RESUMEN
Human monocytic ehrlichiosis, an emerging tick-borne disease, is caused by Ehrlichia chaffeensis. Infections with the pathogen are also common in the canine host. Our previous studies demonstrated that functional disruption within the E. chaffeensis phage head-to-tail connector protein gene results in bacterial attenuation, creating a modified live attenuated vaccine (MLAV). The MLAV confers protective immunity against intravenous and tick transmission challenges one month following vaccination. In this study, we evaluated the duration of MLAV protection. Dogs vaccinated with the MLAV were challenged with wild-type E. chaffeensis via intravenous infection at 4-, 8-, and 12-months post-vaccination. Immunized dogs rapidly cleared the wild-type pathogen infection and tested positive for bacteremia less frequently than unvaccinated controls. While immune responses varied among dogs, vaccinees consistently mounted IgG and CD4+ T-cell responses specific to E. chaffeensis throughout the assessment period. Our findings demonstrate that MLAV-mediated immune protection persists for at least one year against wild-type bacterial infection, marking a major advancement in combating this serious tick-borne disease. The data presented here serve as the foundation for further studies, elucidating the molecular mechanisms underlying virulence and vaccine development and aiding in preventing the diseases caused by E. chaffeensis and other tick-borne rickettsial pathogens.
RESUMEN
Ehrlichia chaffeensis is a tick-transmitted monocytic ehrlichiosis agent primarily causing the disease in people and dogs. We recently described the development and characterization of 55 random mutations in E. chaffeensis, which aided in defining the critical nature of many bacterial genes for its growth in a physiologically relevant canine infection model. In the current study, we tested 45 of the mutants for their infectivity ability to the pathogen's tick vector; Amblyomma americanum. Four mutations resulted in the pathogen's replication deficiency in the tick, similar to the vertebrate host. Mutations causing growth defects in both vertebrate and tick hosts included in genes coding for a predicted alpha/beta hydrolase, a putative dicarboxylate amino acid:cation symporter, a T4SS protein, and predicted membrane-bound proteins. Three mutations caused the bacterial defective growth only in the tick vector, which represented putative membrane proteins. Ten mutations causing no growth defect in the canine host similarly grew well in the tick vector. Mutations in 28 genes/genomic locations causing E. chaffeensis growth attenuation in the canine host were recognized as non-essential for its growth in the tick vector. The tick non-essential genes included genes coding for many metabolic pathway- and outer membrane-associated proteins. This study documents novel vector- and host-specific differences in E. chaffeensis for its functional gene requirements.
Asunto(s)
Ehrlichia chaffeensis , Ehrlichiosis , Garrapatas , Animales , Perros , Garrapatas/microbiología , Amblyomma , Ehrlichia chaffeensis/metabolismo , Infección Persistente , Vertebrados , Ehrlichiosis/veterinaria , Ehrlichiosis/microbiologíaRESUMEN
Tick-borne Anaplasma species are obligate, intracellular, bacterial pathogens that cause important diseases globally in people, agricultural animals, and dogs. Targeted mutagenesis methods are yet to be developed to define genes essential for these pathogens. In addition, vaccines conferring protection against diseases caused by Anaplasma species are not available. Here, we describe a targeted mutagenesis method for deletion of the phage head-to-tail connector protein (phtcp) gene in Anaplasma marginale. The mutant did not cause disease and exhibited attenuated growth in its natural host (cattle). We then assessed its ability to confer protection against wild-type A. marginale infection challenge. Additionally, we compared vaccine protection with the mutant to that of whole cell A. marginale inactivated antigens as a vaccine (WCAV) candidate. Upon infection challenge, non-vaccinated control cattle developed severe disease, with an average 57% drop in packed cell volume (PCV) between days 26-31 post infection, an 11% peak in erythrocytic infection, and apparent anisocytosis. Conversely, following challenge, all animals receiving the live mutant did not develop clinical signs or anemia, or erythrocyte infection. In contrast, the WCAV vaccinees developed similar disease as the non-vaccinees following A. marginale infection, though the peak erythrocyte infection reduced to 6% and the PCV dropped 43%. This is the first study describing targeted mutagenesis and its application in determining in vivo virulence and vaccine development for an Anaplasma species pathogen. This study will pave the way for similar research in related Anaplasma pathogens impacting multiple hosts.
Asunto(s)
Anaplasma marginale , Anaplasmosis , Enfermedades de los Bovinos , Anaplasma , Anaplasma marginale/genética , Anaplasmosis/genética , Anaplasmosis/prevención & control , Animales , Bovinos , Enfermedades de los Bovinos/microbiología , Perros , Humanos , Mutagénesis , Desarrollo de Vacunas , VirulenciaRESUMEN
Ehrlichia ruminantium, a tick-borne rickettsial, causes heartwater in ruminants resulting from vascular damage. Severity of heartwater varies greatly in ruminant species and breeds, age of animals and for diverse geographic E. ruminantium strains. E. ruminantium and a tick vector, Amblyomma variegatum, originating from Africa, are well established in certain Caribbean islands two centuries ago. Besides the possibility of introduction of heartwater through African exotic animal importation, presence of the pathogen, and the tick vector in the Caribbean pose a high risk to ruminants in the USA and other western hemisphere countries. Scientific evidence supporting the heartwater threat to nonendemic regions, however, is lacking. We describe the first infection study in sheep reared in the USA with seven E. ruminantium strains. All infected sheep exhibited clinical signs characteristic of subacute to subclinical disease, which included labored breathing, depression, coughing, and nasal discharges. Gross and microscopic lesions consistent with heartwater disease including edema and hemorrhage were observed in several organs. Pathogen-specific IgG antibody response was detected in animals infected with all seven strains, while molecular analysis confirmed the pathogen presence only when infected with in vitro cultures. This is the first infection study demonstrating severe heartwater in sheep reared in North America.
RESUMEN
Ehrlichia chaffeensis, a tick-transmitted obligate intracellular rickettsial agent, causes human monocytic ehrlichiosis. In recent reports, we described substantial advances in developing random and targeted gene disruption methods to investigate the functions of E. chaffeensis genes. We reported earlier that the Himar1 transposon-based random mutagenesis is a valuable tool in defining E. chaffeensis genes critical for its persistent growth in vivo in reservoir and incidental hosts. The method also aided in extending studies focused on vaccine development and immunity. Here, we describe the generation and mapping of 55 new mutations. To define the critical nature of the bacterial genes, infection experiments were carried out in the canine host with pools of mutant organisms. Infection evaluation in the physiologically relevant host by molecular assays and by xenodiagnoses allowed the identification of many proteins critical for the pathogen's persistent in vivo growth. Genes encoding proteins involved in biotin biosynthesis, protein synthesis and fatty acid biosynthesis, DNA repair, electron transfer, and a component of a multidrug resistance (MDR) efflux pump were concluded to be essential for the pathogen's in vivo growth. Three known immunodominant membrane proteins, i.e., two 28-kDa outer membrane proteins (P28/OMP) and a 120-kDa surface protein, were also recognized as necessary for the pathogen's obligate intracellular life cycle. The discovery of many E. chaffeensis proteins crucial for its continuous in vivo growth will serve as a major resource for investigations aimed at defining pathogenesis and developing novel therapeutics for this and related pathogens of the rickettsial family Anaplasmataceae.
Asunto(s)
Ehrlichia chaffeensis/genética , Ehrlichiosis/microbiología , Genes Bacterianos , Animales , Proteínas Bacterianas/genética , Línea Celular , Perros , Ehrlichia chaffeensis/crecimiento & desarrollo , Ehrlichia chaffeensis/patogenicidad , Ehrlichiosis/transmisión , Biblioteca de Genes , Genoma Bacteriano/genética , Macrófagos/microbiología , Mutagénesis Insercional , Mutación , Garrapatas , Transcripción Genética , Virulencia/genéticaRESUMEN
Rocky Mountain spotted fever (RMSF) is a potentially fatal tick-borne disease in people and dogs. RMSF is reported in the United States and several countries in North, Central, and South America. The causative agent of this disease, Rickettsia rickettsii, is transmitted by several species of ticks, including Dermacentor andersoni, Rhipicephalus sanguineus, and Amblyomma americanum RMSF clinical signs generally include fever, headache, nausea, vomiting, muscle pain, lack of appetite, and rash. If untreated, it can quickly progress into a life-threatening illness in people and dogs, with high fatality rates ranging from 30 to 80%. While RMSF has been known for over a century, recent epidemiological data suggest that the numbers of documented cases and the fatality rates remain high in people, particularly during the last two decades in parts of North America. Currently, there are no vaccines available to prevent RMSF in either dogs or people. In this study, we investigated the efficacies of two experimental vaccines, a subunit vaccine containing two recombinant outer membrane proteins as recombinant antigens (RCA) and a whole-cell inactivated antigen vaccine (WCA), in conferring protection against virulent R. rickettsii infection challenge in a newly established canine model for RMSF. Dogs vaccinated with WCA were protected from RMSF, whereas those receiving RCA developed disease similar to that of nonvaccinated R. rickettsii-infected dogs. WCA also reduced the pathogen loads to nearly undetected levels in the blood, lungs, liver, spleen, and brain and induced bacterial antigen-specific immune responses. This study provides the first evidence of the protective ability of WCA against RMSF in dogs.
Asunto(s)
Antígenos Bacterianos/inmunología , Enfermedades de los Perros , Rickettsia rickettsii/inmunología , Vacunas contra Rickettsia/inmunología , Fiebre Maculosa de las Montañas Rocosas , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Enfermedades de los Perros/inmunología , Enfermedades de los Perros/microbiología , Enfermedades de los Perros/prevención & control , Perros , Proteínas Recombinantes/inmunología , Fiebre Maculosa de las Montañas Rocosas/inmunología , Fiebre Maculosa de las Montañas Rocosas/prevención & control , Fiebre Maculosa de las Montañas Rocosas/veterinariaRESUMEN
The lone star tick, Amblyomma americanum, is an obligatory ectoparasite of many vertebrates and the primary vector of Ehrlichia chaffeensis, the causative agent of human monocytic ehrlichiosis. This study aimed to investigate the comparative transcriptomes of A. americanum underlying the processes of pathogen acquisition and of immunity towards the pathogen. Differential expression of the whole body transcripts in six different treatments were compared: females and males that were E. chaffeensis non-exposed, E. chaffeensis-exposed/uninfected, and E. chaffeensis-exposed/infected. The Trinity assembly pipeline produced 140,574 transcripts from trimmed and filtered total raw sequence reads (approximately 117M reads). The gold transcript set of the transcriptome data was established to minimize noise by retaining only transcripts homologous to official peptide sets of Ixodes scapularis and A. americanum ESTs and transcripts covered with high enough frequency from the raw data. Comparison of the gene ontology term enrichment analyses for the six groups tested here revealed an up-regulation of genes for defense responses against the pathogen and for the supply of intracellular Ca++ for pathogen proliferation in the pathogen-exposed ticks. Analyses of differential expression, focused on functional subcategories including immune, sialome, neuropeptides, and G protein-coupled receptor, revealed that E. chaffeensis-exposed ticks exhibited an upregulation of transcripts involved in the immune deficiency (IMD) pathway, antimicrobial peptides, Kunitz, an insulin-like peptide, and bursicon receptor over unexposed ones, while transcripts for metalloprotease were down-regulated in general. This study found that ticks exhibit enhanced expression of genes responsible for defense against E. chaffeensis.
RESUMEN
Monocytic ehrlichiosis in people caused by the intracellular bacterium, Ehrlichia chaffeensis, is an emerging infectious disease transmitted by the lone star tick, Amblyomma americanum. Tick transmission disease models for ehrlichiosis require at least two hosts and two tick blood feeding episodes to recapitulate the natural transmission cycle. One blood feeding is necessary for the tick to acquire the infection from an infected host and the next feeding is needed to transmit the bacterium to a naïve host. We have developed a model for E. chaffeensis transmission that eliminates the entire tick acquisition stage while still producing high numbers of infected ticks that are also able to transmit infections to naïve hosts. Fully engorged A. americanum nymphs were ventrally needle-infected, possibly into the midgut, and following molting, the unfed adult ticks were used to infect naive deer and dogs. We have also described using the ticks infected by this method the transmission of both wild-type and transposon mutants of E. chaffeensis to its primary reservoir host, white tailed deer and to another known host, dog. The infection progression and IgG antibody responses in deer were similar to those observed with transmission feeding of ticks acquiring infection by natural blood feeding. The pathogen infections acquired by natural tick transmission and by feeding needle-infected ticks on animals were also similar to intravenous infections in causing persistent infections. Needle-infected ticks having the ability to transmit pathogens will be a valuable resource to substantially simplify the process of generating infected ticks and to study infection systems in vertebrate hosts where interference of other pathogens could be avoided.
Asunto(s)
Vectores Artrópodos/microbiología , Ciervos/microbiología , Enfermedades de los Perros/microbiología , Ehrlichia chaffeensis/fisiología , Ehrlichiosis/veterinaria , Ixodidae/microbiología , Animales , Enfermedades de los Perros/transmisión , Perros , Ehrlichiosis/microbiología , Ehrlichiosis/transmisión , Regulación Bacteriana de la Expresión Génica , MutaciónRESUMEN
Tick-borne bacteria, Ehrlichia canis, Anaplasma platys, and Ehrlichia chaffeensis are significant pathogens of dogs worldwide, and coinfections of E. canis and A. platys are common in dogs on the Caribbean islands. We developed and evaluated the performance of a multiplex bead-based assay to detect antibodies to E. canis, A. platys, and E. chaffeensis peptides in dogs from Grenada, West Indies, where E. canis and A. platys infections are endemic. Peptides from outer membrane proteins of P30 of E. canis, OMP-1X of A. platys, and P28-19/P28-14 of E. chaffeensis were coupled to magnetic beads. The multiplex peptide assay detected antibodies in dogs experimentally infected with E. canis and E. chaffeensis, but not in an A. platys experimentally infected dog. In contrast, the multiplex assay and an in-house enzyme-linked immunosorbent assay (ELISA) detected A. platys antibodies in naturally infected Grenadian dogs. Following testing of 104 Grenadian canine samples, multiplex assay results had good agreement with commercially available ELISA and immunofluorescent assay for E. canis antibody-positive dogs ( K values of 0.73 and 0.84), whereas A. platys multiplex results had poor agreement with these commercial assays ( K values of -0.02 and 0.01). Prevalence of seropositive E. canis and A. platys Grenadian dogs detected by the multiplex and commercial antibody assays were similar to previous reports. Although the multiplex peptide assay performed well in detecting the seropositive status of dogs to E. canis and had good agreement with commercial assays, better antigen targets are necessary for the antibody detection of A. platys.
Asunto(s)
Enfermedades de los Perros/diagnóstico , Ehrlichiosis/veterinaria , Anaplasma/inmunología , Anaplasma/aislamiento & purificación , Animales , Anticuerpos Antibacterianos/sangre , Enfermedades de los Perros/sangre , Enfermedades de los Perros/microbiología , Perros , Ehrlichia canis/inmunología , Ehrlichia canis/aislamiento & purificación , Ehrlichia chaffeensis/inmunología , Ehrlichia chaffeensis/aislamiento & purificación , Ehrlichiosis/diagnóstico , Ensayo de Inmunoadsorción Enzimática/veterinaria , Grenada , Valor Predictivo de las PruebasRESUMEN
The variations in prevalence levels of two tick-borne rickettsial pathogens, Ehrlichia chaffeensis and Ehrlichia Ewingii, in a periurban environment were evaluated along with their ecological determinants. Tick life stage and sex, month of tick collection, landscape fragmentation, and ecological covariates specific to pasture and woodland sites were considered as explanatory covariates. Questing lone star ticks (Amblyomma americanum) were collected by flagging for an hour once every week during mid-April through mid-August in years 2013 and 2014. A total of 4357 adult and nymphal ticks (woodland = 2720 and pasture = 1637) were collected and assessed for pathogen prevalence by molecular methods. Female A. americanum ticks were more infected with E. chaffeensis than males or nymphs in woodland areas [â = 6.05%; â = 12.0%; nymphs = 2.09%] and pastures [â = 8.05%; â = 12.03%; nymphs = 3.33%], and the prevalence was influenced by edge density in the landscape. Higher E. ewingii infection was noted among female A. americanum ticks within woodland areas [â = 1.89%; â = 2.14%; nymphs = 1.57%], but no such difference was evident in pastures [â = 1.03%; â = 1.33%; nymphs = 1.12%]. Prevalence of E. ewingii was influenced by edge contrast index, and the percentage of pasture perimeter that was less than 20 meters from woodland areas. This study elucidates the complexity of tick-borne pathogen ecology and points to the need for further studies on the role of reservoir hosts, particularly that played by small vertebrates, which is not fully understood in the region.
Asunto(s)
Ecosistema , Ehrlichia/clasificación , Ehrlichia/aislamiento & purificación , Garrapatas/microbiología , Animales , Ciudades , ADN Bacteriano/genética , Ehrlichia/genética , Femenino , Kansas , Masculino , Ninfa/microbiología , Reacción en Cadena de la Polimerasa , ZoonosisRESUMEN
Dogs acquire infections with the Anaplasmataceae family pathogens, E. canis, E. chaffeensis, E. ewingii, A. platys and A. phagocytophilum mostly during summer months when ticks are actively feeding on animals. These pathogens are also identified as causing diseases in people. Despite the long history of tick-borne diseases in dogs, much remains to be defined pertaining to the clinical and pathological outcomes of infections with these pathogens. In the current study, we performed experimental infections in dogs with E. canis, E. chaffeensis, A. platys and A. phagocytophilum. Animals were monitored for 42 days to evaluate infection-specific clinical, hematological and pathological differences. All four pathogens caused systemic persistent infections detectible throughout the 6 weeks of infection assessment. Fever was frequently detected in animals infected with E. canis, E. chaffeensis, and A. platys, but not in dogs infected with A. phagocytophilum. Hematological differences were evident in all four infected groups, although significant overlap existed between the groups. A marked reduction in packed cell volume that correlated with reduced erythrocytes and hemoglobin was observed only in E. canis infected animals. A decline in platelet numbers was common with E. canis, A. platys and A. phagocytophilum infections. Histopathological lesions in lung, liver and spleen were observed in all four groups of infected dogs; infection with E. canis had the highest pathological scores, followed by E. chaffeensis, then A. platys and A. phagocytophilum. All four pathogens induced IgG responses starting on day 7 post infection, which was predominantly comprised of IgG2 subclass antibodies. This is the first detailed investigation comparing the infection progression and host responses in dogs after inoculation with four pathogens belonging to the Anaplasmataceae family. The study revealed a significant overlap in clinical, hematological and pathological changes resulting from the infections.
Asunto(s)
Anaplasma/inmunología , Anaplasmosis/microbiología , Enfermedades de los Perros/inmunología , Ehrlichia/inmunología , Ehrlichiosis/microbiología , Enfermedades por Picaduras de Garrapatas/veterinaria , Anaplasma/patogenicidad , Animales , Plaquetas/inmunología , Enfermedades de los Perros/microbiología , Perros , Ehrlichia/patogenicidad , Ehrlichiosis/veterinaria , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Hígado/microbiología , Pulmón/microbiología , Recuento de Plaquetas , Bazo/microbiología , Enfermedades por Picaduras de Garrapatas/inmunología , Enfermedades por Picaduras de Garrapatas/microbiología , Garrapatas/microbiologíaRESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Ehrlichia chaffeensis/inmunología , Ehrlichiosis/veterinaria , Vacunas contra Rickettsia/inmunología , Vacunación/veterinaria , Vacunas Atenuadas/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Proliferación Celular , Perros , Ehrlichia chaffeensis/genética , Ehrlichiosis/inmunología , Ehrlichiosis/microbiología , Ehrlichiosis/prevención & control , Insectos Vectores/microbiología , Interferón gamma/biosíntesis , Interleucina-17/biosíntesis , Garrapatas/microbiologíaRESUMEN
Ehrlichia chaffeensis, a tick-borne rickettsial, is responsible for human monocytic ehrlichiosis. In this study, we assessed E. chaffeensis insertion mutations impacting the transcription of genes near the insertion sites. We presented evidence that the mutations within the E. chaffeensis genome at four genomic locations cause polar effects in altering gene expressions. We also reported mutations causing attenuated growth in deer (the pathogen's reservoir host) and in dog (an incidental host), but not in its tick vector, Amblyomma americanum. This is the first study documenting insertion mutations in E. chaffeensis that cause polar effects in altering gene expression from the genes located upstream and downstream to insertion sites and the differential requirements of functionally active genes of the pathogen for its persistence in vertebrate and tick hosts. This study is important in furthering our knowledge on E. chaffeensis pathogenesis.
Asunto(s)
Ehrlichia chaffeensis/genética , Regulación de la Expresión Génica , Especificidad del Huésped/genética , Mutación/genética , Animales , Southern Blotting , Ciervos/microbiología , Perros/microbiología , Ehrlichiosis/sangre , Ehrlichiosis/microbiología , Ehrlichiosis/veterinaria , Inyecciones , Insectos Vectores/microbiología , Mutagénesis Insercional/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Garrapatas/microbiología , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Vacunas Bacterianas/inmunología , Ehrlichia chaffeensis/inmunología , Ehrlichiosis/prevención & control , Ehrlichiosis/veterinaria , Animales , Carga Bacteriana , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/genética , Sangre/microbiología , Ciervos , Perros , Ehrlichia chaffeensis/genética , Ehrlichiosis/inmunología , Genes Bacterianos , Humanos , Mutagénesis Insercional , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunologíaRESUMEN
Ehrlichia chaffeensis, transmitted from Amblyomma americanum ticks, causes human monocytic ehrlichiosis. It also infects white-tailed deer, dogs and several other vertebrates. Deer are its reservoir hosts, while humans and dogs are incidental hosts. E. chaffeensis protein expression is influenced by its growth in macrophages and tick cells. We report here infection progression in deer or dogs infected intravenously with macrophage- or tick cell-grown E. chaffeensis or by tick transmission in deer. Deer and dogs developed mild fever and persistent rickettsemia; the infection was detected more frequently in the blood of infected animals with macrophage inoculum compared to tick cell inoculum or tick transmission. Tick cell inoculum and tick transmission caused a drop in tick infection acquisition rates compared to infection rates in ticks fed on deer receiving macrophage inoculum. Independent of deer or dogs, IgG antibody response was higher in animals receiving macrophage inoculum against macrophage-derived Ehrlichia antigens, while it was significantly lower in the same animals against tick cell-derived Ehrlichia antigens. Deer infected with tick cell inoculum and tick transmission caused a higher antibody response to tick cell cultured bacterial antigens compared to the antibody response for macrophage cultured antigens for the same animals. The data demonstrate that the host cell-specific E. chaffeensis protein expression influences rickettsemia in a host and its acquisition by ticks. The data also reveal that tick cell-derived inoculum is similar to tick transmission with reduced rickettsemia, IgG response and tick acquisition of E. chaffeensis.
Asunto(s)
Ciervos/microbiología , Perros/microbiología , Ehrlichia chaffeensis/aislamiento & purificación , Ehrlichiosis/transmisión , Ehrlichiosis/veterinaria , Macrófagos/microbiología , Garrapatas/microbiología , Animales , Vectores Arácnidos/microbiología , Línea Celular , Ehrlichiosis/sangre , HumanosRESUMEN
AIM OF STUDY: The aim of this study is to explore the ability of the carotenoids (CARs) to offer protection against acute cardiotoxicity and hepatotoxicity induced by doxorubicin (DOX) (25 mg/kg) in tumor bearing Swiss albino mice. MATERIALS AND METHODS: Tumor bearing Swiss albino mice administered with DOX (25 mg/kg, i.p) and two doses of CARs (50 and 100 µg/kg). 24 h after administration of the drugs, histopathological evaluation of tumor, liver and heart tissues carried out. Furthermore, various antioxidant parameters in these tissues were investigated. Serum marker enzymes for tissue injury were examined. RESULTS: Administration of CARs prevented the depletion of antioxidants in the heart and liver, thereby protecting the tissue damage and release of marker enzymes. However, similar antioxidant depletion was not observed in the tumor tissue. CARs prevented DOX induced variation in tissue architecture in heart and liver tissues. However, CARs did not influence DOX induced alterations in the tumor. CONCLUSION: Administration of CARs could prevent DOX induced acute toxicity to heart and liver.
Asunto(s)
Antibióticos Antineoplásicos/efectos adversos , Antioxidantes/farmacología , Carotenoides/farmacología , Doxorrubicina/efectos adversos , Corazón/efectos de los fármacos , Hígado/efectos de los fármacos , Animales , Antibióticos Antineoplásicos/administración & dosificación , Biomarcadores/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Modelos Animales de Enfermedad , Doxorrubicina/administración & dosificación , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Miocardio/metabolismo , Miocardio/patología , Neoplasias/complicaciones , Neoplasias/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacosRESUMEN
Ehrlichia chaffeensis is a tick transmitted pathogen responsible for the disease human monocytic ehrlichiosis. Research to elucidate gene function in rickettsial pathogens is limited by the lack of genetic manipulation methods. Mutational analysis was performed, targeting to specific and random insertion sites within the bacterium's genome. Targeted mutagenesis at six genomic locations by homologous recombination and mobile group II intron-based methods led to the consistent identification of mutants in two genes and in one intergenic site; the mutants persisted in culture for 8 days. Three independent experiments using Himar1 transposon mutagenesis of E. chaffeensis resulted in the identification of multiple mutants; these mutants grew continuously in macrophage and tick cell lines. Nine mutations were confirmed by sequence analysis. Six insertions were located within non-coding regions and three were present in the coding regions of three transcriptionally active genes. The intragenic mutations prevented transcription of all three genes. Transposon mutants containing a pool of five different insertions were assessed for their ability to infect deer and subsequent acquisition by Amblyomma americanum ticks, the natural reservoir and vector, respectively. Three of the five mutants with insertions into non-coding regions grew well in deer. Transposition into a differentially expressed hypothetical gene, Ech_0379, and at 18 nucleotides downstream to Ech_0230 gene coding sequence resulted in the inhibition of growth in deer, which is further evidenced by their failed acquisition by ticks. Similarly, a mutation into the coding region of ECH_0660 gene inhibited the in vivo growth in deer. This is the first study evaluating targeted and random mutagenesis in E. chaffeensis, and the first to report the generation of stable mutants in this obligate intracellular bacterium. We further demonstrate that in vitro mutagenesis coupled with in vivo infection assessment is a successful strategy in identifying genomic regions required for the pathogen's in vivo growth.