RESUMEN
Since CD8+ T cells play an important role in resistance to infection with heartwater, effective vaccines against this disease will likely require identification of antigens that contain CD8+ T cell epitopes responsible for cytotoxic T lymphocyte (CTL) responses. With the use of the fluorescent antigen-transfected target cell (FATT)-CTL assay, IFN-γ ELISPOT and flow cytometry, peptides that induce CTL, proliferation of CD8 + T cells and IFN-γ production were identified as possible target antigens for vaccine development. Of particular relevance was the finding that different peptides from different antigens were able to elicit varied cytotoxic activities by immune peripheral blood mononuclear cells (PBMC) from heartwater immune tick-infected sheep. Several peptides derived from Erum0660, Erum2330, Erum2540, Erum2580 and Erum5000 induced CTL in immune sheep PBMC. Peptide Erum2540-6 was the only peptide that induced significant CTL, CD8+CD45RO+ and CD8+IFN-γ+ by PBMC from all three sheep, and Erum2540 and p2540-20 induced the highest % CTL response in all three outbred sheep. These results suggest that these epitopes may be of major importance in heartwater recombinant vaccine development.
Asunto(s)
Antígenos Bacterianos/inmunología , Ehrlichia ruminantium/inmunología , Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Vacunas Bacterianas/inmunología , Epítopos/inmunología , Femenino , Técnica del Anticuerpo Fluorescente/veterinaria , Hidropericardio/inmunología , Hidropericardio/microbiología , Hidropericardio/prevención & control , Técnicas In Vitro , Activación de Linfocitos/inmunología , Masculino , Reacción en Cadena de la Polimerasa/veterinaria , Ovinos/inmunología , Enfermedades de las Ovejas/inmunología , Enfermedades de las Ovejas/microbiología , Enfermedades de las Ovejas/prevención & controlRESUMEN
Heartwater is a tick-borne non-infectious fatal disease of wild and domestic ruminants caused by the bacterium Ehrlichia ruminantium, transmitted by Amblyomma ticks. Although there is evidence that interferon-gamma (IFN-γ) controls E. ruminantium growth and that cellular immune responses could be protective, an effective recombinant vaccine for this disease is lacking. An overall analysis of which immune pathways are up- or down-regulated in sheep peripheral blood mononuclear cells is expected to lead to a better understanding of the global immune response of sheep to E. ruminantium infection. Therefore, a systems biology oriented approach following the infection with E. ruminantium was investigated from peripheral blood mononuclear cells to aid recombinant vaccine development. In this study, heartwater naïve sheep were infected and challenged by allowing E. ruminantium infected ticks to feed on them. After primary infection, all the animals were treated with antibiotic during the resulting febrile response. Blood was collected daily for E. ruminantium detection by qPCR (pCS20 assay). The pCS20 assay only detected the pathogen in the blood one day prior to and during the febrile stage of infection confirming infection of the sheep. IFN-γ real-time PCR indicated that this cytokine was expressed at specific time points: post infection, during the febrile stage of the disease and after challenge. These were used as a guide to select samples for transcriptome sequencing. This paper focuses on transcripts that are associated with innate activating pathways that were identified to be up- and down-regulated after primary infection and the subsequent challenge. These included the CD14 monocyte marker, toll-like receptor (TLR), nod-like receptor, chemokine, cytosolic and cytokine-cytokine interaction receptor pathways. In particular, TLR4, TLR9 and CD14 were activated together with DNA detection pathways, suggesting that vaccine formulations may be improved if CpG motifs and lipopolysaccharides are included. This data indicates that innate immune activation, perhaps by using adjuvants, should be an important component for consideration during future heartwater recombinant vaccine development.
Asunto(s)
Ehrlichia ruminantium/inmunología , Hidropericardio/inmunología , Inmunidad Innata , Leucocitos Mononucleares/inmunología , Enfermedades de las Ovejas/inmunología , Ovinos/inmunología , Transcriptoma/inmunología , Animales , Femenino , Hidropericardio/patología , Leucocitos Mononucleares/patología , Masculino , Ovinos/microbiología , Enfermedades de las Ovejas/microbiologíaRESUMEN
Diseases induced by obligate intracellular pathogens have a large burden on global human and animal health. Understanding the factors involved in the virulence and fitness of these pathogens contributes to the development of control strategies against these diseases. Based on biological observations, a theoretical model using game theory is proposed to explain how obligate intracellular bacteria interact with their host. The equilibrium in such a game shows that the virulence and fitness of the bacterium is host-triggered and by changing the host's defense system to which the bacterium is confronted, an evolutionary process leads to an attenuated strain. Although, the attenuation procedure has already been conducted in practice in order to develop an attenuated vaccine (e.g., with Ehrlichia ruminantium), there was a lack of understanding of the theoretical basis behind this process. Our work provides a model to better comprehend the existence of different phenotypes and some underlying evolutionary mechanisms for the virulence of obligate intracellular bacteria.
Asunto(s)
Bacterias/inmunología , Bacterias/patogenicidad , Vacunas Bacterianas/inmunología , Teoría del Juego , Modelos Biológicos , Animales , Evolución Biológica , Citoplasma/microbiología , Ehrlichia ruminantium/inmunología , Ehrlichia ruminantium/patogenicidad , Hidropericardio/inmunología , Hidropericardio/prevención & control , Interacciones Huésped-Patógeno , Humanos , Vacunas Atenuadas/inmunología , Virulencia/inmunologíaRESUMEN
The use of cheap and thermoresistant vaccines in poor tropical countries for the control of animal diseases is a key issue. Our work aimed at designing and validating a process for the large-scale production of a ready-to-use inactivated vaccine for ruminants. Our model was heartwater caused by the obligate intracellular bacterium Ehrlichia ruminantium (ER). The conventional inactivated vaccine against heartwater (based on whole bacteria inactivated with sodium azide) is prepared immediately before injection, using a syringe-extrusion method with Montanide ISA50. This is a fastidious time-consuming process and it limits the number of vaccine doses available. To overcome these issues, we tested three different techniques (syringe, vortex and homogenizer) and three Montanide ISA adjuvants (50, 70 and 70M). High-speed homogenizer was the optimal method to emulsify ER antigens with both ISA70 and 70M adjuvants. The emulsions displayed a good homogeneity (particle size below 1 µm and low phase separation), conductivity below 10 µS/cm and low antigen degradation at 4 °C for up to 1 year. The efficacy of the different formulations was then evaluated during vaccination trials on goats. The inactivated ER antigens emulsified with ISA70 and ISA70M in a homogenizer resulted in 80% and 100% survival rates, respectively. A cold-chain rupture assay using ISA70M+ER was performed to mimic possible field conditions exposing the vaccine at 37 °C for 4 days before delivery. Surprisingly, the animal survival rate was still high (80%). We also observed that the MAP-1B antibody response was very similar between animals vaccinated with ISA70+ER and ISA70M+ER emulsions, suggesting a more homogenous antigen distribution and presentation in these emulsions. Our work demonstrated that the combination of ISA70 or ISA70M and homogenizer is optimal for the production of an effective ready-to-use inactivated vaccine against heartwater, which could easily be produced on an industrial scale.
Asunto(s)
Vacunas Bacterianas/inmunología , Vacunas Bacterianas/aislamiento & purificación , Ehrlichia ruminantium/inmunología , Hidropericardio/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Vacunas Bacterianas/administración & dosificación , Estabilidad de Medicamentos , Emulsiones/administración & dosificación , Emulsiones/aislamiento & purificación , Cabras , Hidropericardio/inmunología , Inyecciones Intramusculares , Análisis de Supervivencia , Clima Tropical , Vacunación/métodos , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunología , Vacunas de Productos Inactivados/aislamiento & purificaciónRESUMEN
Ehrlichia ruminantium is an obligate intracellular bacterial pathogen which causes heartwater, a serious tick-borne disease of ruminants throughout sub-Saharan Africa. The development of promising recombinant vaccines has been reported previously, but none has been as effective as immunisation with live organisms. In this study we have used reverse vaccinology to identify proteins that elicit an in vitro cellular immune response similar to that induced by intact E. ruminantium. The experimental strategy involved four successive steps: (i) in silico selection of the most likely vaccine candidate genes from the annotated genome; (ii) cloning and expression of the selected genes; (iii) in vitro screening of the expressed proteins for their ability to induce interferon-gamma (IFN-γ) production in E. ruminantium-immune lymphocytes; and (iv) further examination of the cytokine response profiles of those lymphocytes which tested positive for IFN-γ induction. Based on their overall cytokine induction profiles the recombinant proteins were divided into four distinct groups. Eleven recombinant proteins induced a cytokine profile that was similar to the recall immune response induced by immune peripheral blood mononuclear cells (PBMC) stimulated with intact E. ruminantium. This response comprised the upregulation of cytokines associated with adaptive cellular immune responses as well as innate immunity. A successful vaccine may therefore need to contain a combination of recombinant proteins which induce both immune pathways to ensure protection against heartwater.
Asunto(s)
Proteínas Bacterianas/inmunología , Ehrlichia ruminantium/inmunología , Animales , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/fisiología , Proteínas Bacterianas/farmacología , Vacunas Bacterianas/inmunología , Bovinos/inmunología , Bovinos/microbiología , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/microbiología , Hidropericardio/inmunología , Inmunidad Celular/efectos de los fármacos , Inmunidad Celular/inmunología , Interferón gamma/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Ovinos/inmunología , Ovinos/microbiología , Enfermedades de las Ovejas/inmunología , Enfermedades de las Ovejas/microbiologíaRESUMEN
In order to identify the appropriate strains to use in vaccination trials against heartwater in Burkina Faso, the protective effect of Gardel and Welgevonden strains was assessed against local strains on sheep vaccinated by infection-and-treatment method: Gardel protected significantly against Burkina Faso strains tested (survival rate 59% for immunised sheep vs 13% for control sheep) while Welgevonden did not (survival rate 45% for immunised sheep vs 25% for control sheep). The efficacy of the ISA50 inactivated vaccine, produced under industrial process, was evaluated in sheep during field challenges in two successive years. During year 1, there was a limited protective effect of the Gardel vaccine with 65% of survival rate for the vaccinated group compared to 49% for the control group (N=153, p=0.053). During year 2, the vaccine containing Gardel and a local strain gave an increased protective effect compared to the first trial: 72% of the vaccinated animals survived compared to 47% of the naïve animals (N=173, p<0.001). There was an important genetic diversity of strains in the field with detection of 11 different map1 genotypes in brains from control and vaccinated sheep post mortem. Map1 genotyping of strains detected in brains from control sheep showed that genotype distribution varied according to time and study areas, which could explain the difference in efficacy of the vaccine.
Asunto(s)
Vacunas Bacterianas/inmunología , Ehrlichia ruminantium/genética , Variación Genética , Hidropericardio/prevención & control , Enfermedades de las Ovejas/prevención & control , Animales , Antígenos Bacterianos/inmunología , Técnicas de Tipificación Bacteriana , Burkina Faso , Protección Cruzada , Ehrlichia ruminantium/clasificación , Ehrlichia ruminantium/inmunología , Genotipo , Hidropericardio/inmunología , Ovinos/inmunología , Ovinos/microbiología , Enfermedades de las Ovejas/inmunología , Garrapatas , Vacunas de Productos Inactivados/inmunologíaRESUMEN
A previously identified polymorphic Ehrlichia ruminantium gene, Erum2510, was investigated to determine its ability to induce protective immunity in ruminants following two different DNA immunisation strategies; DNA-only and a DNA prime/recombinant protein (rprotein) boost immunisation. The DNA-only vaccine was also compared to a cocktail of three polymorphic E. ruminantium (Welgevonden) open reading frames (ORFs) adjacent to Erum2510 in the genome. Weak protection was observed in animals immunised with the pCMViUBs_Erum2510 construct alone, while none of the animals immunised with the DNA cocktail were protected. In contrast, all five animals immunised using a DNA prime/rprotein boost strategy survived challenge, thereby indicating that Erum2510 is a good candidate for inclusion in a recombinant vaccine against heartwater. One drawback of using polymorphic genes is a possible lack of cross-protection between genotypes, therefore the genetic diversity of Erum2510 was investigated to establish the degree of polymorphism among different E. ruminantium stocks. Three distinct genotypes were identified indicating that if this gene is used as a vaccine (prime/boost strategy) the vaccine should include a representative Erum2510 gene from each genotype.
Asunto(s)
Vacunas Bacterianas/inmunología , Ehrlichia ruminantium/genética , Hidropericardio/prevención & control , Animales , Clonación Molecular , Ehrlichia ruminantium/inmunología , Genotipo , Hidropericardio/inmunología , Inmunización Secundaria , Sistemas de Lectura Abierta , Filogenia , Polimorfismo Genético , Ovinos , Vacunas Sintéticas/inmunologíaRESUMEN
Four E. ruminantium 1H12 open reading frames and their proteins known to protect sheep against heartwater needle challenge were encapsulated into, or adsorbed onto poly(d,l-lactide-co-glycolide) microparticles. Microspheres with smooth surface and smaller than 5 µm diameters were produced, with high adsorption and encapsulation efficiencies. Gel electrophoresis showed that neither encapsulation nor adsorption affected the stability of the DNA or proteins. Cationic microparticles released â¼40% of plasmid DNA on day 1 while PLGA 50:50-COOH microparticles co-encapsulating plasmid DNA and polyvinyl alcohol only started to release from days 12-28. Recombinant proteins were released from PLGA 85:15 and homopolymer R 203 S microparticles in a biphasic manner with a high initial burst release (â¼45-80%). In contrast, PLGA 50:50 microparticles had low (15-65%) initial burst release followed by (25-80%) release by days (days 28-42). A cocktail of these microparticles could therefore be used as single-dose auto-booster vaccine.
Asunto(s)
Ehrlichia ruminantium/genética , Hidropericardio/prevención & control , Vacunas de ADN/química , Adsorción , Animales , Proteínas Bacterianas/química , ADN Bacteriano/química , Composición de Medicamentos , Hidropericardio/inmunología , Cinética , Ácido Láctico/química , Sistemas de Lectura Abierta , Plásmidos/aislamiento & purificación , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Ovinos/inmunologíaRESUMEN
Sera from 497 sheep and 555 goats collected in a cross sectional study from different geographical locations in north-eastern Tanzania were examined for antibodies to Ehrlichia ruminantium using MAP 1-B ELISA technique. E. ruminantium antibodies were found in 68.6% (341/497) of sheep and 64.7% (359/555) of goats. Overall seroprevalence was 66.5% (700/1052). Infection rates were higher in sheep than goats (P < 0.05), in pastoral than in agro-pastoral production systems (P < 0.05) and in female sheep than males (P < 0.05). (131/143) 91.6% of the farms/flocks tested revealed sero-positive animals. E.ruminantium infections were found in all the geographical villages and districts tested. The infection rates per administrative district varied from 36.4% (Muheza) to 90% (Mkinga) in goats and from 11.9% (Muheza) to 94.6% (Mkinga) in sheep. The results shows E. ruminantium infection was prevalent and widely but unevenly distributed throughout the eight districts under study. These findings should be taken into consideration when future disease control and livestock upgrading programs are implemented.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Ehrlichia ruminantium/inmunología , Enfermedades de las Cabras/sangre , Hidropericardio/epidemiología , Enfermedades de las Ovejas/sangre , Animales , Ensayo de Inmunoadsorción Enzimática/veterinaria , Enfermedades de las Cabras/epidemiología , Enfermedades de las Cabras/inmunología , Cabras , Hidropericardio/sangre , Hidropericardio/inmunología , Factores de Riesgo , Estudios Seroepidemiológicos , Ovinos , Enfermedades de las Ovejas/epidemiología , Enfermedades de las Ovejas/inmunología , Tanzanía/epidemiologíaRESUMEN
Heterologous prime/boost immunisation strategies using the Ehrlichia ruminantium 1H12 pCMViUBs_ORFs [Pretorius A, Collins NE, Steyn HC, Van Strijp F, Van Kleef M, Allsopp BA. Protection against heartwater by DNA immunisation with four Ehrlichia ruminantium open reading frames. Vaccine 2007;25(12):2316-24] were investigated in this study. All the animals immunised twice with a recombinant (r) DNA cocktail of four 1H12 pCMViUBs_ORFs followed by a r1H12 protein and those immunised 3x with 1H12 plasmid rDNA showed 100% protection against a virulent E. ruminantium Welgevonden needle challenge. In addition, 90% of the sheep immunised twice with rDNA and boosted with r1H12 lumpy skin disease virus (LSDV) survived. Only the lymphocytes isolated from the r1H12 protein boost group showed specific proliferation and increased interferon (IFN)-gamma expression. In contrast, only 20% protection was obtained in animals immunised with the rDNA prime/r1H12 protein boost when subjected to natural tick challenge in the field. Thus this heterologous prime/boost immunisation strategy had not conferred any significant protection against a field challenge.
Asunto(s)
Transmisión de Enfermedad Infecciosa/prevención & control , Ehrlichia ruminantium/inmunología , Hidropericardio/prevención & control , Inmunización Secundaria/métodos , Enfermedades de las Ovejas/prevención & control , Vacunas de ADN/inmunología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proliferación Celular , Vectores de Enfermedades , Fiebre/etiología , Vectores Genéticos , Hidropericardio/inmunología , Interferón gamma/biosíntesis , Virus de la Dermatosis Nodular Contagiosa/genética , Linfocitos/inmunología , Ovinos , Enfermedades de las Ovejas/inmunología , Análisis de Supervivencia , Garrapatas/microbiología , Factores de Tiempo , Vacunas de Subunidad/inmunologíaAsunto(s)
Babesiosis/prevención & control , Hidropericardio/prevención & control , Theileriosis/prevención & control , Enfermedades por Picaduras de Garrapatas/prevención & control , Vacunas Atenuadas/inmunología , Animales , Animales Domésticos , Babesiosis/inmunología , Babesiosis/parasitología , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/inmunología , Ehrlichia ruminantium , Hidropericardio/inmunología , Hidropericardio/microbiología , Vacunas Antiprotozoos/administración & dosificación , Vacunas Antiprotozoos/inmunología , Theileria/clasificación , Theileria/inmunología , Theileriosis/inmunología , Theileriosis/parasitología , Enfermedades por Picaduras de Garrapatas/inmunología , Enfermedades por Picaduras de Garrapatas/microbiología , Enfermedades por Picaduras de Garrapatas/parasitología , Vacunas Atenuadas/administración & dosificaciónRESUMEN
The attenuated Ehrlichia ruminantium (Welgevonden) stock provides protection against a virulent homologous needle challenge in Merino sheep and Boer goats against heartwater. In this study, cryopreserved stabilates were tested for their suitability as a vaccine in Merino sheep. Vaccination did not produce disease and upon challenge with the virulent homologous stock all animals were fully protected. The vaccination protected all except one animal out of 5 challenged 12 months after immunization. The intramuscular route gave better protection than the subcutaneously applied vaccine. The attenuated vaccine was further evaluated in highly susceptible Angora goats. Although the attenuated vaccine showed an unexpectedly high degree of virulence, animals were fully protected against a lethal needle challenge.
Asunto(s)
Vacunas Bacterianas/administración & dosificación , Ehrlichia ruminantium/inmunología , Enfermedades de las Cabras/prevención & control , Hidropericardio/prevención & control , Enfermedades de las Ovejas/prevención & control , Vacunas Atenuadas/administración & dosificación , Animales , Animales Domésticos , Vacunas Bacterianas/inmunología , Criopreservación , Enfermedades de las Cabras/inmunología , Enfermedades de las Cabras/microbiología , Cabras , Hidropericardio/inmunología , Hidropericardio/microbiología , Masculino , Ovinos , Enfermedades de las Ovejas/inmunología , Enfermedades de las Ovejas/microbiología , Resultado del Tratamiento , Vacunación/veterinaria , Vacunas Atenuadas/inmunología , VirulenciaRESUMEN
Heartwater (cowdriosis) is a disease of ruminants caused by a rickettsial pathogen Ehrlichia ruminantium and transmitted by ticks of the genus Amblyomma. The purpose of this work was to evaluate the protective efficacies of inactivated and attenuated vaccines to protect sheep against heartwater in The Gambia. An inactivated vaccine, prepared from E. ruminantium (Gardel stock), and a live attenuated vaccine from E. ruminantium (Senegal stock), were evaluated in two independent on-station trials. A local stock of E. ruminantium (Kerr Seringe) was used as challenge material. Inactivated and live attenuated vaccines provided 43% and 100% protection, respectively, against virulent needle challenge. In a subsequent field trial, the attenuated vaccine protected 75% of sheep against virulent tick challenge, which was fatal for all control sheep. Quantification by real-time PCR showed that an immunising dose of approximately 23,000 attenuated E. ruminantium organisms was sufficient. Moreover, restriction fragment length polymorphism (RFLP) analysis indicated that the local Kerr Seringe genotype caused mortality amongst control sheep, whereas fatalities in the vaccinated group could be attributed to a different genotype.
Asunto(s)
Vacunas Bacterianas/inmunología , Ehrlichia ruminantium/inmunología , Hidropericardio/prevención & control , Enfermedades de las Ovejas/prevención & control , Ovinos/inmunología , Animales , Vacunas Bacterianas/genética , ADN Bacteriano/sangre , ADN Bacteriano/inmunología , Ehrlichia ruminantium/genética , Gambia , Genotipo , Hidropericardio/sangre , Hidropericardio/genética , Hidropericardio/inmunología , Inmunización , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Ovinos/microbiología , Enfermedades de las Ovejas/sangre , Enfermedades de las Ovejas/genética , Enfermedades de las Ovejas/inmunología , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunologíaRESUMEN
Overwhelming evidence has accumulated of the effectiveness of immunization with live attenuated vaccines to control tick-borne diseases of livestock. Despite several disadvantages, vaccination with live attenuated organisms against tropical theileriosis, babesiosis and possibly heartwater constitutes one of the most cost-effective intervention strategies. Although great advances have been made through genomics and proteomics research, this has not yet translated into effective non-living vaccines. As a result, there is a continuing necessity to use available live vaccines in tick and tick-borne disease-control strategies adapted to conditions prevailing in many parts of the world.
Asunto(s)
Vacunas Bacterianas , Enfermedades de los Bovinos/prevención & control , Vacunas Antiprotozoos , Enfermedades por Picaduras de Garrapatas/prevención & control , Vacunas Atenuadas , Animales , Vectores Arácnidos/microbiología , Vectores Arácnidos/parasitología , Babesia bovis/inmunología , Babesiosis/inmunología , Babesiosis/prevención & control , Bovinos , Enfermedades de los Bovinos/inmunología , Ehrlichia ruminantium/inmunología , Hidropericardio/inmunología , Hidropericardio/prevención & control , Theileria parva/inmunología , Theileriosis/inmunología , Theileriosis/prevención & control , Enfermedades por Picaduras de Garrapatas/inmunología , Garrapatas/microbiología , Garrapatas/parasitologíaRESUMEN
This work evaluates the effect of purification process and storage conditions (buffer formulation and temperature) on the efficacy of Ehrlichia ruminantium (ER) elementary bodies to be used as an inactivated vaccine candidate against heartwater. In vitro assays revealed that, to avoid major losses in ER integrity and corresponding antigenic properties, a buffer with pH between 5.6 and 8 and an osmolality above 100 mOsmol/kg H(2)O is recommended. Amongst the tested formulations, both PBS and NaCl have shown to stabilize ER antigens at -20 degrees C. To assess the protective properties of the different vaccine formulations, in vivo experiments were performed using a goat model. The results obtained showed that the preparation of ER antigens using a novel membrane-based purification strategy and a simple vaccine formulation (NaCl, -20 degrees C) induced equivalent protection to the conventional vaccine based on ER antigens prepared by a multistep centrifugation methodology and stored at -20 degrees C in PBS buffer.
Asunto(s)
Vacunas Bacterianas/química , Vacunas Bacterianas/uso terapéutico , Ehrlichia/inmunología , Hidropericardio/inmunología , Hidropericardio/prevención & control , Algoritmos , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/aislamiento & purificación , Bovinos , Química Farmacéutica , Cricetinae , Ehrlichia/crecimiento & desarrollo , Células Endoteliales/inmunología , Cabras/inmunología , Concentración de Iones de Hidrógeno , Microscopía Fluorescente , Concentración Osmolar , Temperatura , Ultrafiltración , Vacunas de Productos Inactivados/química , Vacunas de Productos Inactivados/aislamiento & purificación , Vacunas de Productos Inactivados/uso terapéuticoRESUMEN
We have reported previously that a recombinant DNA vaccine consisting of four Ehrlichia ruminantium (Welgevonden) open reading frames (ORFs) known as the 1H12 cocktail provided protection against a virulent E. ruminantium (Welgevonden) needle challenge in sheep. In this study, we have investigated the vaccine effectiveness of two other cocktails of E. ruminantium (Welgevonden) ORFs, as well as single ORFs from the 1H12 cocktail, to protect sheep against a virulent needle challenge with the homologous strain. Each individual 1H12 ORF provided protection, but all the animals vaccinated with the other cocktails succumbed to the challenge.
Asunto(s)
Ehrlichia ruminantium/inmunología , Hidropericardio/inmunología , Inmunización/métodos , Sistemas de Lectura Abierta/genética , Vacunas de ADN/inmunología , Animales , Islas de CpG/genética , Ehrlichia ruminantium/genética , Hidropericardio/prevención & control , Linfocitos/inmunología , Ovinos , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genéticaRESUMEN
Using the MAP1-B enzyme-linked immunosorbent assay, we tested 1,318 serum samples collected from sheep and goats at 28 sites in the five divisions of The Gambia to determine the Ehrlichia ruminantium seroprevalence rates and to assess the risk for heartwater. About half (51.6%) of 639 sheep were positive, with seroprevalence rates per site varying between 6.9% and 100%. The highest seroprevalence was detected in the western part of the country (88.1% in the Western Division and 62.1% in the Lower River Division). Sheep in the two easterly divisions (Central River and Upper River divisions) showed the lowest seroprevalence of 29.3% and 32.4%, respectively, while those in the North Bank Division showed an intermediate prevalence of 40.6%. In goats, less than one-third (30.3%) of 679 animals tested were positive. The highest seroprevalence was detected in goats in the North Bank Division (59%) and Western Division (44.1%). Goats in the Lower River Division showed an intermediate level of 21.9%, whereas the lowest rates were found in the eastern part of the country (4.8% in the Central River Division and 2.3% in the Upper River Division). At nearly all sites, seroprevalence rates were higher in sheep than in goats. The results show a gradient of increasing heartwater risk for susceptible small ruminants from the east to the west of The Gambia. These findings need to be taken into consideration when future livestock-upgrading programs are implemented.
Asunto(s)
Ehrlichia ruminantium , Enfermedades de las Cabras/epidemiología , Hidropericardio/epidemiología , Estudios Seroepidemiológicos , Enfermedades de las Ovejas/epidemiología , Análisis de Varianza , Animales , Antígenos Bacterianos/sangre , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/sangre , Proteínas Bacterianas/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Gambia/epidemiología , Enfermedades de las Cabras/sangre , Enfermedades de las Cabras/microbiología , Cabras , Hidropericardio/diagnóstico , Hidropericardio/inmunología , Hidropericardio/microbiología , Proteínas de la Membrana/sangre , Proteínas de la Membrana/inmunología , Ovinos , Enfermedades de las Ovejas/sangre , Enfermedades de las Ovejas/inmunología , Enfermedades de las Ovejas/microbiologíaRESUMEN
Ehrlichia ruminantium, the agent of cowdriosis transmitted by Amblyomma ticks, presents an extensive genetic and antigenic diversity of key importance for vaccine formulation. Two means of nested polymerase chain reaction (PCR) targeting were developed to conduct molecular epidemiology studies in the Caribbean and Africa. The first used a conserved DNA fragment for detection of the pathogen in animals and vectors, and the second relied on the polymorphic map1 gene for genotyping. As compared to a PCR, the nested PCR showed a 2-Log10 improvement of sensitivity and allowed amplification from ticks, blood, brain, and lungs from infected animals, providing a more accurate picture of the tick infection rate. In Guadeloupe, this rate reached 36% (N = 212) instead of 1.7% (N = 224), as previously estimated. Genetic typing was done by restriction fragment length polymorphism or sequencing of map1 amplification products. Molecular epidemiology studies conducted in field sites selected for vaccination trials with inactivated vaccine, revealed the circulation of genetically divergent strains in limited geographical areas. It is known, then, that genetic clustering based on map1 has no predictive value regarding the protective value of a given strain against a new strain. However, tracing the strains by this technique revealed the extent of E. ruminantium diversity that one can expect in a given region, and the method allows differentiation between an inadequate immune response and the challenge by a breakthrough strain on animals dying despite vaccination. Up to now, genetic typing does not avoid cross-protection studies, which were conducted in parallel, although on a more limited scale. The importance of pathogen diversity studies for optimization of vaccine design is discussed as well as the research for new polymorphic genes. These genes may allow better predictions on cross-protection, given the recent completion of the sequence of the full genome of two E. ruminantium strains.
Asunto(s)
ADN Bacteriano/análisis , Ehrlichia ruminantium/genética , Hidropericardio/genética , Hidropericardio/inmunología , Garrapatas/microbiología , Animales , Animales Domésticos , Vectores de Enfermedades , Variación Genética , Genotipo , Hidropericardio/prevención & control , Epidemiología Molecular , Técnicas de Amplificación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , VacunasRESUMEN
IFN-gamma is considered as a key factor in protection against heartwater of ruminants, caused by the obligate intracellular bacterium Ehrlichia ruminantium. In this study, a better definition of the molecular masses of IFN-gamma inducing proteins of the Gardel strain of E. ruminantium was obtained by the use of continuous flow electrophoresis (CFE) and sensitized polyclonal lymphocytes. Out of 15 E. ruminantium CFE fractions tested within the 14-39 kDa region, eight were commonly reacted to by all goats. Interestingly, half of these fractions fall within the 23-29 kDa region, shown previously to contain polymorphic B-cell epitopes. Thus, the results suggest that this region also contains T-cell epitopes potentially involved in protection. Also, several proteins were found to be more immunogenic than the serologically immunodominant MAP1 protein. Finally, high activity within the 15-19 kDa region was observed, which confirms previous work done with CD4+ T-cell lines obtained from cattle immunized with a South African strain of E. ruminantium. The proteins falling within the molecular weight ranges defined in this study may have potential as vaccine antigens.