RESUMEN
BACKGROUND: Nanoparticles (NPs) are viable candidates as carriers of exogenous materials into cells via transfection and can be used in the DNA vaccination strategy against leptospirosis. OBJECTIVES: We evaluated the efficiency of halloysite clay nanotubes (HNTs) and amine-functionalised multi-walled carbon nanotubes (NH2-MWCNTs) in facilitating recombinant LemA antigen (rLemA) expression and protecting Golden Syrian hamsters (Mesocricetus auratus) against Leptospira interrogans lethal infection. METHODS: An indirect immunofluorescent technique was used to investigate the potency of HNTs and NH2-MWCNTs in enhancing the transfection and expression efficiency of the DNA vaccine in Chinese hamster ovary (CHO) cells. Hamsters were immunised with two doses of vaccines HNT-pTARGET/lemA, NH2-MWCNTs-pTARGET/lemA, pTARGET/lemA, and empty pTARGET (control), and the efficacy was determined in terms of humoral immune response and protection against a lethal challenge. FINDINGS: rLemA DNA vaccines carried by NPs were able to transfect CHO cells effectively, inducing IgG immune response in hamsters (p < 0.05), and did not exhibit cytotoxic effects. Furthermore, 83.3% of the hamsters immunised with NH2-MWCNTs-pTARGET/lemA were protected against the lethal challenge (p < 0.01), and 66.7% of hamsters immunised with HNT-pTARGET/lemA survived (p < 0.05). MAIN CONCLUSIONS: NH2-MWCNTs and HNTs can act as antigen carriers for mammalian cells and are suitable for DNA nanovaccine delivery.
Asunto(s)
Antígenos Bacterianos/administración & dosificación , Proteínas Bacterianas/administración & dosificación , Vacunas Bacterianas/administración & dosificación , Leptospirosis/prevención & control , Factores de Transcripción/administración & dosificación , Vacunas de ADN/administración & dosificación , Animales , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Cricetinae , Modelos Animales de Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Leptospira interrogans/inmunología , Leptospirosis/inmunología , Nanopartículas , Factores de Transcripción/inmunología , Vacunas de ADN/inmunologíaRESUMEN
Leptospirosis is a major public health problem with an incidence of over one million human cases each year. It is a globally distributed, zoonotic disease and is associated with significant economic losses in farm animals. Leptospirosis is caused by pathogenic Leptospira spp. that can infect a wide range of domestic and wild animals. Given the inability to control the cycle of transmission among animals and humans, there is an urgent demand for a new vaccine. Inactivated whole-cell vaccines (bacterins) are routinely used in livestock and domestic animals, however, protection is serovar-restricted and short-term only. To overcome these limitations, efforts have focused on the development of recombinant vaccines, with partial success. Reverse vaccinology (RV) has been successfully applied to many infectious diseases. A growing number of leptospiral genome sequences are now available in public databases, providing an opportunity to search for prospective vaccine antigens using RV. Several promising leptospiral antigens were identified using this approach, although only a few have been characterized and evaluated in animal models. In this review, we summarize the use of RV for leptospirosis and discuss the need for potential improvements for the successful development of a new vaccine towards reducing the burden of human and animal leptospirosis.
Asunto(s)
Vacunas Bacterianas/inmunología , Leptospira/inmunología , Animales , Antígenos Bacterianos/inmunología , Humanos , Leptospira/genética , Leptospirosis/inmunología , Leptospirosis/microbiología , Vacunas Sintéticas/inmunologíaRESUMEN
Bovine herpesvirus (BoHV) glycoprotein E (gE) is a non-essential envelope glycoprotein and the deletion of gE has been used to develop BoHV-1 and BoHV-5 differential vaccine strains. The DIVA (Differentiation of Infected from Vaccinated Animals) strategy, using marker vaccines based on gE-negative BoHV strains, allows the identification of vaccinated or infected animals in immunoassays designed to detect anti-gE antibodies. In this study a codon optimized synthetic sequence of gE containing highly conserved regions from BoHV-1 and BoHV-5 was expressed in Pichia pastoris. Following expression, the recombinant gE (rgE) was secreted and purified from the culture medium. The rgE was identified by Western blotting (WB) using sera from cattle naturally infected with BoHV-1 and/or BoHV-5, or sera from bovines experimentally infected with wild-type BoHV-5. Sera collected from cattle vaccinated with a BoHV-5 gI/gE/US9¯ marker vaccine failed to recognise rgE. Expression of rgE, based on a sequence containing highly conserved regions from BoHV-1 and BoHV-5, in P. pastoris enabled the production of large quantities of rgE suitable for use in immunoassays for the differentiation vaccinated or infected cattle.
Asunto(s)
Expresión Génica , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 5 , Rinotraqueítis Infecciosa Bovina , Pichia/metabolismo , Proteínas del Envoltorio Viral , Proteínas Virales , Animales , Bovinos , Herpesvirus Bovino 1/metabolismo , Vacunas contra Herpesvirus/farmacología , Rinotraqueítis Infecciosa Bovina/sangre , Rinotraqueítis Infecciosa Bovina/diagnóstico , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/aislamiento & purificación , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificación , Proteínas Virales/metabolismoRESUMEN
Immunisation with the C-terminal region of leptospiral immunoglobulin-like A protein (LigANI) has shown promising results against leptospirosis. We evaluated the humoral immune response and protection induced by LigANI associated with carboxyl multi-walled carbon nanotubes (COOH-MWCNTs), CpG oligodeoxynucleotides (CpG ODNs), or Alhydrogel. Animals immunised with CpG ODNs were unable to develop a humoral immune response, whereas immunisation with LigANI and COOH-MWCNTs produced a high level of IgG antibodies, similar to that with LigANI and Alhydrogel, but it was not protective. The use of carbon nanotubes as an adjuvant in subunit vaccines against leptospirosis is a novel approach for improving specific IgG production.
Asunto(s)
Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Inmunoglobulina G/inmunología , Leptospira interrogans/química , Leptospirosis/inmunología , Nanotubos de Carbono , Adyuvantes Inmunológicos , Animales , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Cricetinae , Inmunidad Humoral , Inmunoglobulina G/sangre , Leptospira interrogans/inmunología , Mesocricetus , Proteínas Recombinantes/inmunología , Vacunas de Subunidad/inmunologíaRESUMEN
Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of the Leptospira genus. Vaccination with bacterins has severe limitations. Here, we evaluated the N-terminal region of the leptospiral immunoglobulin-like B protein (LigBrep) as a vaccine candidate against leptospirosis using immunisation strategies based on DNA prime-protein boost, DNA vaccine, and subunit vaccine. Upon challenge with a virulent strain ofLeptospira interrogans, the prime-boost and DNA vaccine approaches induced significant protection in hamsters, as well as a specific IgG antibody response and sterilising immunity. Although vaccination with recombinant fragment of LigBrep also produced a strong antibody response, it was not immunoprotective. These results highlight the potential of LigBrep as a candidate antigen for an effective vaccine against leptospirosis and emphasise the use of the DNA prime-protein boost as an important strategy for vaccine development.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Leptospira/inmunología , Leptospirosis/prevención & control , Vacunación/métodos , Adyuvantes Inmunológicos , Animales , Biopsia , Chlorocebus aethiops , Secuencia Conservada , Cricetinae , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunidad Humoral/inmunología , Inmunoglobulina A/genética , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulinas/genética , Inmunoglobulinas/inmunología , Riñón/patología , Leptospirosis/inmunología , Pulmón/patología , Mesocricetus , Análisis de Supervivencia , Vacunas de ADN/inmunología , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/microbiología , Células VeroRESUMEN
Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of theLeptospira genus. Vaccination with bacterins has severe limitations. Here, we evaluated the N-terminal region of the leptospiral immunoglobulin-like B protein (LigBrep) as a vaccine candidate against leptospirosis using immunisation strategies based on DNA prime-protein boost, DNA vaccine, and subunit vaccine. Upon challenge with a virulent strain ofLeptospira interrogans, the prime-boost and DNA vaccine approaches induced significant protection in hamsters, as well as a specific IgG antibody response and sterilising immunity. Although vaccination with recombinant fragment of LigBrep also produced a strong antibody response, it was not immunoprotective. These results highlight the potential of LigBrep as a candidate antigen for an effective vaccine against leptospirosis and emphasise the use of the DNA prime-protein boost as an important strategy for vaccine development.
Asunto(s)
Animales , Cricetinae , Femenino , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Leptospira/inmunología , Leptospirosis/prevención & control , Vacunación/métodos , Adyuvantes Inmunológicos , Biopsia , Chlorocebus aethiops , Secuencia Conservada , Ensayo de Inmunoadsorción Enzimática , Inmunidad Humoral/inmunología , Inmunoglobulina A/genética , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulinas/genética , Inmunoglobulinas/inmunología , Riñón/patología , Leptospirosis/inmunología , Pulmón/patología , Mesocricetus , Análisis de Supervivencia , Células Vero , Vacunas de ADN/inmunología , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/microbiologíaRESUMEN
Leptospirosis is a zoonotic disease caused by pathogenic spirochetes from the genus Leptospira, which includes 20 species and more than 300 serovars. Canines are important hosts of pathogenic leptospires and can transmit the pathogen to humans via infected urine. Here, we report the phenotypic and molecular characterization of Leptospira interrogans isolated from Canis familiaris in Southern Brazil. The isolated strain was characterized by variable-number tandem-repeats analysis as L. interrogans, serogroup Icterohaemorrhagiae. In addition, the isolate was recognized by antibodies from human and canine serum samples previously tested by microscopic agglutination test. Ultimately, the expression of membrane-associated antigens (LipL32 and leptospiral immunoglobulin-like proteins) from pathogenic leptospires using monoclonal antibodies was detected by indirect immunofluorescence assay. In conclusion, identification of new strains of Leptospira can help in the diagnosis and control of leptospirosis.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/análisis , Enfermedades de los Perros/microbiología , Perros/microbiología , Leptospira interrogans/química , Leptospira interrogans/genética , Leptospirosis/veterinaria , Lipoproteínas/análisis , Repeticiones de Minisatélite , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/análisis , Brasil , Leptospira interrogans/aislamiento & purificación , Leptospirosis/microbiología , Tipificación MolecularRESUMEN
We studied the feasibility of using halloysite clay nanotubes (HNTs) and carboxyl-functionalised multi-walled carbon nanotubes (COOH-MWCNTs) as antigen carriers to improve immune responses against a recombinant LipL32 protein (rLipL32). Immunisation using the HNTs or COOH-MWCNTs significantly increased the rLipL32-specific IgG antibody titres (p < 0.05) of Golden Syrian hamsters. None of the vaccines tested conferred protection against a challenge using a virulent Leptospira interrogans strain. These results demonstrated that nanotubes can be used as antigen carriers for delivery in hosts and the induction of a humoral immune response against purified leptospiral antigens used in subunit vaccine preparations.
Asunto(s)
Silicatos de Aluminio/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/inmunología , Inmunoglobulina G/análisis , Leptospira interrogans/inmunología , Lipoproteínas/inmunología , Nanotubos de Carbono , Animales , Antígenos/administración & dosificación , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/inmunología , Dióxido de Carbono/inmunología , Arcilla , Cricetinae , Estudios de Factibilidad , Inmunidad Humoral/inmunología , Leptospira interrogans/clasificación , Mesocricetus , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunologíaRESUMEN
We studied the feasibility of using halloysite clay nanotubes (HNTs) and carboxyl-functionalised multi-walled carbon nanotubes (COOH-MWCNTs) as antigen carriers to improve immune responses against a recombinant LipL32 protein (rLipL32). Immunisation using the HNTs or COOH-MWCNTs significantly increased the rLipL32-specific IgG antibody titres (p < 0.05) of Golden Syrian hamsters. None of the vaccines tested conferred protection against a challenge using a virulent Leptospira interrogans strain. These results demonstrated that nanotubes can be used as antigen carriers for delivery in hosts and the induction of a humoral immune response against purified leptospiral antigens used in subunit vaccine preparations.
Asunto(s)
Carbohidratos de la Dieta/análisis , Fibras de la Dieta/análisis , Calidad de los Alimentos , Inspección de Alimentos/métodos , Frutas/química , Modelos Biológicos , Malus/química , Calibración , Productos Agrícolas/química , Productos Agrícolas/crecimiento & desarrollo , Productos Agrícolas/metabolismo , Dinamarca , Carbohidratos de la Dieta/metabolismo , Fibras de la Dieta/metabolismo , Almacenamiento de Alimentos , Alimentos Modificados Genéticamente , Frutas/crecimiento & desarrollo , Frutas/metabolismo , Análisis de los Mínimos Cuadrados , Modelos Lineales , Malus/crecimiento & desarrollo , Malus/metabolismo , Análisis de Regresión , Reproducibilidad de los Resultados , Solubilidad , Espectroscopía Infrarroja CortaRESUMEN
Leptospiral immunoglobulin-like (Lig) proteins are of great interest due to their ability to act as mediators of pathogenesis, serodiagnostic antigens, and immunogens. Purified recombinant LigA protein is the most promising subunit vaccine candidate against leptospirosis reported to date, however, as purified proteins are weak immunogens the use of a potent adjuvant is essential for the success of LigA as a subunit vaccine. In the present study, we compared xanthan pv. pruni (strain 106), aluminium hydroxide (alhydrogel), and CpG ODN as adjuvants in a LigA subunit vaccine preparation. Xanthan gum is a high molecular weight extracellular polysaccharide produced by fermentation of Xanthomonas spp., a plant-pathogenic bacterium genus. Preparations containing xanthan induced a strong antibody response comparable to that observed when alhydrogel was used. Upon challenge with a virulent strain of L. interrogans serovar Copenhageni, significant protection (Fisher test, P < 0.05) was observed in 100%, 100%, and 67% of hamsters immunized with rLigANI-xanthan, LigA-CpG-xanthan, and rLigANI-alhydrogel, respectively. Furthermore, xanthan did not cause cytotoxicity in Chinese hamster ovary (CHO) cells in vitro. The use of xanthan as an adjuvant is a novel alternative for enhancing the immunogenicity of vaccines against leptospirosis and possibly against other pathogens.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Vacunas Bacterianas/inmunología , Leptospirosis/inmunología , Leptospirosis/prevención & control , Polisacáridos Bacterianos/inmunología , Vacunas de Subunidad/inmunología , Animales , Formación de Anticuerpos/efectos de los fármacos , Antígenos Bacterianos/inmunología , Células CHO , Muerte Celular/efectos de los fármacos , Cricetinae , Cricetulus , Modelos Animales de Enfermedad , Femenino , Humedad , Inmunidad Humoral/efectos de los fármacos , Inmunización , Leptospira interrogans/fisiología , Leptospirosis/microbiología , Mesocricetus , Nitrógeno/análisis , Polisacáridos Bacterianos/farmacología , Ácido Pirúvico/análisisRESUMEN
The C-terminal region of the Leptospiral immunoglobulin-like A protein (LigA) contains six carboxy-terminal Ig-like repeat domains (LigANI). Subunit vaccine preparations based on recombinant LigANI produced in Escherichia coli, are promising vaccine candidates, albeit with variable efficacy. In the present study, LigANI was expressed in the methylotrophic yeast Pichia pastoris using a 12 L bioreactor to produce mannosylated LigANI (mLigANI) for use in a vaccine preparation against leptospirosis. Hamsters immunized with a mLigANI vaccine preparation produced a significant IgG antibody response (P < 0.001) and were protected (83.3 %; P < 0.001) against lethal challenge with 36× LD50 of a virulent strain of L. interrogans serovar Copenhageni. A vaccine preparation based on demannosylated mLigANI (nmLigANI) elicited an immune response in hamsters, but did not afford protection. The production of mLigANI in bioreactor by P. pastoris yielded ~50 mg L(-1) of recombinant protein. P. pastoris is a potential platform for the production of leptospiral antigens on an industrial scale. The results demonstrate that LigANI secreted by P. pastoris on mannosylated form (mLigANI) protect hamsters as subunit vaccine of L. interrogans lethal infection.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Reactores Biológicos , Leptospira/química , Leptospirosis/prevención & control , Proteínas Recombinantes/inmunología , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cricetinae , Femenino , Leptospira/genética , Leptospirosis/inmunología , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de SupervivenciaRESUMEN
Pathogenic Leptospira spp. are the etiological agents of leptospirosis, an important disease of both humans and animals. In urban settings, L. interrogans serovars are the predominant cause of disease in humans. The purpose of this study was to characterize a novel Leptospira isolate recovered from an abandoned swimming pool. Molecular characterization through sequencing of the rpoB gene revealed 100% identity with L. interrogans and variable-number tandem-repeat (VNTR) analysis resulted in a banding pattern identical to L. interrogans serogroup Icterohaemorrhagiae, serovar Copenhageni or Icterohaemorrhagiae. The virulence of the strain was determined in a hamster model of lethal leptospirosis. The lethal dose 50% (LD50) was calculated to be two leptospires in female hamsters and a histopathological examination of infected animals found typical lesions associated with severe leptospirosis, including renal epithelium degeneration, hepatic karyomegaly, liver-plate disarray and lymphocyte infiltration. This highly virulent strain is now available for use in further studies, especially evaluation of vaccine candidates.
RESUMEN
Toward developing an effective vaccine capable of conferring heterologous protection, the putative lipoprotein LemA, which presents an M3 epitope similar to that of Listeria, was evaluated as a vaccine candidate in the hamster model of leptospirosis. LemA is conserved (>70% pairwise identity) among the pathogenic Leptospira spp., indicating its potential in stimulating a cross-protective immune response. Using different vaccination strategies, including prime-boost, DNA vaccine, and a subunit preparation, recombinant LemA conferred different levels of protection in hamsters. Significant protection against mortality was observed for the prime-boost and the DNA vaccine strategies, which showed 87.5% (P < 0.01) and 62.5% (P < 0.05) efficacy, respectively. Although the subunit vaccine preparation protected 50.0% of immunized hamsters, the level of protection was not significant. None of the hamsters in the control groups survived challenge with a virulent strain of Leptospira interrogans serogroup Icterohaemorrhagiae. Characterization of the immune response found that the strongest antibody response was stimulated by the subunit vaccine preparation, followed by the prime-boost strategy. The DNA vaccine failed to elicit an antibody response in immunized hamsters.
Asunto(s)
Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Leptospira interrogans serovar icterohaemorrhagiae/inmunología , Leptospirosis/inmunología , Factores de Transcripción/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/administración & dosificación , Cricetinae , Modelos Animales de Enfermedad , Inmunización Secundaria , Leptospirosis/prevención & control , Factores de Transcripción/administración & dosificación , Vacunación , Vacunas de ADN/inmunología , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
The leptospiral immunoglobulin-like (Lig) proteins LigA and LigB possess immunoglobulin-like domains with 90-amino-acid repeats and are adhesion molecules involved in pathogenicity. They are conserved in pathogenic Leptospira spp. and thus are of interest for use as serodiagnostic antigens and in recombinant vaccine formulations. The N-terminal amino acid sequences of the LigA and LigB proteins are identical, but the C-terminal sequences vary. In this study, we evaluated the protective potential of five truncated forms of LigA and LigB proteins from Leptospira interrogans serovar Canicola as DNA vaccines using the pTARGET mammalian expression vector. Hamsters immunized with the DNA vaccines were subjected to a heterologous challenge with L. interrogans serovar Copenhageni strain Spool via the intraperitoneal route. Immunization with a DNA vaccine encoding LigBrep resulted in the survival of 5/8 (62.5%) hamsters against lethal infection (P < 0.05). None of the control hamsters or animals immunized with the other vaccine preparations survived. The vaccine induced an IgG antibody response and, additionally, conferred sterilizing immunity in 80% of the surviving animals. Our results indicate that the LigBrep DNA vaccine is a promising candidate for inclusion in a protective leptospiral vaccine.
Asunto(s)
Antígenos Bacterianos/inmunología , Leptospira interrogans/inmunología , Leptospirosis/prevención & control , Vacunas de ADN/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/química , Proteínas Bacterianas/inmunología , Cricetinae , Femenino , Inmunoglobulina G/inmunología , Leptospirosis/inmunología , Leptospirosis/microbiología , Mesocricetus , Ratones , Ratones Endogámicos BALB C , Vacunación , Vacunas de ADN/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
Leptospirosis is an important global zoonotic disease caused by pathogenic Leptospira spp. species. Swine leptospirosis has a major economic impact because pigs are sources of animal protein and by-products. The signs of swine leptospirosis are abortion, stillbirth, birth of weak or ill piglets, appearing 14-60 days after infection. The reference method for diagnosis of leptospirosis is the microscopic agglutination test (MAT), in which serum samples are reacted with live antigen suspensions of leptospiral serovars. However, MAT is laborious and time consuming as a diagnostic procedure when dealing with a large number of samples; therefore, efforts are being made to develop novel, sensitive, and specific diagnostic tests for leptospirosis. In this study, a recombinant LipL32 based on enzyme-linked immunosorbent assay (rLipL32/ELISA) was evaluated as a screening test for the detection of pathogenic leptospiral-specific antibodies. A total of 86 swine serum samples tested by MAT were used to develop rLipL32/ELISA. Compared to positive and negative sera tested by MAT, rLipL32/ELISA showed 100 % sensitivity, 85.1 % specificity, and 91.86 % accuracy. No positive reaction for other bacterial diseases (enzootic pneumonia and brucellosis) was observed. The rLipL32/ELISA reported in this study is a specific, sensitive, and convenient test for the detection of antibodies against swine leptospiral infection and can be used as a rapid screening test in epidemiological surveys.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Técnicas Bacteriológicas/métodos , Leptospira/inmunología , Leptospirosis/veterinaria , Lipoproteínas , Enfermedades de los Porcinos/diagnóstico , Medicina Veterinaria/métodos , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Leptospira/genética , Leptospirosis/diagnóstico , Lipoproteínas/genética , Tamizaje Masivo/métodos , Proteínas Recombinantes/genética , Sensibilidad y Especificidad , Pruebas Serológicas , PorcinosRESUMEN
Pathogenic Leptospira spp. are the etiological agents of leptospirosis, an important disease of both humans and animals. In urban settings, L. interrogans serovars are the predominant cause of disease in humans. The purpose of this study was to characterize a novel Leptospira isolate recovered from an abandoned swimming pool. Molecular characterization through sequencing of the rpoB gene revealed 100% identity with L. interrogans and variable-number tandem-repeat (VNTR) analysis resulted in a banding pattern identical to L. interrogans serogroup Icterohaemorrhagiae, serovar Copenhageni or Icterohaemorrhagiae. The virulence of the strain was determined in a hamster model of lethal leptospirosis. The lethal dose 50% (LD50) was calculated to be two leptospires in female hamsters and a histopathological examination of infected animals found typical lesions associated with severe leptospirosis, including renal epithelium degeneration, hepatic karyomegaly, liver-plate disarray and lymphocyte infiltration. This highly virulent strain is now available for use in further studies, especially evaluation of vaccine candidates.
Asunto(s)
Ratas , Secuencia de Bases , Genoma Bacteriano , Técnicas In Vitro , Mucosa Intestinal , Leptospira interrogans serovar icterohaemorrhagiae/genética , Leptospira interrogans serovar icterohaemorrhagiae/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Área Urbana , Enfermedad de Weil , Cricetinae , Técnicas Histológicas , Métodos , Piscinas , VirulenciaRESUMEN
Leptospirosis is a globally prevalent zoonosis caused by pathogenic Leptospira spp.; several serologic variants have reservoirs in synanthropic rodents. The capybara is the largest living rodent in the world, and it has a wide geographical distribution in Central and South America. This rodent is a significant source of Leptospira since the agent is shed via urine into the environment and is a potential public health threat. In this study, we isolated and identified by molecular techniques a pathogenic Leptospira from capybara in southern Brazil. The isolated strain was characterized by partial rpoB gene sequencing and variable-number tandem-repeats analysis as L. interrogans, serogroup Icterohaemorrhagiae. In addition, to confirm the expression of virulence factors, the bacterial immunoglobulin-like proteins A and B expression was detected by indirect immunofluorescence using leptospiral specific monoclonal antibodies. This report identifies capybaras as an important source of infection and provides insight into the epidemiology of leptospirosis.
Asunto(s)
Leptospira/clasificación , Leptospira/patogenicidad , Tipificación Molecular , Roedores/microbiología , Factores de Virulencia/biosíntesis , Animales , Brasil , ADN Bacteriano/química , ADN Bacteriano/genética , ARN Polimerasas Dirigidas por ADN/genética , Técnica del Anticuerpo Fluorescente , Leptospira/genética , Leptospira/aislamiento & purificación , Repeticiones de Minisatélite , Análisis de Secuencia de ADNRESUMEN
Leptospirosis is an important neglected infectious disease that occurs in urban environments, as well as in rural regions worldwide. Rodents, the principal reservoir hosts of pathogenic Leptospira spp., and other infected animals shed the bacteria in their urine. During occupational or even recreational activities, humans that come into direct contact with infected animals or with a contaminated environment, particularly water, are at risk of infection. Prevention of urban leptospirosis is largely dependent on sanitation measures that are often difficult to implement, especially in developing countries. Vaccination with inactivated whole-cell preparations (bacterins) has limited efficacy due to the wide antigenic variation of the pathogen. Intensive efforts towards developing improved recombinant vaccines are ongoing. During the last decade, many reports on the evaluation of recombinant vaccines have been published. Partial success has been obtained with some surface-exposed protein antigens. The combination of protective antigens and new adjuvants or delivery systems may result in the much-needed effective vaccine.
Asunto(s)
Antígenos Bacterianos/inmunología , Vacunas Bacterianas/inmunología , Leptospira/inmunología , Leptospirosis/inmunología , Leptospirosis/prevención & control , Vacunas Sintéticas/inmunología , Animales , Antígenos Bacterianos/genética , Vacunas Bacterianas/genética , Callithrix , Cricetinae , Modelos Animales de Enfermedad , Gerbillinae , Cobayas , Humanos , Leptospira/genética , Leptospirosis/microbiología , Mesocricetus , RatasRESUMEN
Leptospirosis is the most widespread zoonosis in the world. Current vaccines are based on whole-cell preparations that cause severe side effects and do not induce satisfactory immunity. In light of the leptospiral genome sequences recently made available, several studies aimed at identification of protective recombinant immunogens have been performed; however, few such immunogens have been identified. The aim of this study was to evaluate 27 recombinant antigens to determine their potential to induce an immune response protective against leptospirosis in the hamster model. Experiments were conducted with groups of female hamsters immunized with individual antigen preparations. Hamsters were then challenged with a lethal dose of Leptospira interrogans. Thirteen antigens induced protective immune responses; however, only recombinant proteins LIC10325 and LIC13059 induced significant protection against mortality. These results have important implications for the development of an efficacious recombinant subunit vaccine against leptospirosis.
Asunto(s)
Antígenos Bacterianos/inmunología , Vacunas Bacterianas/inmunología , Leptospira interrogans/inmunología , Leptospira interrogans/patogenicidad , Leptospirosis/prevención & control , Animales , Antígenos Bacterianos/genética , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/genética , Cricetinae , Modelos Animales de Enfermedad , Femenino , Leptospirosis/inmunología , Subunidades de Proteína/genética , Subunidades de Proteína/inmunología , Análisis de Supervivencia , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
The search for a vaccine capable of conferring heterologous protection, through the identification of conserved and cross-protective antigens, remains an ongoing priority in leptospirosis research. In the present study, an in silico analysis was used to identify potentially protective lipoproteins from Leptospira interrogans serovar Copenhageni. Eight putative lipoproteins were selected (LIC10009, LIC10054, LIC10091, LIC11058, LIC11567, LIC13059, LIC13305, and LIC20172), cloned and expressed in Escherichia coli and purified by affinity chromatography. The recombinant proteins were used to inoculate mice and the subsequent humoral immune response was evaluated by ELISA. Seven of the potential lipoproteins induced a significant IgG response. Furthermore, all of the recombinant proteins were recognized by antibodies present in the sera of severe leptospirosis patients. These putative lipoproteins exhibited potential for further evaluation as prospective vaccine candidates.