Impact of vaccination with the Anaplasma phagocytophilum MSP4 chimeric antigen on gene expression in the rabbit host.

There are currently no vaccines available to prevent and control of Anaplasma phagocytophilum, an intracellular bacterial pathogen transmitted by ticks that occurs in many regions of the world and causes disease in a wide range of domestic and wild hosts, including humans. Vaccines induce long-lasting immunity and could prevent or reduce transmission of this pathogen. Understanding how vaccines induce a protective response can be difficult due to the complexity of the immune system, which operates at many levels throughout the organism. New perspectives in vaccinology, based on systems biology approaches, integrate many scientific disciplines to fully understand the biological responses to vaccination, where a transcriptomic approach could reveal relevant information of the host immune system, allowing profiling for rational design of vaccine formulations, administration, and potential protection. In the present study we report the gene expression profiles by RNA-seq followed by functional analysis using whole blood samples from rabbits immunized with a recombinant chimeric protein containing peptides from the MSP4 protein of A. phagocytophilum, which showed satisfactory results in terms of potential protection. Transcriptomic analysis revealed differential expression of 720 genes, with 346 genes upregulated and 374 genes downregulated. Overrepresentation of biological and metabolic pathways correlated with immune response, protein signaling, cytoskeleton organization and protein synthesis were found. These changes in gene expression could provide a complete and unique picture of the biological response to the epitope candidate vaccine against A. phagocytophilum in the host.

Development of a SONIX Vaccine to Protect Against Ehrlichiosis.

The obligately Gram-negative intracellular bacterium Ehrlichia that resides in mononuclear phagocytes is the etiologic agent of human monocytotropic ehrlichiosis (HME). HME is an emerging and often life-threatening, tick-transmitted infectious disease in the USA. Currently, three different Ehrlichia species can cause ehrlichiosis in humans in the USA-Ehrlichia chaffeensis, Ehrlichia ewingii, and Ehrlichia muris subspecies eauclairensis. Ehrlichia also causes diseases in companion animals and domesticated ruminants. Ehrlichia are vector-borne diseases and transmitted by tick bites. As yet there are no commercially available vaccines to protect against these pathogens. Previously we developed structure-based vaccines and subunit vaccines to protect against ehrlichiosis in animal models. Though the vaccines are efficient in inducing protection, there is a delay in clearing the pathogens in challenge studies. In this chapter we demonstrate the development of a SONIX vaccine that is more potent than conventional vaccines. The vaccination strategy may be useful in Emergency Use Authorization (EUA) scenarios during public health emergencies.

Clinical and immunological responses in sheep after inoculation with Himar1-transformed Anaplasma phagocytophilum and subsequent challenge with a virulent strain of the bacterium.

In Norway, the tick-transmitted bacterium Anaplasma phagocytophilum is estimated to cause tick-borne fever (TBF) in 300 000 lambs on pastures each year, resulting in economic and animal welfare consequences. Today, prophylactic measures mainly involve the use of acaricides, but a vaccine has been requested by farmers and veterinarians for decades. Several attempts have been made to produce a vaccine against A. phagocytophilum including antigenic surface proteins, inactivated whole cell vaccines and challenge followed by treatment. In the current study, a virulent wild type strain of A. phagocytophilum named Ap.Norvar1 (16S rRNA sequence partial identical to sequence in GenBank acc.no M73220) was subject to genetic transformation with a Himar1-transposon, which resulted in three bacterial mutants, capable of propagation in a tick cell line (ISE6). In order to test the immunogenicity and pathogenicity of the live, mutated bacteria, these were clinically tested in an inoculation- and challenge study in sheep. One group was inoculated with the Ap.Norvar1 as an infection control. After inoculation, the sheep inoculated with mutated bacteria and the Ap.Norvar1 developed typical clinical signs of infection and humoral immune response. After challenge with Ap.Norvar1, 28 days later all groups inoculated with mutated bacteria showed clinical signs of tick-borne fever and bacteremia while the group initially inoculated with the Ap.Norvar1, showed protection against clinical disease. The current study shows a weak, but partial protection against infection in animals inoculated with mutated bacteria, while animals that received Ap.Norvar1 both for inoculation and challenge, responded with homologues protection.

An Entry-Triggering Protein of Ehrlichia Is a New Vaccine Candidate against Tick-Borne Human Monocytic Ehrlichiosis.

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes human monocytic ehrlichiosis, an emerging disease transmitted by the Lone Star tick, Amblyomma americanum. E. chaffeensis outer membrane protein entry triggering protein of Ehrlichia (EtpE) is necessary for bacterial entry into human cells. We investigated the role of EtpE in transmission of the bacteria from tick to human cells and whether or not vaccination with EtpE can prevent transmission of ehrlichiae from ticks to mammals. An antiserum against the recombinant C terminus of EtpE (rEtpE-C), which binds a mammalian cell-surface receptor and triggers bacterial entry, significantly inhibited E. chaffeensis transmission from infected tick cells to human monocytes in culture. Each of five specific-pathogen-free dogs were vaccinated with rEtpE-C along with an immunostimulating complex or were sham vaccinated with the complex alone. Dogs vaccinated with rEtpE-C developed high antibody titers against rEtpE-C and produced interferon-γ-secreting cells, as assessed with the ELISpot assay. All 10 dogs were challenged with A. americanum adult ticks infected as nymphs by syringe inoculation with E. chaffeensis Upon challenge, both the vaccinated and control dogs became infected by day 1 post-tick attachment, but the majority of rEtpE-C-vaccinated dogs rapidly cleared the infection from the bloodstream as soon as day 7, whereas most of sham-vaccinated dogs remained infected at day 35. Peripheral blood leukocytes from vaccinated dogs had significantly elevated interferon-γ mRNA levels and secreted significantly elevated interferon-γ soon after tick attachment. Thus, the EtpE-C vaccine represents the first ehrlichial protein vaccine demonstrated to reduce bacterial infection in mammals upon challenge with infected ticks.IMPORTANCE The incidence of tick-borne diseases has risen dramatically in the past two decades and continues to rise. Discovered in 1986 and designated a nationally notifiable disease in 1998 by the Centers for Disease Control and Prevention, human monocytic ehrlichiosis, which is caused by the bacterium Ehrlichia chaffeensis, is one of the most prevalent, life-threatening, emerging tick-borne zoonoses in the United States. We investigated the role of the E. chaffeensis protein EtpE in transmission of the bacterium from tick to human cells and in vaccinated dogs with EtpE to assess the efficacy of vaccination against E. chaffeensis-infected tick challenge. Our results help fill gaps in our understanding of E. chaffeensis-derived protective antigens that could be used in a candidate vaccine for immunization of humans to counter tick-transmitted ehrlichiosis.

Immunization against Anaplasma phagocytophilum Adhesin Binding Domains Confers Protection against Infection in the Mouse Model.

Anaplasma phagocytophilum causes granulocytic anaplasmosis, a debilitating infection that can be fatal in the immunocompromised. It also afflicts animals, including dogs, horses, and sheep. No granulocytic anaplasmosis vaccine exists. Because A. phagocytophilum is an obligate intracellular bacterium, inhibiting microbe-host cell interactions that facilitate invasion can disrupt infection. The binding domains of A. phagocytophilum adhesins A. phagocytophilum invasion protein A (AipA), A. phagocytophilum surface protein (Asp14), and outer membrane protein A (OmpA) are essential for optimal bacterial entry into host cells, but their relevance to infection in vivo is undefined. In this study, C57BL/6 mice were immunized with a cocktail of keyhole limpet hemocyanin-conjugated peptides corresponding to the AipA, Asp14, and OmpA binding domains in alum followed by challenge with A. phagocytophilum The bacterial peripheral blood burden was pronouncedly reduced in immunized mice compared to controls. Examination of pre- and postchallenge sera from these mice revealed that immunization elicited antibodies against AipA and Asp14 peptides but not OmpA peptide. Nonetheless, pooled sera from pre- and postchallenge groups, but not from control groups, inhibited A. phagocytophilum infection of HL-60 cells. Adhesin domain immunization also elicited interferon gamma (IFN-γ)-producing CD8-positive (CD8+) T cells. A follow-up study confirmed that immunization against only the AipA or Asp14 binding domain was sufficient to reduce the bacterial peripheral blood load in mice following challenge and elicit antibodies that inhibit A. phagocytophilum cellular infection in vitro These data demonstrate that AipA and Asp14 are critical for A. phagocytophilum to productively infect mice, and immunization against their binding domains elicits a protective immune response.

Prevention of transmission of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum by Ixodes spp. ticks to dogs treated with the Seresto® collar (imidacloprid 10% + flumethrin 4.5%).

The capability of imidacloprid 10% + flumethrin 4.5% (Seresto®) collars to prevent transmission of Borrelia burgdorferi sensu lato (Bbsl) and Anaplasma phagocytophilum (Ap) by naturally infected ticks was evaluated in two studies with 44 dogs. In each study, one group served as non-treated control, whereas the other groups were treated with the Seresto® collar. All dogs were exposed to naturally Bbsl- and Ap-infected hard ticks (Ixodes ricinus, Ixodes scapularis). In study 1, tick infestation was performed on study day (SD) 63 (2 months post-treatment [p.t.]); in study 2, it was performed on SD 32 (one month p.t.) respectively SD 219 (seven months p.t.). In situ tick counts were performed 2 days after infestation. Tick counts and removals followed 6 (study 1) or 5 days (study 2) later. Blood sampling was performed for the detection of specific Bbsl and Ap antibodies and, in study 1, for the documentation of Ap DNA by PCR. Skin biopsies were examined for Bbsl by PCR and culture (only study 1). The efficacy against Ixodes spp. was 100% at all time points. In study 1, two of six non-treated dogs became infected with Bbsl, and four of six tested positive for Ap; none of the treated dogs tested positive for Bbsl or Ap. In study 2, ten of ten non-treated dogs became infected with Bbsl and Ap; none of the treated dogs tested positive for Bbsl or Ap; 100% acaricidal efficacy was shown in both studies. Transmission of Bbsl and Ap was successfully blocked for up to 7 months.

Assessing the clinical and bacteriological outcomes of vaccination with recombinant Asp14 and OmpA against A. phagocytophilum in sheep.

Anaplasma phagocytophilum is a tick borne bacterium, causing disease in sheep and other mammals, including humans. The bacterium has great economic and animal welfare implications for sheep husbandry in Northern Europe. With the prospect of a warmer and more humid climate, the vector availability will likely increase, resulting in a higher prevalence of A. phagocytophilum. The current preventive measures, as pyrethroids acting on ticks or long acting antibiotics controlling bacterial infection, are suboptimal for prevention of the disease in sheep. Recently, the increased awareness on antibiotic- and pyrethorid resistance, is driving the search for a new prophylactic approach in sheep against A. phagocytophilum. Previous studies have used an attenuated vaccine, which gave insufficient protection from challenge with live bacteria. Other studies have focused on bacterial membrane surface proteins like Asp14 and OmpA. An animal study using homologous proteins to Asp14 and OmpA of A. marginale, showed no protective effect in heifers. In the current study, recombinant proteins of Asp14 (rAsp14) and OmpA (rOmpA) of A. phagocytophilum were produced and prepared as a vaccine for sheep. Ten lambs were vaccinated twice with an adjuvant emulsified with rAsp14 or rOmpA, three weeks apart and challenged with a live strain of A. phagocytophilum (GenBank acc.nr M73220) on day 42. The control group consisted of five lambs injected twice with PBS and adjuvant. Hematology, real time qPCR, immunodiagnostics and flow cytometric analyses of peripheral blood mononuclear cells were performed. Vaccinated lambs responded with clinical signs of A.phagocytophilum infection after challenge and bacterial load in the vaccinated group was not reduced compared to the control group. rAsp14 vaccinated lambs generated an antibody response against the vaccine, but a clear specificity for rAsp14 could not be established. rOmpA-vaccinated lambs developed a strong specific antibody response on days 28 after vaccination and 14 days post-challenge. Immunofluorescent staining and flow cytometric analysis of peripheral blood mononuclear monocytes revealed no difference between the three groups, but the percentage of CD4+, CD8+, γδ TcR+, λ-Light chain+, CD11b+, CD14+ and MHC II+ cells, within the groups changed during the study, most likely due to the adjuvant or challenge with the bacterium. Although an antigen specific antibody response could be detected against rOmpA and possibly rAsp14, the vaccines seemed to be ineffective in reducing clinical signs and bacterial load caused by A. phagocytophilum. This is the first animal study with recombinant Asp14 and OmpA aimed at obtaining clinical protection against A. phagocytophilum in sheep.

Ehrlichia spp. infection in rural dogs from remote indigenous villages in north-eastern Brazil.

BACKGROUND: Ehrlichia canis is a tick-borne bacterium that causes severe, life-threatening disease in dogs, being more prevalent in tropical and subtropical countries. Randomized studies conducted in Brazil indicate that the prevalence of E. canis infection in dogs ranges from 0.7% to over 50.0%. In a study conducted in northern Brazil, the prevalence was higher in dogs from urban areas, as compared to dogs from rural areas. In the present study, we investigated the exposure to Ehrlichia spp. infection in dogs from remote indigenous villages located in a rural area in north-eastern Brazil. METHODS: From March to June 2015, 300 privately owned dogs were blood sampled and tested by a rapid ELISA and by a conventional PCR in order to detect anti-Ehrlichia spp. antibodies and E. canis DNA, respectively. Additionally, dogs were also tested for anti-Anaplasma spp. antibodies and Anaplasma platys DNA, using the same diagnostic approaches. Positivity was correlated with tick infestation and dogs' data (gender, age and level of restriction). RESULTS: Overall, 212 (70.7%) dogs were positive for at least one test targeting Ehrlichia spp. In particular, 173 (57.7%) dogs were positive only by rapid ELISA, 5 (1.7%) only by PCR and 34 (11.4%) were simultaneously positive by both tests. In the same way, 39 (13.0%) dogs presented detectable E. canis DNA in their blood, whereas 18 (6.0%) dogs were A. platys DNA-positive. Coupling serological and PCR data, 63 (21.0%) dogs were simultaneously positive to Ehrlichia spp. and Anaplasma spp. Positivity rates for both Ehrlichia spp. and Anaplasma spp. were higher among dogs more than 1 year of age. Sick dogs were more positive to Ehrlichia spp. as compared to healthy dogs. CONCLUSIONS: Dogs from rural areas in north-eastern Brazil are highly exposed to Ehrlichia spp. infection and positivity rates do not necessarily correlate with current tick infestation load, since only one infected tick bite is needed to get the infection. This reinforces the importance of keeping dogs free of ticks, in order to reduce as much as possible the risk of infection by E. canis and other tick-borne pathogens such as Babesia vogeli, which are usually co-endemic.

Tick-Borne Diseases in Turkey: A Review Based on One Health Perspective.

The importance of tick-borne diseases is increasing all over the world, including Turkey. Global warming, environmental and ecological changes and the existence of suitable habitats increase the impact of ticks and result in frequent emergence or re-emergence of tick-borne diseases (TBDs) with zoonotic characteristics. In Turkey, almost 19 TBDs have been reported in animals and men, involving four protozoa (babesiosis, theileriosis, cytauxzoonosis, hepatozoonosis), one filarial nematode (acanthocheilonemasis), ten bacterial agents (anaplasmosis, ehrlichiosis, aegyptianellosis, tick-borne typhus, Candidatus Rickettsia vini, Lyme borreliosis, tick-borne relapsing fever [TBRF], tularaemia, bartonellosis, and hemoplasmosis), and four viral infections (tick-borne encephalitis [TBE], Crimean-Congo Haemorrhagic Fever [CCHF], louping-ill [LI], and lumpy skin disease [LSD]). The growing number of TBD cases, in particular the fatal viral epidemics in humans, have led to increased public awareness and concern against TBDs in recent years. The World Health Organization (WHO) has developed a new political concept, called the "One Health" initiative, which is especially relevant for developing strategies against tick infestations and TBD control in humans and animals. It would be beneficial for Turkey to adopt this new strategy and establish specific research and control programs in coordination with international organizations like WHO, the World Organization for Animal Health (OIE), the Food and Agriculture Organization (FAO), the Centers for Disease Control and Prevention (CDC), and the European Center for Disease Prevention and Control (ECDC) to combat TBDs based on the "One Health Initiative" concept. In this article, we review the occurrence of primary TBDs in man and animals in Turkey in light of the "One Health" perspective.

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.

Vaccination with an Attenuated Mutant of Ehrlichia chaffeensis Induces Pathogen-Specific CD4+ T Cell Immunity and Protection from Tick-Transmitted Wild-Type Challenge in the Canine Host.

Ehrlichia chaffeensis is a tick-borne rickettsial pathogen and the causative agent of human monocytic ehrlichiosis. Transmitted by the Amblyomma americanum tick, E. chaffeensis also causes disease in several other vertebrate species including white-tailed deer and dogs. We have recently described the generation of an attenuated mutant strain of E. chaffeensis, with a mutation in the Ech_0660 gene, which is able to confer protection from secondary, intravenous-administered, wild-type E. chaffeensis infection in dogs. Here, we extend our previous results, demonstrating that vaccination with the Ech_0660 mutant protects dogs from physiologic, tick-transmitted, secondary challenge with wild-type E. chaffeensis; and describing, for the first time, the cellular and humoral immune responses induced by Ech_0660 mutant vaccination and wild-type E. chaffeensis infection in the canine host. Both vaccination and infection induced a rise in E. chaffeensis-specific antibody titers and a significant Th1 response in peripheral blood as measured by E. chaffeensis antigen-dependent CD4+ T cell proliferation and IFNγ production. Further, we describe for the first time significant IL-17 production by peripheral blood leukocytes from both Ech_0660 mutant vaccinated animals and control animals infected with wild-type E. chaffeensis, suggesting a previously unrecognized role for IL-17 and Th17 cells in the immune response to rickettsial pathogens. Our results are a critical first step towards defining the role of the immune system in vaccine-induced protection from E. chaffeensis infection in an incidental host; and confirm the potential of the attenuated mutant clone, Ech_0660, to be used as a vaccine candidate for protection against tick-transmitted E. chaffeensis infection.

Efficacy of sarolaner in the prevention of Borrelia burgdorferi and Anaplasma phagocytophilum transmission from infected Ixodes scapularis to dogs.

The efficacy of sarolaner (Simparica™, Zoetis) to prevent transmission primarily of Borrelia burgdorferi and secondarily of Anaplasma phagocytophilum from infected wild-caught Ixodes scapularis to dogs was evaluated in a placebo-controlled laboratory study. Twenty-four purpose-bred laboratory Beagles seronegative for B. burgdorferi and A. phagocytophilum antibodies were allocated randomly to one of three treatment groups: placebo administered orally on Days 0 and 7, or sarolaner at 2mg/kg administered orally on Day 0 (28 days prior to tick infestation) or on Day 7 (21 days prior to tick infestation). On Day 28, each dog was infested with approximately 25 female and 25 male wild caught adult I. scapularis that were determined to have prevalence of 57% for B. burgdorferi and 6.7% for A. phagocytophilum by PCR. In situ tick counts were conducted on Days 29 and 30. On Day 33, all ticks were counted and removed. Acaricidal efficacy was calculated based on the reduction of geometric mean live tick counts in the sarolaner-treated groups compared to the placebo-treated group for each tick count. Blood samples collected from each dog on Days 27, 49, 63, 77, 91 and 104 were tested for the presence of B. burgdorferi and A. phagocytophilum antibodies using the SNAP(®) 4Dx(®) Plus Test, and quantitatively assayed for B. burgdorferi antibodies using an ELISA test. Skin biopsies collected on Day 104 were tested for the presence of B. burgdorferi by bacterial culture and PCR. Geometric mean live tick counts for placebo-treated dogs were 14.8, 12.8, and 19.1 on Days 29, 30, and 33, respectively. The percent reductions in mean live tick counts at 1, 2, and 5 days after infestation were 86.3%, 100%, and 100% for the group treated with sarolaner 21 days prior to infestation, and 90.9%, 97.1%, and 100% for the group treated with sarolaner 28 days prior to infestation. Geometric mean live tick counts for both sarolaner-treated groups were significantly lower than those for the placebo group on all count days (P<0.0001). There were no adverse reactions to treatment with sarolaner. Transmission of B. burgdorferi to all eight placebo-treated dogs was confirmed by positive antibody (6 of 8 dogs), PCR (7 of 8 dogs), and/or culture (7 of 8 dogs). Similarly, transmission of A. phagocytophilum was confirmed by the presence of antibodies in four placebo-treated dogs. In contrast, treatment with a single dose of sarolaner prevented transmission of B. burgdorferi from infected ticks to dogs infested 21 or 28 days after treatment as demonstrated by negative antibody, PCR, and culture results. Prevention of transmission of A. phagocytophilum was demonstrated by negative antibody results in all sarolaner-treated dogs.

A novel combination of fipronil and permethrin (Frontline Tri-Act®/Frontect®) reduces risk of transmission of Babesia canis by Dermacentor reticulatus and of Ehrlichia canis by Rhipicephalus sanguineus ticks to dogs.

BACKGROUND: The ability of Frontline Tri-Act®/Frontect®, a topical ectoparasiticide containing fipronil and permethrin for dogs, to prevent the transmission of Babesia canis as well as Ehrlichia canis was evaluated by infesting dogs with infected vector ticks. METHODS: For the Babesia canis study, 16 dogs were randomly allocated to two groups. Eight dogs were treated on day 0 with a topical spot-on formulation containing 6.76 % w/v fipronil plus 50.48 % w/v permethrin and eight dogs served as the untreated control group. Dermacentor reticulatus ticks, with a B. canis infection rate ranging between 2 and 10 %, were placed onto dogs on days 7, 14, 21 and 28. In situ tick counts were performed on Days 9, 16 and 23. Ticks were counted and removed on Day 30. Infection of the dogs with B. canis was monitored by rectal temperature readings, clinical examinations and blood smears as well as PCR and IFA (indirect fluorescent antibody assay). For the Ehrlichia canis study, another 16 dogs were allocated to two groups. Eight dogs were treated with the fipronil and permethrin combination on days 0 and 28 and eight dogs served as untreated controls. Rhipicephalus sanguineus ticks, carrying an infection rate of 13 % for E. canis, were released in the sleeping kennels of the dogs on days 7, 14, 21, 28, 35, 42, 49 and 56. Ticks were counted in situ on the dogs on a weekly basis. All ticks were removed and counted on the final assessment day 58. Infection of the dogs with E. canis was monitored by rectal temperature, clinical examinations, and testing of blood samples by PCR, IFA and platelet counts. RESULTS: B. canis was transmitted by D. reticulatus ticks to all eight untreated control dogs and to one treated dog, which was confirmed by blood smears, PCR and IFA. E.canis was transmitted by R. sanguineus ticks to all eight untreated control dogs. Two of the dogs in the treated group were found positive based on PCR and/or IFA. CONCLUSIONS: Frontline Tri-Act®/Frontect® significantly lowered the risk for dogs to acquire a B. canis infection by 87.5 % over a challenge period of 28 days. The risk for dogs to acquire E. canis was reduced by 75 % over a period of 56 days.

Comparison of the acute phase protein and antioxidant responses in dogs vaccinated against canine monocytic ehrlichiosis and naive-challenged dogs.

BACKGROUND: Canine monocytic ehrlichiosis (CME) is a tick-borne disease with a global distribution, caused by Ehrlichia canis. The inflammatory response to E. canis infection includes changes in certain acute phase proteins (APP) and in biomarkers of the oxidative status. APP responses are considered part of the innate immune response to CME. The aim of this study was to evaluate the APP and oxidative marker responses in dogs vaccinated against CME with an attenuated vaccine and subsequently challenged with a wild E. canis strain. METHODS: The study included 3 groups of 4 beagle dogs. Group 1 dogs were inoculated subcutaneously with an attenuated E. canis vaccine on day 0, and again on day 213. Group 2 initially served as controls for group 1 during the vaccination phase and then vaccinated once on day 213. Group 3 consisted of naïve dogs which constituted the control group for the challenge phase. All 12 dogs were infected intravenously with a wild strain of E. canis on day 428 of the study. APP levels were serially measured during two periods: days 0-38 post-vaccination (groups 1 and 2) and days 0-39 post-challenge (groups 1, 2, 3). RESULTS: Changes in C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin, albumin, paraoxonase-1 (PON-1) and total antioxidant capacity (TAC) were of significantly smaller magnitude in vaccinated dogs and appeared later on a time scale compared to unvaccinated dogs challenged with a wild strain. Alterations in the level of APP during the vaccination phase of the study were of lower extent compared to those in the challenged unvaccinated dogs during the post-challenge phase. Positive APP levels correlated positively with the rickettsial load, body temperature and negatively with the thrombocyte counts (p < 0.05). CONCLUSIONS: Vaccination with an attenuated E. canis strain and challenge with a wild strain resulted in considerably reduced responses of positive and negative APP, and oxidative biomarker responses in vaccinated compared to unvaccinated dogs, reflecting a milder innate inflammatory response conferred by protection of the vaccine.

Attenuated Mutants of Ehrlichia chaffeensis Induce Protection against Wild-Type Infection Challenge in the Reservoir Host and in an Incidental Host.

Ehrlichia chaffeensis, a tick-borne rickettsial organism, causes the disease human monocytic ehrlichiosis. The pathogen also causes disease in several other vertebrates, including dogs and deer. In this study, we assessed two clonally purified E. chaffeensis mutants with insertions within the genes Ech_0379 and Ech_0660 as vaccine candidates in deer and dogs. Infection with the Ech_0379 mutant and challenge with wild-type E. chaffeensis 1 month following inoculation with the mutant resulted in the reduced presence of the organism in blood compared to the presence of wild-type infection in both deer and dogs. The Ech_0660 mutant infection resulted in its rapid clearance from the bloodstream. The wild-type infection challenge following Ech_0660 mutant inoculation also caused the pathogen's clearance from blood and tissue samples as assessed at the end of the study. The Ech_0379 mutant-infected and -challenged animals also remained positive for the organism in tissue samples in deer but not in dogs. This is the first study that documents that insertion mutations in E. chaffeensis that cause attenuated growth confer protection against wild-type infection challenge. This study is important in developing vaccines to protect animals and people against Ehrlichia species infections.

Recombinant Ehrlichia P29 protein induces a protective immune response in a mouse model of ehrlichiosis.

Ehrlichioses are emerging tick-borne bacterial diseases of humans and animals for which no vaccines are available. The diseases are caused by obligately intracellular bacteria belonging to the genus Ehrlichia. Several immunoreactive proteins of ehrlichiae have been identified based on their reactivity with immune sera from human patients and animals. These include the major outer membrane proteins, ankyrin repeat proteins and tandem repeat proteins (TRP). Polyclonal antibodies directed against the tandem repeats (TRs) of Ehrlichia chaffeensis TRP32, TRP47 and TRP120 have been shown to provide protection in mice. In the present study, we evaluated E. muris P29, which is the ortholog of E. chaffeensis TRP47 and E. canis TRP36, as a subunit vaccine in a mouse model of ehrlichiosis. Our study indicated that unlike E. chaffeensis TRP47 and E. canis TRP36, orthologs of E. muris (P29) and E. muris-like agent (EMLA) do not contain tandem repeats. Immunization of mice with recombinant E. muris P29 induced significant protection against a challenge infection. The protection induced by E. muris P29 was associated with induction of strong antibody responses. In contrast to development of P29-specific IgG antibodies following immunization, development of P29-specific IgG antibodies, but not IgM antibodies, was impaired during persistent E. muris infection. Furthermore, our study indicated that CD4+ T cells target P29 during E. muris infection and differentiate into IFN-γ-producing Th1 effector/memory cells. In conclusion, our study indicated that orthologs of E. muris P29 showed considerable variation in the central tandem repeat region among different species, induction of P29-specific IgG antibody response was impaired during persistent E. muris infection, and rP29 induced protective immune responses.

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.

Mimicry epitope from Ehrlichia canis for interphotoreceptor retinoid-binding protein 201-216 prevents autoimmune uveoretinitis by acting as altered peptide ligand.

We report here identification of novel mimicry epitopes for interphotoreceptor retinoid-binding protein (IRBP) 201-216, a candidate ocular antigen that causes experimental autoimmune uveoretinitis (EAU) in A/J mice. One mimicry epitope from Ehrlichia canis (EHC), designated EHC 44-59, induced cross-reactive T cells for IRBP 201-216 capable of producing T helper (Th)1 and Th17 cytokines, but failed to induce EAU in A/J mice. In addition, animals first primed with suboptimal doses of IRBP 201-216 and subsequently immunized with EHC 44-59 did not develop EAU; rather, the mimicry epitope prevented the disease induced by IRBP 201-216. However, alteration in the composition of EHC 44-59 by substituting alanine with valine at position 49, similar to the composition of IRBP 201-216, enabled the mimicry epitope to acquire uveitogenicity. The data provide new insights as to how microbes containing mimicry sequences for retinal antigens can prevent ocular inflammation by acting as naturally occurring altered peptide ligands.

Imidacloprid 10 % / flumethrin 4.5 % collars (Seresto®, Bayer) successfully prevent long-term transmission of Ehrlichia canis by infected Rhipicephalus sanguineus ticks to dogs.

The objective of this study was to determine the empirical efficacy of imidacloprid 10 %/flumethrin 4.5 % (Seresto®) collars in preventing long-term transmission of Ehrlichia canis by infected Rhipicephalus sanguineus ticks to dogs. The study was a parallel group design, single centre, randomised, non-blinded, controlled, long-term efficacy study. The treatment group of 8 dogs was fitted with Seresto® collars, the untreated control group of 8 dogs received no collars. Ehrlichia canis-infected ticks were released into the dogs sleeping quarters at 14-day intervals up to Day +378. Control group dogs infected with E. canis were continuously replaced to keep the control sample size constant, and a total of 39 control dogs were required. The final clinical examination and blood sampling occurred on Day +420. The primary assessment criterion was the number of dogs infected with E. canis, as confirmed by IFA and PCR, and the secondary criterion was the acaricidal efficacy based on tick counts. All scheduled blood samples taken were subject to analyses for both PCR and IFA, but only positive cases are discussed. Up to Day +378, none of the collar-treated dogs were infected with E. canis, whereas 34 of the 35 untreated dogs enrolled before Day +371 were infected. The acaricidal efficacy of the collar ranged from 90 % to 100 % for the duration of the assessment period.