RESUMEN

The objectives of this study were to provide the buffalo research community with an updated SNP map for the Axiom Buffalo Genotyping (ABG) array with genomic positions for SNP currently unmapped and to map all cattle QTL from the CattleQTLdb onto the buffalo reference assembly. To update the ABG array map, all SNP probe sequences from the ABG array were re-aligned against the UOA_WB_1 assembly. With the new map, the number of mapped markers increased by approximately 10% and went from 106 778 to 116 708, which reduced the average marker spacing by approximately 2 kb. A comparison of results between signatures of autozygosity study using the ABG and the new map showed that, when the additional markers were used there was an increase in the autozygosity peaks and additional peaks in BBU5 and BBU11 could be identified. After sequence alignment and quality control, 64 650 (UMD3.1) and 76 530 (ARS_UCD1.2) cattle QTL were mapped onto the buffalo genome. The mapping of the bovine QTL database onto the buffalo genome should be useful for genome-wide association studies in buffalo and, given the high homology between the two species, the positions of cattle QTL on the buffalo genome can serve as a stepping stone towards a water buffalo QTL database.

Asunto(s)

Búfalos/genética , Estudio de Asociación del Genoma Completo/veterinaria , Genotipo , Sitios de Carácter Cuantitativo , Animales , Bovinos/genética

RESUMEN

Characterization of autozygosity is relevant to monitor genetic diversity and manage inbreeding levels in breeding programs. Identification of autozygosity hotspots can unravel genomic regions targeted by selection for economically important traits and can help identify candidate genes for selection. In this study, we estimated the inbreeding levels of a Brazilian population of Murrah buffalo undergoing selection for milk production traits, particularly milk yield. We also studied the distribution of runs of homozygosity (ROH) islands and identified putative genes and quantitative trait loci (QTL) under selection. We genotyped 422 Murrah buffalo for 51,611 SNP; 350 of these had ROH longer than 10 Mb, indicating the occurrence of inbreeding in the last 5 generations. The mean length of the ROH per animal was 4.28 ± 1.85 Mb. Inbreeding coefficients were calculated from the genomic relationship matrix, the pedigree, and the ROH, with estimates varying between 0.242 and 0.035. Inbreeding estimates from the pedigree had a low correlation with the genomic estimates, and estimates from the genomic relationship matrix were much higher than those from the pedigree or the ROH. Signatures of selection were identified in 6 genomic regions, located on chromosomes 1, 2, 3, 5, 16, and 18, encompassing a total of 190 genes and 174 QTL. Many of the genes (e.g., APRT and ACSF3) and QTL identified are related to milk production traits, such as milk yield, milk fat yield and percentage, and milk protein yield and percentage. Other genes are associated with reproduction and immune response traits as well as morphological aspects of the buffalo species. Inbreeding levels in this population are still low but are increasing due to selection and should be managed to avoid future losses due to inbreeding depression. The proximity of genes linked to milk production traits with genes associated with reproduction and immune system traits suggests the need to include these latter genes in the breeding program to avoid negatively affecting them due to selection for production traits.

Asunto(s)

Búfalos/genética , Genómica , Leche/metabolismo , Reproducción , Animales , Brasil , Búfalos/fisiología , Femenino , Genotipo , Homocigoto , Endogamia , Masculino , Linaje , Fenotipo , Sitios de Carácter Cuantitativo/genética

RESUMEN

BACKGROUND: Asian buffaloes (Bubalus bubalis) have an important socio-economic role. The majority of the population is situated in developing countries. Due to the scarce resources in these countries, very few species-specific biotechnology tools exist and a lot of cattle-derived technologies are applied to buffaloes. However, the application of cattle genomic tools to buffaloes is not straightforward and, as results suggested, despite genome sequences similarity the genetic polymorphisms are different. RESULTS: The first SNP chip genotyping platform designed specifically for buffaloes has recently become available. Herein, a genome-wide association study (GWAS) and gene network analysis carried out in buffaloes is presented. Target phenotypes were six milk production and four reproductive traits. GWAS identified SNP with significant associations and suggested candidate genes that were specific to each trait and also genes with pleiotropic effect, associated to multiple traits. CONCLUSIONS: Network predictions of interactions between these candidate genes may guide further molecular analyses in search of disruptive mutations, help select genes for functional experiments and evidence metabolism differences in comparison to cattle. The cattle SNP chip does not offer an optimal coverage of buffalo genome, thereafter the development of new buffalo-specific genetic technologies is warranted. An annotated reference genome would greatly facilitate genetic research, with potential impact to buffalo-based dairy production.

Asunto(s)

Búfalos/genética , Animales , Industria Lechera , Estudio de Asociación del Genoma Completo , Genotipo , Polimorfismo de Nucleótido Simple/genética

RESUMEN

This study investigates the genetic association of the SNP present in the ACTA1 gene with performance traits, organs and carcass of broilers to help marker-assisted selection of a paternal broiler line (TT) from EMBRAPA Swine and Poultry, Brazil. Genetic and phenotypic data of 1,400 broilers for 68 traits related to body performance, organ weights, weight of carcass parts, and yields as a percentage of organs and carcass parts were used. The maximum likelihood method, considering 4 analytical models, was used to analyze the genetic association between the SNP and these important economic traits. The association analysis was performed using a mixed animal model including the random effect of the animal (polygenic), and the fixed effects of sex (2 levels), hatch (5 levels) and SNP (3 levels), besides the random error. The traits significantly associated (P<0.05) with the SNP were analyzed, along with body weight at 42 days of age (BW42), by the restricted maximum likelihood method using the multi-trait animal model to estimate genetic parameters. The analysis included the residual and additive genetic random effects and the sex-hatch fixed effect. The additive effects of the SNP were associated with breast meat (BMY), liver yield (LIVY), body weight at 35 days of age (BW35); drumstick skin (DSW), drumstick (DW) and breast (BW) weights. The heritability estimates for these traits, in addition to BW42, ranged from 0.24±0.06 to 0.45±0.08 for LIVY and BW35, respectively. The genetic correlation ranged from 0.02±0.18 for LIVY and BMY to 0.97±0.01 for BW35 and BW42. Based on the results of this study, it can be concluded that ACTA1 gene is associated with performance traits BW35, LIV and BMY, DW, BW and DW adjusted for body weight at 42 days of age. Therefore, the ACTA1 gene is an important molecular marker that could be used together with others already described to increase the economically important traits in broilers.

Asunto(s)

Actinas/genética , Proteínas Aviares/genética , Pollos/fisiología , Polimorfismo de Nucleótido Simple , Actinas/metabolismo , Animales , Proteínas Aviares/metabolismo , Peso Corporal , Brasil , Pollos/genética , Pollos/crecimiento & desarrollo , Femenino , Marcadores Genéticos , Funciones de Verosimilitud , Masculino , Carne/análisis , Tamaño de los Órganos

RESUMEN

The objective of this study was to identify associations between known polymorphisms in genes related to adipose tissue and sexual precocity in Nellore cattle. A total of 1689 precocious and non-precocious heifers belonging to farms participating in Conexão Delta G breeding program were studied. SNPs from the Illumina High-Density Bovine SNP BeadChip were used. This chip contains 777,000 SNPs located within the region of the candidate genes at a distance of up to 5 kb, considering that linkage disequilibrium (LD) exists at this distance. Linear models were used for statistical analysis. The fastPHASE and GenomeStudio programs were used for haplotype reconstruction and LD analysis based on r2 statistics. Fifty-seven candidate genes and 443 SNPs were analyzed: among the latter, 370 SNPs formed 83 haplotypes, while the remaining SNPs were studied separately. Statistical analysis showed that only three haplotypes, one haplotype consisting of two SNPs located in the FABP4 gene and two haplotypes consisting of four and two SNPs located in the PPP3CA gene, had a significant effect on sexual precocity at P < 0.05. It can be concluded that the FABP4 and PPP3CA genes influence sexual precocity and may therefore be used in selection programs designed to improve sexual precocity in Nellore cattle.

Asunto(s)

Bovinos/genética , Estudios de Asociación Genética , Metabolismo de los Lípidos/genética , Pubertad Precoz/genética , Animales , Cromosomas de los Mamíferos/genética , ADN/aislamiento & purificación , Femenino , Frecuencia de los Genes , Técnicas de Genotipaje , Haplotipos , Masculino , Polimorfismo de Nucleótido Simple

RESUMEN

The diacylglycerol-O-transferase 1 gene is a positional and functional candidate for milk composition traits. The objective of this study was to evaluate the segregation of the variable number of tandem repeat polymorphisms in the regulatory region of diacylglycerol-O-transferase 1 gene in a water buffalo herd, and to assess the association of this mutation with milk production traits. For this purpose, 196 Murrah buffalo cows were genotyped by PCR. The association of the marker with total milk, fat, and protein yields at 305 d of lactation, milk fat and protein percentage, and somatic cell scores were evaluated by single-trait analyses using a generalized mixed model. Two segregating alleles were identified in the population. The allele with 2 repeats affected fat percentage favorably. The present results suggest that this polymorphism is an interesting marker to include in the genetic evaluation of buffaloes.

Asunto(s)

Leche/química , Repeticiones de Minisatélite , Polimorfismo Genético , Alelos , Animales , Búfalos/genética , Diacilglicerol O-Acetiltransferasa/genética , Diacilglicerol O-Acetiltransferasa/metabolismo , Grasas de la Dieta/análisis , Femenino , Genotipo , Lactancia , Proteínas de la Leche/análisis , Mutación , Fenotipo , Regiones Promotoras Genéticas

RESUMEN

The hormone ghrelin is produced in the stomach wall, has an orexigenic function, stimulates growth hormone secretion, and affects the energy balance of the animal. Therefore, the ghrelin gene (GHRL) is considered to be a good candidate marker for the identification of traits of great economic importance in cattle, such as those associated with feed intake, growth, and carcass quality. The use of molecular genetic markers associated with such traits permits the earlier and more accurate identification of superior animals, thus reducing the interval between generations, and increasing the genetic gain. Six SNPs were found in the GHRL gene, located in intron 3, intron 4, and exon 5. The positions of the SNPs on the gene and the substitutions were: g.2184A>G, g.2347T>C, g.4469T>C, g.4548A>G, g.4663T>C, and g.4729T>C (GenBank accession No. JX565585). After analysis of linkage disequilibrium, association tests were performed between four SNPs with the traits year weight for males, yearling weight for females, dry matter intake, loin eye area, and rump fat thickness (P ≤ 0.05). Therefore, GHRL is an important candidate gene that may be used to identify genetic variations that influence traits of economic importance in beef cattle.

Asunto(s)

Estudios de Asociación Genética , Ghrelina/genética , Carne , Carne Roja , Animales , Peso Corporal/genética , Bovinos , Femenino , Genotipo , Haplotipos , Desequilibrio de Ligamiento , Masculino , Fenotipo , Polimorfismo de Nucleótido Simple

RESUMEN

The aim of this study was to identify single-nucleotide polymorphisms (SNPs) in buffaloes associated with milk yield and content, in addition to somatic cell scores based on the cross-species transferability of SNPs from cattle to buffalo. A total of 15,745 SNPs were analyzed, of which 1562 showed 1% significance and 4742 with 5% significance, which were associated for all traits studied. After application of Bonferroni's correction for multiple tests of the traits analyzed, we found 2 significant SNPs placed on cattle chromosomes BTA15 and BTA20, which are homologous to buffalo chromosomes BBU16 and BBU19, respectively. In this genome association study, we found several significant SNPs affecting buffalo milk production and quality. Furthermore, the use of the high-density bovine BeadChip was suitable for genomic analysis in buffaloes. Although extensive chromosome arm homology was described between cattle and buffalo, the exact chromosomal position of SNP markers associated with these economically important traits in buffalo can be determined only through buffalo genome sequencing.

Asunto(s)

Búfalos/genética , Estudio de Asociación del Genoma Completo/veterinaria , Lactancia , Polimorfismo de Nucleótido Simple , Animales , Bovinos , Cromosomas de los Mamíferos , Femenino , Marcadores Genéticos , Genotipo , Sitios de Carácter Cuantitativo

RESUMEN

To define the best strategies for genomic association studies and genomic selection, it is necessary to determine the extent of linkage disequilibrium (LD) and the genetic structure of the study population. The current study evaluated the transference of genomic information contained in the Illumina BovineHD BeadChip from cattle to buffaloes, and assessed the extent of the LD in buffaloes. Of the 688,593 bovine single nucleotide polymorphism (SNP) that were successfully genotyped from the 384 buffalo samples, only 16,580 markers were polymorphic, and had minor allele frequencies greater than 0.05. A total of 16,580 polymorphic SNPs were identified, which were uniformly distributed throughout the autosomes, because the density and mean distance between markers were similar for all autosomes. The average minor allele frequency for the 16,580 SNPs was 0.23. The overall mean LD for pairs of adjacent markers was 0.29 and 0.71, when measured as for r2 and |D'|, respectively. The 16,580 polymorphic SNPs were matched to Bos taurus chromosome in the current bovine genome assembly (Btau 4.2), and could be utilized in association studies. In conclusion, the Illumina BovineHD BeadChip contains approximately 16,580 polymorphic markers for the water buffalo, which are broadly distributed across the genome. These data could be used in genomic association and genomic selection studies; however, it might be necessary to develop a panel with specific SNP markers for water buffaloes.

Asunto(s)

Búfalos/genética , Frecuencia de los Genes , Genoma , Genómica/métodos , Polimorfismo de Nucleótido Simple , Animales , Bovinos , Cromosomas de los Mamíferos , Estudios de Asociación Genética , Desequilibrio de Ligamiento

RESUMEN

The osteopontin gene may influence the fertility of water buffaloes because it is a protein present in sperm. The aim of this work was to identify polymorphisms in this gene and associate them with fertility parameters of animals kept under extensive grazing. A total of 306 male buffaloes older than 18 months, from two farms, one in the state of Amapá and the other in the state of Pará, Brazil were used in the study. Seven SNPs were identified in the regions studied. The polymorphisms were in gene positions 1478, 1513 and 1611 in the region 5'upstrem and positions 6690, 6737, 6925 and 6952 in the region amplified in intron 5. The SNPs were associated with the traits, namely scrotal circumference, scrotal volume, sperm motility, sperm concentration and sperm pathology. There were significant SNPs (p < 0.05) for all the traits. The SNP 6690 was significant for scrotal circumference, sperm concentration, sperm motility and sperm pathology and the SNP 6737 for scrotal volume. The genotype AA of SNP 6690 presented the highest averages for scrotal circumference, sperm concentration and motility and the lowest total number of sperm pathologies. For the scrotal volume trait, the animals with the largest volume were correlated with the presence of the genotype GG of SNP 6737. These results indicate a significance of the osteopontin gene as it seems to exert a substantial influence on the semen production traits of male buffaloes.

Asunto(s)

Búfalos/fisiología , Osteopontina/metabolismo , Polimorfismo de Nucleótido Simple , Semen/fisiología , Alelos , Animales , Brasil , Búfalos/genética , Regulación de la Expresión Génica/fisiología , Genotipo , Desequilibrio de Ligamiento , Masculino , Osteopontina/genética

RESUMEN

The composition of the crotalic venom and the immunochemistry and/or pathophysiological characterization and main components were well studied. However, few studies have been carried out to investigate the effect of toxins of this venom on the development of the immune response. The objective of this work was to find out if venom or crotoxin of Crotalus durissus terrificus was able to modulate the immune response through its ability to change the mediators involved in the immune response by an unrelated antigen. We observed in the murine model, that venom as well as crotoxin have inhibitory effect on splenic cells proliferation induced by Con-A. Moreover, CB did not inhibit the proliferative response, suggesting that the integrity of crotoxin complex is necessary for the development of this phenomenon. Moreover, we showed that the effect on cellular proliferation was unrelated to cytotoxicity activity. We also observed that venom or crotoxin inhibited cytokine release induced in HSA immunised mice, mainly IL-2, IL-4 and IL-10, however, crotoxin did not inhibit the release of IFN-gamma. The involvement of T or B cells in the suppressive effect of venom was evaluated through the transference of purified splenic cells from venom-mice to normal mice that also produced low IgG1 anti-HSA levels, indicating the participation of these cells in this process. Mechanism of action of the crotalic venom on development of immune response to an unrelated antigen is much more complex, therefore it must not only involve the interaction of distinct cellular populations, but activation or inhibition of signalling proteins, need to be further investigated.

Asunto(s)

Venenos de Crotálidos/toxicidad , Crotalus , Crotoxina/toxicidad , Tolerancia Inmunológica/efectos de los fármacos , Inmunidad Celular/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Interferón gamma/metabolismo , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Receptores de Concanavalina A/metabolismo , Albúmina Sérica/inmunología , Factores de Tiempo

RESUMEN

In Brazil, the Crotalus durissus terrificus subspecie is the most studied, particularly concerning its crotoxin. Crotoxin is the major toxic component of the South American rattlesnake Crotalus durissus venom. It is composed of two different subunits, CA called crotapotin and CB weakly toxic phospholipase A2 with high enzymatic activity. In this paper, we decided to make a study of the main toxic characteristics of crotoxin (CTX) and CB fraction from the other subspecies, Crotalus durissus cascavella and of Crotalus durissus collilineatus, in comparison with those of C. d. terrificus. Ours results have shown that the venoms presented similar chromatographic profiles and the purified fractions were free of contaminants. Regarding the toxic activities, the DL50 of the crotoxins showed no significant differences between the subspecies. The smaller toxicity of CB indicated that the toxicity of the crotoxin complex depends on the interaction between CA and CB. CTX and fraction CB of the three species of Crotalus showed negligible proteolytic activity. C. d. terrificus CTX presented higher PLA2 activity when compared with the others two subspecies. The oedema induced by CB developed later than the CTX and reached its peak 3 h after the injection. The myotoxic activity was determined by assaying serum CK levels. Mice injected with CTX of C. d. terrificus presented greater myotoxic activity compared to the others. The myotoxic activity of CB from the three subspecies was lower than the activity of the crotoxin, reinforcing the idea that the fraction CA increases the toxicity of CB.

Asunto(s)

Crotalus , Crotoxina/toxicidad , Fosfolipasas A/toxicidad , Análisis de Varianza , Animales , Brasil , Caseínas/metabolismo , Cromatografía por Intercambio Iónico , Creatina Quinasa/sangre , Crotoxina/metabolismo , Edema/inducido químicamente , Electroforesis en Gel de Poliacrilamida , Dosificación Letal Mediana , Ratones , Ratones Endogámicos BALB C , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Factores de Tiempo , Pruebas de Toxicidad Aguda

RESUMEN

BACKGROUND: Crotoxin (CTX) is a potent neurotoxin from Crotalus durissus terrificus snake venom (CdtV) composed of two subunits: one without catalytic activity (crotapotin), and a basic phospolipase A2. Recent data have demonstrated that CdtV or CTX inhibit some immune and inflammatory reactions. AIM: The aim of this paper was to investigate the mechanisms involved in these impaired responses. MATERIALS AND METHODS: Male Swiss mice were bled before and at different intervals of time after subcutaneous injection of CTX or bovine serum albumin (BSA) (control animals). The effect of treatments on circulating leukocyte mobilisation and on serum levels of interleukin (IL)-6, IL-10, interferon (IFN)-gamma and corticosterone were investigated. Spleen cells from treated animals were also stimulated in vitro with concanavalin A to evaluate the profile of IL-4, IL-6, IL-10 or IFN-gamma secretion. Cytokine levels were determined by immunoenzymatic assay and corticosterone levels by radioimmunoassay. To investigate the participation of endogenous corticosteroid on the effects evoked by CTX, animals were treated with metyrapone, an inhibitor of glucocorticoid synthesis, previous to CTX treatment. RESULTS: Marked alterations on peripheral leukocyte distribution, characterised by a drop in the number of lymphocytes and monocytes and an increase in the number of neutrophils, were observed after CTX injection. No such alteration was observed in BSA-treated animals. Increased levels of IL-6, IL-10 and corticosterone were also detected in CTX-injected animals. IFN-gamma levels were not modified after treatments. In contrast, spleen cells obtained from CTX-treated animals and stimulated with concanavalin A secreted less IL-10 and IL-4 in comparison with cells obtained from control animals. Metyrapone pretreatment was effective only to reverse the neutrophilia observed after CTX administration. CONCLUSIONS: Our results suggest that CTX may contribute to the deficient inflammatory and immune responses induced by crude CdtV. CTX induces endogenous mechanisms that are responsible, at least in part, for these impaired responses.

Asunto(s)

Crotoxina/inmunología , Neurotoxinas/inmunología , Fosfolipasas A/inmunología , Animales , Crotalus , Crotoxina/administración & dosificación , Crotoxina/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Glucocorticoides/sangre , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Interleucina-10/sangre , Interleucina-4/biosíntesis , Interleucina-6/biosíntesis , Interleucina-6/sangre , Recuento de Leucocitos , Masculino , Metirapona/farmacología , Ratones , Ratones Endogámicos BALB C , Neurotoxinas/administración & dosificación , Neurotoxinas/antagonistas & inhibidores , Fosfolipasas A/administración & dosificación , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A2 , Bazo/efectos de los fármacos , Bazo/inmunología

RESUMEN

Bothropstoxin-I (BthTX-I), the principal myotoxin of Bothrops jararacussu venom, is devoid of phospholipase A(2) (PLA(2)) activity but capable of blocking neuromuscular transmission in mouse nerve-muscle preparations. In this study, the ability of crotoxin antiserum and heparin in preventing the neurotoxic and myotoxic effects of BthTX-I was investigated. Phrenic nerve-diaphragm preparations (PND) stimulated indirectly with supramaximal stimuli (0.2 ms, 0.1 Hz) were incubated with BthTX-I (20 microg/ml) alone or with BthTX-I preincubated with antiserum or heparin for 30 min at 37 degrees C prior to testing. Control preparations were incubated with Tyrode solution, antiserum or heparin alone. BthTX-I (20 microg/ml) produced 50% neuromuscular blockade in the PND preparations in 31+/-4min, with complete blockade occurring in 120 min. The antiserum and heparin significantly prevented the neuromuscular blockade caused by BthTX-I (84 +/- 4% and 100% protection, respectively). Light microscopy examination of the muscles at the end of the 120 min incubation showed that BthTX-I damaged 48 +/- 6% of the fibers. Preincubating the toxin with antivenom significantly reduced the extent of this damage (only 15 +/- 4% of fibers affected, corresponding to 69% protection, P<0.01) whereas heparin offered no protection (34 +/- 7% of fibers affected, not significantly different from that seen with toxin alone). These results show that the antivenom was more effective in neutralizing the myotoxic effects of BthTX-I than was heparin.

Asunto(s)

Antivenenos/farmacología , Venenos de Crotálidos/antagonistas & inhibidores , Crotoxina/antagonistas & inhibidores , Inmunoglobulina G/análisis , Músculo Esquelético/efectos de los fármacos , Nervio Frénico/efectos de los fármacos , Animales , Antivenenos/inmunología , Antivenenos/uso terapéutico , Bothrops , Venenos de Crotálidos/inmunología , Venenos de Crotálidos/toxicidad , Crotoxina/inmunología , Crotoxina/toxicidad , Estimulación Eléctrica , Electroforesis en Gel de Poliacrilamida , Heparina/uso terapéutico , Inmunoglobulina G/sangre , Inmunoglobulina G/aislamiento & purificación , Técnicas In Vitro , Inyecciones Subcutáneas , Masculino , Ratones , Músculo Esquelético/patología , Músculo Esquelético/fisiología , Bloqueo Neuromuscular , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/patología , Unión Neuromuscular/fisiología , Pruebas de Neutralización , Nervio Frénico/fisiología , Conejos , Factores de Tiempo

RESUMEN

Lachesis muta muta and Bothrops atrox snakes are responsible for most accidents occurring in the Amazon. The clinical features of the accidents are similar; however, there are still controversies about the efficacy of Bothrops antivenoms for treating L. m. muta accidents. In this work, we evaluated the antigenic cross-reactivity between these venoms using polyclonal and monoclonal antibodies and the efficacy of B. atrox and L. m. muta experimental antivenoms in cross-neutralizing the main toxic activities of each venom. Electrophoretic patterns differed consistently between the species. However, antigenic cross-reactivity was extensive except for a few bands. Several species-specific monoclonal antibodies were obtained by immunization of Balb/c mice with L. m. muta whole venom or B. atrox and L. m. muta specific antigens. The monoclonal antibodies specific to L. m. muta recognized different bands of this venom and the antibodies specific to B. atrox recognized a complex pattern on whole venom by Western blotting. These antibodies are important tools for developing an immunoassay able to discriminate patients bitten by these snakes. The experiments involving cross-neutralization of the main activities of the venoms showed that hemorrhage and blood incoagulability induced by B. atrox venom were similarly neutralized by both B. atrox and L. m. muta antivenoms. However, B. atrox antivenom partially neutralized the hemorrhage and completely failed in neutralizing coagulopathy induced by L. m. muta venom. Therefore, antigenic variation between B. atrox and L. m. muta venoms does occur and the use of specific antivenom is suggested for patients bitten by Lachesis snakes.

Asunto(s)

Anticuerpos Monoclonales/aislamiento & purificación , Antivenenos/farmacología , Bothrops , Venenos de Crotálidos/antagonistas & inhibidores , Venenos de Crotálidos/toxicidad , Animales , Antivenenos/inmunología , Coagulación Sanguínea/efectos de los fármacos , Reacciones Cruzadas , Venenos de Crotálidos/inmunología , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos BALB C , Conejos , Especificidad de la Especie

RESUMEN

The recent development of acellular pertussis vaccines has been a significant improvement in the conventional whole-cell diphtheria-pertussis-tetanus toxoid vaccines, but high production costs will limit its widespread use in developing countries. Since Mycobacterium bovis BCG vaccination against tuberculosis is used in most developing countries, a recombinant BCG-pertussis vaccine could be a more viable alternative. We have constructed recombinant BCG (rBCG) strains expressing the genetically detoxified S1 subunit of pertussis toxin 9K/129G (S1PT) in fusion with either the beta-lactamase signal sequence or the whole beta-lactamase protein, under control of the upregulated M. fortuitum beta-lactamase promoter, pBlaF*. Expression levels were higher in the fusion with the whole beta-lactamase protein, and both were localized to the mycobacterial cell wall. The expression vectors were relatively stable in vivo, since at two months 85% of the BCG recovered from the spleens of vaccinated mice maintained kanamycin resistance. Spleen cells from rBCG-S1PT-vaccinated mice showed elevated gamma interferon (IFN-gamma) and low interleukin-4 (IL-4) production, as well as increased proliferation, upon pertussis toxin (PT) stimulation, characterizing a strong antigen-specific Th1-dominant cellular response. The rBCG-S1PT strains induced a low humoral response against PT after 2 months. Mice immunized with rBCG-S1PT strains displayed high-level protection against an intracerebral challenge with live Bordetella pertussis, which correlated with the induction of a PT-specific cellular immune response, reinforcing the importance of cell-mediated immunity in the protection against B. pertussis infection. Our results suggest that rBCG-expressing pertussis antigens could constitute an effective, low-cost combined vaccine against tuberculosis and pertussis.

Asunto(s)

Mycobacterium bovis/genética , Toxina del Pertussis , Vacuna contra la Tos Ferina/inmunología , Vacunas Sintéticas/inmunología , Factores de Virulencia de Bordetella/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Encéfalo/microbiología , Interferón gamma/biosíntesis , Interleucina-4/biosíntesis , Masculino , Ratones , Ratones Endogámicos BALB C , Mycobacterium bovis/inmunología , Tos Ferina/prevención & control

RESUMEN

The subspecies of the South American rattlesnake, Crotalus durissus are classified according to their external morphological features and geographical distribution. We have determined some biological activities of C. durissus cascavella, C. durissus collilineatus and C. durissus terrificus venoms. C. durissus terrificus had a significantly higher clotting activity on bovine plasma and fibrinogen, human fibrinogen and rabbit plasma. C. durissus cascavella presented a statistically higher phospholipase A2 (PLA2) activity in regard to C. durissus collilineatus. Their myotoxic and proteolytic activity, median lethal doses, or median platelet aggregating doses (on rabbit and human platelets) could not differentiate the three subspecies examined. However, the electrophoretic profile and the dose-response curve for edematogenic activity for C.d. cascavella venom were different from the others. With regard to the inorganic element content of the venoms, higher levels of Br, Cl and Mg, and a lower level of Zn, were found in C.d. cascavella venom. Crotamine-like activity could not be detected in C.d. cascavella venom. Furthermore, equine antivenom specific for C. durissus terrificus venom cross-reacted equally with the antigens of the three venom pools by ELISA and Western blotting. These results indicate that the venoms from the three studied subspecies of C. durissus were very similar, except for minor differences in paw edema-inducing activity, electrophoretic profile, phospholipase A2 activity, crotamine-like activity and inorganic element contents of C.d. cascavella venom.

Asunto(s)

Venenos de Crotálidos/farmacología , Crotalus , Animales , Antivenenos/inmunología , Coagulación Sanguínea , Bromo/análisis , Bovinos , Cloro/análisis , Venenos de Crotálidos/análisis , Venenos de Crotálidos/metabolismo , Edema/inducido químicamente , Factor X/metabolismo , Fibrinógeno/metabolismo , Humanos , Magnesio/análisis , Enfermedades Musculares/inducido químicamente , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Agregación Plaquetaria , Protrombina/metabolismo , Conejos , América del Sur , Especificidad de la Especie

RESUMEN

Envenomation by Thalassophryne nattereri fishes are an important medical problem in northeast of Brazil, causing in human victims considerable pain and edema followed by necrosis. Venom obtained from fresh captured specimens of this fish was tested in vitro or in animal models for a better characterization of its toxic activities. Intradermal injection of the venom in the foot pad of mice induced local edema and hemorrhage followed a few hours later by necrosis. Subcutaneous injection of the venom induced systemic effects consisting in jerking motions, paralysis of hind limbs, erection of hair, rotational movements and violent convulsions followed by death. Dead animals showed hyperemia of the small intestine and lungs. The venom showed distinct edematous, necrotizing and hemolytic activities, a low level of hemorrhagic, myotoxic and proteolytic activities and no detectable phospholipase A2 activity. SDS-PAGE analysis of the crude venom showed at least 17 components with the major band located around Mw = 19,000. Almost all proteins stained by amido black were also revealed by Western blotting with antibodies to T. nattereri venom. Fractionation of the venom by either gel filtration or cation exchange chromatography resulted in a few distinct peaks but in both situations the biological activities were located in only one of the peaks which corresponded to basic proteins with approximately Mw = 47,000. Heating of the venom at 56 degrees C for 60 min completely destroyed its biological activities. All venom toxic activities except edema were completely neutralized after in vitro incubation with anti-T. nattereri serum.

Asunto(s)

Venenos de los Peces/aislamiento & purificación , Venenos de los Peces/toxicidad , Animales , Antivenenos/farmacología , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Venenos de los Peces/antagonistas & inhibidores , Venenos de los Peces/química , Peces Venenosos , Ratones , Pruebas de Neutralización

RESUMEN

The effect of venom of South American rattlesnake Crotalus durissus terrificus (cdt) on the humoral and cellular immune response was studied in BALB/c mice that were immunized with soluble antigens [human serum albumin (HSA) or chicken ovoalbumin (OVA)] or sensitized to DNFB 1 hr after venom injection. Pretreatment of the animals with cdt venom induced a significant reduction in the level of anti-OVA and anti-HSA IgG antibodies. The effect of crotoxin, a major neurotoxic component of cdt venom, its acidic non-toxic subunit (CA) and its basic phospholipase A2 subunit (CB) was also studied. The whole crotoxin molecule was as able as cdt venom to induce a significant decrease in the level of anti-OVA and anti-HSA IgG antibodies. However, the CA and CB subunits of crotoxin did not change the antibody level to either antigen, suggesting that the suppressive effect of crotoxin requires the intact molecule. Both cdt venom and the whole crotoxin molecule were able to induce a significant decrease in the level of anti-HSA IgG1 antibodies. The levels of other IgG isotypes and IgE were barely detectable and could not be estimated. In spite of their suppressive effect on the humoral immune response neither cdt venom nor crotoxin had any effect on the cellular immune response as estimated by contact sensitivity reaction to DNFB. It is suggested that cdt venom and its crotoxin component have an inhibitory effect on the humoral but not on the cellular immune response.

Asunto(s)

Formación de Anticuerpos/efectos de los fármacos , Venenos de Crotálidos/toxicidad , Inmunidad Celular/efectos de los fármacos , Mordeduras de Serpientes/inmunología , Animales , Anticuerpos/sangre , Anticuerpos/inmunología , Formación de Anticuerpos/inmunología , Antígenos/inmunología , Dinitrofluorobenceno/inmunología , Inmunidad Celular/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Albúmina Sérica/inmunología

RESUMEN

The biological activities of the venom of three species of spiders of the genus Loxosceles were studied (L. gaucho, L. laeta and L. intermedia). The dermonecrotic and lethal activities are shared by all three Loxosceles venoms. Only low levels of proteolytic, myotoxic and phospholipase A2 activities were demonstrable even when a large amount of venom was used. No direct hemolytic activity was detected. L. intermedia venom was the most lethal (LD50 0.48 mg/kg), the L. laeta venom was the least lethal (LD50 1.45 mg/kg) whereas L. gaucho venom showed an intermediate value (LD50 0.74 mg/kg). The anti-Loxosceles serum used (anti-arachnidic serum) was able to neutralize the most important activities (i.e., dermonecrotic and lethal activities) of the three venoms. SDS-PAGE and immunoblotting using the anti-arachnidic serum showed that almost all venom antigens were recognized by this antiserum. The possible mechanisms of action of the Loxosceles venom are discussed.

Asunto(s)

Venenos de Araña/toxicidad , Arañas , Animales , Sueros Inmunes/inmunología , Dosificación Letal Mediana , Ratones , Pruebas de Neutralización , Conejos , Venenos de Araña/análisis , Venenos de Araña/inmunología