4, G5, G6, G7, G8 dogs were housed in equivalent kennels. Tick infestation, clinical status and Ehrlichia seroprevalence were assessed during 1 year (duration of the study). Dog treated with fipronil showed neither canine monocytic ehrlichiosis (CME) nor tick infestations. In all groups of untreated control animals, R. sanguineus tick infestations were frequent, particularly in kennels (G5, G6 and G7) as well as morbidity and mortality due to CME. E. canis infection rates were low for fipronil treated animals: 2.7% (1/37) for G3 and 5.5% (1/18) for G8 group. Among control animals, seroprevalence was maximum (100%) in dogs kept in kennels (G5, G6 and G7 groups) and high among native dogs in Djibouti (G1 group): 69.7% (46/66) and in Dakar (G4 group): 50% (16/32). Dogs belonging to expatriate citizens (G2 group) were less likely to be infected: 21.7% (13/60). The comparison of serological results among French army dogs and French citizen dogs that were introduced in Djibouti for an average of 10 months shows a statistically significant (P<0.001) difference. Among fipronil treated animals (G3 group), 2 dogs out of 55 seroconverted (3.6%) compared to 13 out of 60 dogs (21.7%) in the control G2 group. The results of our study indicate the preventative efficacy of a fipronil monthly treatment to avoid CME in endemic areas. Epidemiological data concerning animals that live in the same endemic areas are an example of the serious consequences (in terms of mortality and morbidity) that are related to the absence of efficient methods for tick-control. In order to protect dogs that are in transit in endemic areas against tick-transmitted diseases, the use of an adapted acaricide product is recommended.