ABSTRACT
Abstract Crotalid envenomation is a neglected collective health problem involving many countries in America, which need secure and inexpensive snake anti-venom treatments. Here, high antibody titers (IgY) were raised in the Ostrich (Struthio camelus) egg yolk by immunizing with the venom of Venezuelan venomous Crotalus snakes. Ostriches were immunized with a pool of venoms from common rattlesnake (Crotalus durissus cumanensis), Uracoan rattlesnake (Crotalus vegrandis), Guayana rattlesnake (Crotalus durissus ruruima) and black rattlesnake (Crotalus pifanorum). The anti-snake venom antibodies were prepared from egg yolk by the water dilution method, enriched by the addition of caprylic acid (CA) and precipitation with ammonium sulfate at 30% (W/V). The purity and molecular mass of the final product was satisfactory, yielding a single ∼ 175 kDa band in SDS-PAGE gels ran under non-reducing conditions. In the immunoblot analysis, specific binding of the antivenom was observed with most venom proteins. The LD50 was 16.5 g/mouse (825 μg/kg body weight). High titers of IgY against Crot/pool venom were shown by ELISA. The median effective dose (ED50) was 19.66 mg/2LD50. IgY antibodies neutralized efficiently the Crot/pool venom lethality. As far as we know, this is the first anti-snake venom produced in ostriches, which could make this technology an affordable alternative for low-income countries, since it is likely to produce manteniabout 2-4 g of IgY per ostrich egg. Hence, almost 400 g of IgY can be purified from only one ostrich during a year. In addition, there are enormous differences in the cost of investment in the maintenance of horses, from the points of view of infrastructure, feeding and veterinary care, in which the cost can reach USD 100 per animal per day, compared to a maintenance cost of USD 146 per month per producing bird. These results are encouraging and could easily be extrapolated to the manufacturing of other antivenoms and antitoxins as well, as they could be applied to the manufacturing of potential diagnostic tools.
Resumen El envenenamiento por crotálidos es un problema de salud colectiva desatendido, que involucra a muchos países del continente americano, los cuales necesitan tratamientos seguros y económicos. En este trabajo, se obtuvieron títulos altos de anticuerpos (IgY) producidos en yema de huevo de avestruz (Struthio camelus) mediante la inmunización con el veneno de serpientes venezolanas del genero Crotalus. Se inmunizaron avestruces con una colección de veneno de serpientes de cascabel común (Crotalus durissus cumanensis), cascabel de Uracoa (Crotalus vegrandis), cascabel de Guayana (Crotalus durissus ruruima) y cascabel negra (Crotalus pifanorum). Los anticuerpos anti-veneno de serpiente se prepararon a partir de yema de huevo por el método de dilución en agua, enriquecidos mediante la adición de ácido caprílico (CA), seguido de una precipitación con sulfato de amonio al 30% (P/V). La pureza y masa molecular de los anticuerpos (IgY) se definieron mediante ensayos de SDS-PAGE nativos y las masas moleculares se establecieron electroforéticamente, obteniéndose una única banda de IgY de ∼ 175 kDa. El análisis de inmunotransferencia mostró la unión específica del antiveneno con la mayoría de las proteínas del veneno. La DL50 fue de 16,5 μg/ratón (825 μg / kg de peso corporal); Se mostraron títulos altos de IgY contra el veneno de Crot / pool mediante ELISA. La dosis mediana efectiva (DE50) fue de 19,66 mg/2 LD50. Los anticuerpos IgY neutralizaron eficazmente la letalidad del veneno de Crot / pool. Hasta donde sabemos, se trata del primer antídoto de serpiente producido en avestruces, lo que podría abaratar la producción de este tratamiento en países del tercer mundo. Ya que es probable que se obtengan alrededor de 2-4 g de IgY por huevo de avestruz. Por lo tanto, se podrían purificar casi 400 g de IgY de un solo avestruz durante un año. Asimismo, debido a las enormes diferencias en el costo de inversión en el mantenimiento de los caballos desde el punto de vista de infraestructura, alimentación y atención veterinaria, en los que el costo puede llegar a los 100 USD por día, frente a los 146 USD por mes de mantenimiento de la producción de aves. Estos resultados abren un campo terapéutico, para la fabricación de otros antivenenos contra un amplio espectro de toxinas y también como probables herramientas de diagnóstico.
ABSTRACT
Objective: To identify unique immunogenic epitopes of Zika virus non-structural 1 (NS1) antigen and produce immunoglobulin Y (IgY) for potential use in he diagnosis of of Zika virus infection. Methods: Immunogenic epitopes were identified using in silico B-cell epitope prediction. A synthetic peptide analog of the predicted epitope was used to induce antipeptide IgY production in hens which was purified using affinity chromatography. Presence of purified IgY and its binding specificity were performed by gel electrophoresis and ELISA, respectively. Results: Out of the nine continuous epitopes identified, the sequence at position 193-208 (LKVREDYSLECDPAVI) was selected and used to produce anti-peptide IgY. The produced IgY was found to bind to the synthetic analog of the Zika virus NS1 immunogenic epitope but not to other flaviviruses and random peptides from other pathogens. Conclusions: In this study, we identified an immunogenic epitope unique to Zika virus that can be used to develop a serodiagnostic tool that specifically detect Zika virus infection.
ABSTRACT
BACKGROUND Malaria is a disease that affects many tropical and subtropical countries, including Brazil. The use of tests for malaria detection is one of the fundamental strategies recommended by the World Health Organization for the control and eradication of the disease. The lack of diagnostic tests leads to an increase in transmission and non-reporting cases. OBJECTIVES This work described an electrochemical immunosensor for detecting Plasmodium vivax lactate dehydrogenase antigen (Ag-PvLDH). METHODS The device has developed by immobilising egg yolk IgY antibodies (Ab-PvLDH) on a gold electrode surface using cysteamine as linker. The immunosensor fabrication was followed by differential pulse voltammetry, and contact angle measurements were performed to characterise the modified gold electrode surface. FINDINGS The results for Ag-PvLDH determination exhibit a linear response at 10-50 µg mL-1 concentration range, with a limit of detection of 455 ng mL-1. The excellent selectivity of the device was confirmed. MAIN CONCLUSIONS The developed immunosensor showed a good performance, therefore, it can be considered an alternative test to detect malaria caused by P. vivax.
ABSTRACT
Resumen Los sistemas de defensa anti-oxidante utilizados por el parásito intracelular Leishmania braziliensis durante el proceso de infección permiten eliminar especies reactivas de oxígeno y nitrógeno a expensas de equivalentes reductores derivados de la tripanotiona, evitando daños celulares del patógeno. Con el objetivo de identificar potenciales blancos moleculares para el desarrollo de fármacos contra este parásito, se realizó la detección de la enzima triparedoxina peroxidasa citoplasmática de L. braziliensis (LbTXNPxII), la cual es esencial para disminuir concentraciones tóxicas de peróxido de hidrógeno en el contexto de infección. Para esto se generaron anticuerpos policlonales en modelo aviar, partiendo de la clonación, expresión y purificación de la proteína recombinante 6xHis-SUMO-LbTXNPxII (37kDa) en el sistema heterólogo Escherichia coli. La proteína purificada se utilizó como antígeno para la producción de anticuerpos IgY, cuya implementación en estudios in situ permitió detectar y localizar la enzima LbTXNPxII endógena (22kDa) en el citoplasma de promastigotes fijados y verificar su interacción molecular con la nicotinamida/ nicotinato mononucleótido adenilil transferasa, enzima involucrada en la síntesis del NAD. De este modo, se reporta el desarrollo de una herramienta bioquímica para la identificación y estudio de la enzima LbTXNPxII y su participación en vías del metabolismo energético y de defensa anti-oxidante.
Abstract The antioxidant defense systems used by the intracellular parasite Leishmania braziliensis during the infection process make it possible to eliminate reactive oxygen and nitrogen species at the expense of reducing equivalents derived from trypanothione, avoiding cellular damage of the pathogen. In order to identify potential molecular targets for the development of drugs against this parasite, the cytoplasmic tryparedoxin peroxidase of L. braziliensis (LbTXNPxII), which is essential to reduce toxic concentrations of hydrogen peroxide in the context of infection, was carried out. In this regard, polyclonal antibodies were generated in an avian model, starting from the cloning, expression, and purification of the recombinant protein 6xHis-SUMO-LbTXNPxII (37kDa) in the heterologous system of Escherichia coli. The purified protein was used as an antigen for the production of IgY antibodies, whose implementation in in situ experiments allowed the detection and localization of the endogenous LbTXNPxII enzyme (22kDa) in the cytoplasm of fixed promastigotes, as well as the verification of its molecular interaction with nicotinamide/nicotinate mononucleotide adenylyltransferase, an enzyme involved in the synthesis of NAD. Thus, the development of a biochemical tool for the identification and study of the LbTXNPxII enzyme and its participation in energy metabolism and antioxidant defense pathways is reported.
Resumo Os sistemas de defesa antioxidante utilizados pelo parasita intracelular Leishmania braziliensis durante o processo de infecção, permitem a eliminação de espécies reativas de oxigênio e nitrogênio em detrimento de equivalentes redutores derivados de tripanotiona, evitando o dano celular do patógeno. Com o objetivo de identificar potenciais alvos moleculares para o desenvolvimento de drogas contra esse parasita, foi detectada a enzima citoplasmática triparedoxina peroxidase de L. braziliensis (LbTXNPxII), essencial para reduzir as concentrações tóxicas de peróxido de hidrogênio no contexto de infecção. Para isso, anticorpos policlonais foram gerados em modelo aviário, a partir da clonagem, expressão e purificação da proteína recombinante 6xHis-SUMO-LbTXNPxII (37kDa) no sistema heterólogo de Escherichia coli. A proteína purificada foi utilizada como antígeno para a produção de anticorpos IgY, cuja implementação em experimentos in situ permitiu a detecção e localização da enzima LbTXNPxII endógena (22kDa) no citoplasma de promastigotas fixos e verificar sua interação molecular com nicotinamida/nicotinato mononucleotídeo adenililtransferase, enzima envolvida na síntese de NAD. Assim, é relatado o desenvolvimento de uma ferramenta bioquímica para a identificação e estudo da enzima LbTXNPxII e sua participação no metabolismo energético e nas vias de defesa antioxidante.
ABSTRACT
Early childhood caries (ECC) is defined as a condition of one or more of decayed, missing, and filling in teeth (dmft) of children aging less than 71 months or even younger. Prevalence and severity of ECC is still high in some countries. Aim of this study to evaluate the effect of streptococcal IgY on the quantity of Streptococcus mutansin high caries risk children. 20 children aged 3-5 years were selected from the Department of Pedodontics of which 10 children were with high caries experience and 10 were controls who were caries free. Saliva swabs were taken from the occlusal surface of primary second molars and microbial count was counted using mitis salivarius bactaracinagar. High risk caries were given one No decayTMtablet morning and evening for 15 days. Swabs were taken before intervention and than 1 month,2 months and 3 months post intervention. Streptococcus mutanscount was significantly reduced in patients who have taken no decay tablets. Within the limits of the study, we found that streptococcal IgY was able to reduced streptococcus mutanscount after 2 months.There was no statistically significant difference in Streptococcus mutanscount between high caries risk children and caries free children after 3 months
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BACKGROUND: Hot start can greatly improve specificity, sensitivity and yield of PCR. Non-specific amplification can occur in PCR when reaction mixture is prepared at room temperature, because Taq DNA polymerase is active and the primers can hybridize non-specifically. Hot start Taq DNA polymerases remain inactive at room temperature and are activated after heating at 95°C preventing non-specific amplification. Monoclonal antibodies against Taq DNA polymerase is the first line of reagents used for turn on regular Taq DNA polymerase into Hot start one. The goal of this research was to produce and evaluate Hot Start antibodies derived from chicken eggs. RESULTS: We performed affinity purification of yolk immunoglobulin (IgY) and obtained polyclonal Hot Start antibodies. The yield of specific antibodies was 0.36 mg per egg or 0.2% of total yolk antibodies. The protocol for real time measurement and Hot start IgY activity assessment was developed. We found that Hot start IgY can reversibly block Taq DNA polymerase activity at 50°C and have no negative impact neither on the Taq DNA polymerase activity after denaturation nor on the reverse transcriptase. We estimated that 1.0 µg of Hot start IgY effectively blocks 5 U activity of Taq DNA polymerase. CONCLUSIONS: Egg derived Hot Start polyclonal antibodies are the cheapest source of Hot start antibodies, from one immune egg we can isolate 0.36 mg IgY, this quantity is enough for producing 1800 U activity of Hot start Taq DNA Polymerase.
Subject(s)
Egg Yolk/metabolism , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Temperature , Immunoglobulins/isolation & purification , Immunoglobulins/biosynthesis , Immunoglobulins/immunology , Enzyme-Linked Immunosorbent Assay , Polymerase Chain Reaction , Taq Polymerase , Egg Yolk/immunology , Antibodies, Monoclonal/isolation & purificationABSTRACT
The venom of bamboo vipers (Trimeresurus stejnegeri - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived antivenom is a specific treatment against snakebites, but its production costs are high and there are some inevitable side effects. The aim of the present work is to help in the development of an affordable and more endurable therapeutic strategy for snakebites. Methods: T. stejnegeri venom proteins were inactivated by glutaraldehyde in order to immunize hens for polyclonal immunoglobulin (IgY) antibodies production. After IgY binding assays, two antibody libraries were constructed expressing single-chain variable fragment (scFv) antibodies joined by the short or long linker for use in phage display antibody technology. Four rounds of biopanning were carried out. The selected scFv antibodies were then further tested for their binding activities and neutralization assays to TS proteins. Results: Purified IgY from egg yolk showed the specific binding ability to TS proteins. The dimensions of these two libraries contain 2.4 × 107 and 6.8 × 107 antibody clones, respectively. An increase in the titers of eluted phage indicated anti-TS clones remarkably enriched after 2nd panning. The analysis based on the nucleotide sequences of selected scFv clones indicated that seven groups of short linkers and four groups of long linkers were identified. The recombinant scFvs showed significant reactivity to TS venom proteins and a cross-reaction to Trimeresurus mucrosquamatus venom proteins. In in vivo studies, the data demonstrated that anti-TS IgY provided 100% protective effects while combined scFvs augmented partial survival time of mice injected with a lethal amount of TS proteins. Conclusion: Chickens were excellent hosts for the production of neutralization antibodies at low cost. Phage display technology is available for generation of monoclonal antibodies against snake venom proteins. These antibodies could be applied in the development of diagnostic kits or as an alternative for snakebite envenomation treatment in the near future.(AU)
Subject(s)
Animals , Snake Venoms , Antivenins , Chickens , Trimeresurus , Antibodies , BacteriophagesABSTRACT
Immunoglobulin Y (IgY) is an effective orally administered antibody used to protect against various intestinal pathogens, but which cannot tolerate the acidic gastric environment. In this study, IgY was microencapsulated by alginate (ALG) and coated with chitooligosaccharide (COS). A response surface methodology was used to optimize the formulation, and a simulated gastrointestinal (GI) digestion (SGID) system to evaluate the controlled release of microencapsulated IgY. The microcapsule formulation was optimized as an ALG concentration of 1.56% (15.6 g/L), COS level of 0.61% (6.1 g/L), and IgY/ALG ratio of 62.44% (mass ratio). The microcapsules prepared following this formulation had an encapsulation efficiency of 65.19%, a loading capacity of 33.75%, and an average particle size of 588.75 µm. Under this optimum formulation, the coating of COS provided a less porous and more continuous microstructure by filling the cracks on the surface, and thus the GI release rate of encapsulated IgY was significantly reduced. The release of encapsulated IgY during simulated gastric and intestinal digestion well fitted the zero-order and first-order kinetics functions, respectively. The microcapsule also allowed the IgY to retain 84.37% immune-activity after 4 h simulated GI digestion, significantly higher than that for unprotected IgY (5.33%). This approach could provide an efficient way to preserve IgY and improve its performance in the GI tract.
ABSTRACT
ABSTRACT: Advances in the fields of glycobiology and immunology have provided many insights into the role of carbohydrate-protein interactions in the immune system. Jacalin of Artocarpus integrifolia (JCA) and structural mannoprotein of Saccharomyces uvarum (MPS) are molecules with immunomodulatory properties. JCA is an IgA human lectin binding molecule that causes the mitogenic stimulation of immune cells, production of cytokines, chemotaxis, and activation of leukocytes. Studies on the immunomodulatory properties of JCA and MPS in mammals and fish suggest that they have an action on antibody production. The aim of this study was to investigate the possible action of JCA and MPS on the production of specific antibodies in laying hens. For this, laying hens were inoculated with an intra abdominal injection of sheep red blood cells (SRBC) with either JCA (0.075 µg, 0.75 µg, and 7.5 µg) or MPS (20 µg and 100 µg). Levels of anti-SRBC antibodies of the IgY, IgM, and IgA classes were evaluated by ELISA. Results showed that JCA and MPS have immunomodulatory effects on levels of anti-SRBC IgM, IgA, and IgY. An immunostimulatory effect of JCA was observed in primary immune response on anti-SRBC IgY, while an inhibitory effect of JCA and MPS was observed in secondary immune response on the production of IgM and IgA anti-SRBC. These results suggested that MPS and JCA have immunomodulatory effects on antibody production and could be used in future studies on humoral immune response in poultry.
RESUMO: Avanços nos campos glicobiologia e imunologia forneceram muitas informações sobre o papel das interações da proteína-carboidrato na modulação do sistema imunológico. A jacalina extraída de Artocarpus integrifolia (JCA) e a manoproteína da parede celular de Saccharomyces uvarum (MPS) são moléculas com propriedades imunomoduladoras. JCA é uma lectina com afinidade pela IgA humana e tem ação mitogênica sobre células do sistema imunológico estimulando a produção de citocinas, a quimiotaxia e a ativação de leucócitos. Estudos sobre as propriedades imunomoduladoras de JCA e MPS em mamíferos e peixes sugerem que essas moléculas podem ter um efeito sobre a produção de anticorpos. O objetivo deste estudo foi investigar a ação da JCA e MPS sobre a produção de anticorpos específicos em galinhas poedeiras. Para isso, galinhas poedeiras foram inoculadas por via intra-abdominal com eritrócitos de carneiro (SRBC) em associação com o JCA (0,075 µg, 0,75 µg, e 7,5 µg) ou MPS (20 µg e 100 µg). Os níveis de anticorpos anti-SRBC das classes IgY, IgM, e IgA foram avaliados por ELISA. Os resultados mostraram que a JCA e a MPS têm um efeito imunomodulador sobre a produção IgY, IgM, ou IgA anti-SRBC. Um efeito imunoestimulador da JCA foi observado sobre a produção de anticorpos IgY na resposta imune primária, enquanto um efeito imuno inibitório da JCA e da MPS sobre a produção de IgM e IgA anti-SRBC na resposta imune secundária. Estes resultados sugerem que o MPS e JCA tem efeito modulador sobre a produção de anticorpos e podem ser utilizados em estudos futuros sobre a imunidade humoral em aves comerciais.
ABSTRACT
Despite common occurrence and importance of canine distemper disease the majority of tests currently available for diagnosis are hampered by either low sensitivity or specificity. In this study it was evaluated antigenic and immunogenic characteristics of a conserved region of nucleocapsid protein of canine distemper virus (rCDV NP) expressed in Escherichia coli employing a codon optimized synthetic gene. The expression of rCDVNP in Star strain (mean 300μg/mL, purified) was confirmed by SDS-PAGE and Western blot analysis by using His-Tag monoclonal antibodies. Western blot and ELISA, employing positive and negative control dog sera, demonstrated the rCDVNP antigenicity. The rCDVNP was inoculated in hens and immunoglobulin Y (IgY) was purified from the egg yolk. The mean yield of IgY was 28.55mg/mL. IgY reacted with the recombinant protein as demonstrated by Western blot and ELISA assays. In summary, our findings demonstrated that rCDVNP is antigenic since CDV positive dog sera recognized the protein in vitro. Additionally, the rCDVNP proved to be immunogenic in hens being possible to isolate a high concentration of specific IgY antibodies from the egg yolk. Taken together, these results indicate that the rCDVNP along with the specific IgY could be useful tools for development of the canine distemper immunodiagnostic assays.(AU)
Apesar da ocorrência comum e importância da cinomose canina, a maioria dos testes atualmente disponíveis para diagnóstico são prejudicados pela baixa sensibilidade ou especificidade. Neste estudo foram avaliadas características antigênicas e imunogênicas de uma região conservada da proteína do nucleocapsídeo do virus da cinomose canina (rCDV NP) expressa em Escherichia coli empregando um gene sintético e codons otimizados. A expressão na cepa Star (média de 300μg/mL, purificada) foi confirmada por SDS-PAGE e Western blot utilizando anticorpos monoclonais anti-His-Tag. A antigenicidade da rCDVNP foi demonstrada por western blot e ELISA empregando soros de cães positivos e negativos. A rCDVNP foi inoculada em galinhas e imunoglobulina Y (gY) foi obtida e purificada a partir da gema. A produção média de IgY foi 28.55mg/mL. Anticorpos IgY reagiram com a proteína recombinante, quando analisados por Western blot e ELISA. Em resumo, nossos achados demonstram que a rCDVNP produzida é antigênica, uma vez que os anticorpos de soro de cães positivos para CDV reconheceram a proteína in vitro. Além disso, a rCDVNP foi imunogênica em galinhas, sendo possível isolar anticorpos IgY específicos a partir da gema do ovo em altas concentrações. Tomados em conjunto, estes resultados indicam que a rCDVNP juntamente com a IgY específica podem ser ferramentas úteis para elaborar ensaios de imunodiagnóstico de cinomose canina.(AU)
Subject(s)
Animals , Dogs , Distemper Virus, Canine/genetics , Distemper Virus, Canine/immunology , Dogs/microbiology , Escherichia coli/genetics , Antigen-Antibody ReactionsABSTRACT
RESUMO: Toxoplasma gondii é um protozoário apicomplexa que infecta animais de sangue quente, podendo ser considerado um dos principais parasitas capazes de infectar os seres humanos. Galinhas domésticas podem ser facilmente infectadas por protozoários, uma vez que estas podem ingerir oocistos encontrados no solo, sendo consideradas boas indicadoras de contaminação ambiental por T. gondii. O objetivo deste estudo foi determinar a presença de anticorpos anti-T. gondii em galinhas domésticas criadas extensivamente e avaliar os fatores de risco associados ao protozoário, na zona rural de Santa Maria, RS, Brasil. No período de março de 2013 a fevereiro 2014 foram coletadas 597 amostras de sangue de galinhas domésticas em 74 propriedades, oriundas de nove estratos que representam cada distrito da zona rural. Para avaliar os fatores de risco, nessas propriedades foi aplicado um questionário epidemiológico aos moradores. As amostras de soro foram testadas por imunofluorescência indireta, e 49,2% (294/597) foram positivas para anticorpos anti-T. gondii, com títulos variando de 16 a 4096. Das 74 propriedades analisadas, em 63 (85,1%) houve relatos que os gatos têm acesso ao deposito de alimentos, com associação significativa quando associado à presença de galinhas positivas (p=0,04) e o OR de 4,07. A variável "abate de animais" (aves e bovinos), em 51 (68,9%) das propriedades foi relatado o abate de bovinos e aves na propriedade, com valor de p significativo (p=0,05). A maioria das propriedades 59 (79,7%) foi relatada a presença de gatos domésticos, o que poderia estar associada com a alta soroprevalência encontrada em galinhas e a taxa de contaminação ambiental. A elevada prevalência de anticorpos encontrada neste estudo, além da alta frequência de propriedades com casos positivos, sugere uma grande contaminação ambiental nos distritos pesquisados, sendo assim um risco potencial para a saúde humana e animal.
ABSTRACT: Toxoplasma gondii is an apicomplex protozoan that infects warm-blooded animals and can be considered a major parasite capable of infecting humans. Domestic chickens can be easily infected by protozoa, since they can ingest oocysts found in the soil and are considered good indicators of environmental contamination by T. gondii. The aim of this study was to determine the presence of anti-T. gondii antibodies in free range chickens and to evaluate the risks factors associated with the protozoan in rural area of Santa Maria, RS, Brazil. From March 2013 to February 2014, 597 blood samples from domestic chickens were collected from 74 farms, from nine layers representing each district in the rural area. To evaluate the risk factors, in these farms an epidemiological questionnaire was applied to the residents. Serum samples were tested by indirect immunofluorescence and 49.2% (294/597) were positive for anti-T.gondii antibodies, with titres varying from 16 to 4096. Of the 74 analyzed farms, 63 (85.1%) reported that cats had access to food deposits, with a significant association when positive chickens were present (p = 0.04) and the OR of 4.07. The variable "slaughter of animals" (poultry and cattle) in 51 (68.9%) of the farms was reported the slaughter of cattle and birds in the farm, with significant p value (p = 0.05). Most farms 59 (79.7%) reported the presence of domestic cats, which could be associated with the high seroprevalence found in chickens and the rate of environmental contamination. The high prevalence of antibodies found in this study, in addition to the high frequency of farms with positive cases, suggests a great environmental contamination in the studied districts, thus being a potential risk to human and animal health.
Subject(s)
Animals , Chickens/microbiology , Toxoplasmosis/microbiology , Risk Factors , Fluorescent Antibody Technique, Indirect/veterinaryABSTRACT
ABSTRACT: Gallus gallus domesticus' immune system is a promising tool for generation of antibody-based immunobiologics. Immunoglobulin y (IgY) is extracted from egg yolk and has equivalent functions to mammal's igg antibody. Avian immune system can be stimulated to produce a high-quality antibody repertoire. In this review, we present an overview of avian immune system emphasizing igy and its applications as an immunobiologic.
RESUMO: O sistema imunológico deGallus gallus domesticus é uma ferramenta promissora para a geração de imunobiológico a partir de anticorpos. A imunoglobulina Y (IgY) é extraída da gema do ovo e apresenta funções equivalentes ao anticorpo IgG dos mamíferos. O sistema imune aviário pode ser estimulado para produzir um repertório de anticorpos de alta qualidade. Nesta revisão apresentamos aspectos gerais do sistema imune aviário enfatizando o IgY e suas aplicações como um imunobiológico.
ABSTRACT
Objective:Prepared the specific chicken egg yolk immunoglobulins (IgY) against the cell wall protein of Trichophyton mentagrophytes (tmCWP) and detected its biological activities,which was to establish the basis for the preventment and treatment in dermatophytes disease.Methods: In this work,tmCWP was extracted and purified by cold alkali method,and being used as immunogen to immunized healthy laying hens.The IgY was extracted from the egg yolk by polyethylene glycol method and purified by saturated ammonium sulfate method,respectively.The concentration of the extracted IgY was detected by Bradford method.The purity and molecular weight of the specific anti-tmCWP IgY were analysed by SDS-PAGE.The titer of IgY was obtained by ELISA.The immunoreactivity of IgY was performed by Western blot.Results: The purity of the extracted IgY reached to 87.27％.ELISA indicated that the titer of the specific anti-tmCWP IgY gradual rised 20 days after primary immunization and reached to the highest value (1∶32 000) after 45 days.Western blot revealed that the specific IgY showed a good immunoreactivity and a specifically combination capacity.Conclusion: In our work,the tmCWP could be regarded as the immunogen to prepare the specific anti-tmCWP IgY,which could provide a novel thought for the therapy of Trichophyton mentagrophytes infection.
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Aim To evaluate the potency of anti-D. acutus venom IgY neutralizing the main activities of D. acutus venom.Methods After mixing the different a-mounts of IgY with snake venom and incubating togeth-er,the main activities of snake venom were assayed by biochemical methods.Results The in vitro assays in-dicated that anti-D.acutus venom IgY obviously neu-tralized the activities of PLA2 ,5′-nucleotidase,hyalu-ronidase,metalloprotease and serine proteinase (fi-brinogenase)in D.acutus venom.Mouse experiments showed that the ED50 value of IgY for mouse was 1 131.09 μg.Conclusion Anti-D.acutus venom IgY antibodies have good effects in neutralizing D.acutus venom without the toxicities themselves.
ABSTRACT
Background The five-paced pit viper (Deinagkistrodon acutus), endemic to China and northern Vietnam, is responsible for most snakebites in the Chinese territory. Antivenom produced from horses is the main treatment for snakebites, but it may cause numerous clinical side effects and have other disadvantages involved in their production such as the welfare of animals. The present study was conducted aiming to develop an alternative antibody (IgY) from the egg yolk of leghorn chickens immunized with snake venom. Methods IgY from the egg yolk of white leghorn chickens previously immunized intramuscularly with D. acutus venom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot. Finally, IgY neutralization assays to test its efficacy against hemorrhagic, edema-forming and myotoxic activities of D. acutus venom were conducted on mice. Results For the first time, IgY antibodies against D. acutus venom were raised successfully in egg yolk of chickens injected with D. acutus venom multiple times. By three steps, including caprylic acid extraction, ammonium sulfate precipitation and affinity chromatography, IgY antibodies were isolated and purified from egg yolk, which exhibited a single protein band on SDS-PAGE and two bands (about 65 kDa and 35 kDa, respectively) under reducing conditions, and presented a high titer (1:40,000) tested by ELISA. Immunoblot analysis confirmed that these IgY were polyclonal antibodies since they bound to components of D. acutus venom. Furthermore, immunodiffusion assay showed that anti-D. acutus venom IgY cross-reacted with the venoms of Trimeresurus albolabris and D. saxatilis Emelianov, but did not react to the venoms of Bungarus multicinctus and Naja atra. In the neutralizing lethal assay, the median effective dose of anti-D. acutus venom IgY was 14.14 mg/kg of mouse body weight under the challenge dose (3 LD50 of D. acutus venom). In neutralizing the hemorrhagic, edema-forming and myotoxic activities of D. acutus venom, IgY showed the characteristic dose-dependent neutralization effects against all these toxic activities of D. acutus venom. Conclusion Anti-D. acutus venom IgY antibodies with high purity and titer were for the first time raised successfully in egg yolk of chickens immunized with D. acutus venom. They were effective in neutralizing the lethal effects, and the hemorrhagic, edema-forming and myotoxic acitivities of D. acutus venom. IgY could be an effective source to develop a treatment against snake bites in humans or animals in the future.(AU)
Subject(s)
Animals , Snake Venoms , Antivenins , Immunodiffusion , Crotalinae , Naja naja , AntibodiesABSTRACT
Abstract Background The five-paced pit viper (Deinagkistrodon acutus), endemic to China and northern Vietnam, is responsible for most snakebites in the Chinese territory. Antivenom produced from horses is the main treatment for snakebites, but it may cause numerous clinical side effects and have other disadvantages involved in their production such as the welfare of animals. The present study was conducted aiming to develop an alternative antibody (IgY) from the egg yolk of leghorn chickens immunized with snake venom. Methods IgY from the egg yolk of white leghorn chickens previously immunized intramuscularly with D. acutus venom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot. Finally, IgY neutralization assays to test its efficacy against hemorrhagic, edema-forming and myotoxic activities of D. acutus venom were conducted on mice. Results For the first time, IgY antibodies against D. acutus venom were raised successfully in egg yolk of chickens injected with D. acutus venom multiple times. By three steps, including caprylic acid extraction, ammonium sulfate precipitation and affinity chromatography, IgY antibodies were isolated and purified from egg yolk, which exhibited a single protein band on SDS-PAGE and two bands (about 65 kDa and 35 kDa, respectively) under reducing conditions, and presented a high titer (1:40,000) tested by ELISA. Immunoblot analysis confirmed that these IgY were polyclonal antibodies since they bound to components of D. acutus venom. Furthermore, immunodiffusion assay showed that anti-D. acutus venom IgY cross-reacted with the venoms of Trimeresurus albolabris and D. saxatilis Emelianov, but did not react to the venoms of Bungarus multicinctus and Naja atra. In the neutralizing lethal assay, the median effective dose of anti-D. acutus venom IgY was 14.14 mg/kg of mouse body weight under the challenge dose (3 LD50 of D. acutus venom). In neutralizing the hemorrhagic, edema-forming and myotoxic activities of D. acutus venom, IgY showed the characteristic dose-dependent neutralization effects against all these toxic activities of D. acutus venom. Conclusion Anti-D. acutus venom IgY antibodies with high purity and titer were for the first time raised successfully in egg yolk of chickens immunized with D. acutus venom. They were effective in neutralizing the lethal effects, and the hemorrhagic, edema-forming and myotoxic acitivities of D. acutus venom. IgY could be an effective source to develop a treatment against snake bites in humans or animals in the future.
ABSTRACT
Objective:To prepare the egg yolk immunoglobulin Y ( IgY) against human Sucrase and study its stability,in vitro activity. Methods:Hy-line laying hens were immunized with human Sucrase protein,IgY was isolated and purified from egg yolks of im-munized hens using water dilution and salting out method. Indirect ELISA was used to evaluate the titer and stability of IgY. The purity and specificity of IgY were analysed by SDS-PAGE and Western blot respectively. The inhibitory effects of IgY on α-glucosidase was studied by PNPG method. Results:Indirect ELISA results showed IgY could be detected on the tenth day after the first immunization, and the peak titer of IgY was 1:12 800 after the 40th day of immunization. SDS-PAGE showed that the heavy chain and light chain of IgY were 65 kD and 25 kD respectively, and the IgY against human Sucrase could specifically recognize the protein of human Sucrase. The IgY maintained primary titer when it was kept between 29-69℃ for 15 min,and pH 4-7,37℃,4 h. The titer of IgY was maintained 50% after digestion by pepsin and trypsin respectively for 2 hours. IgY had a higher resistence to pepsin than trypsin after longer digestion time. IgY showed an inhibitory effect on α-glucosidase in concentration dependent manner. The half inhibitory concentration (IC50) was 0. 540 mg/ml. Conclusion:The IgY against human Sucrase has been successfully obtained,which established foundations for its study of Type 2 diabetes mellitus rat models in vivo.
ABSTRACT
Background Snakebite incidence in southwestern China is mainly attributed to one of the several venomous snakes found in the country, the white-lipped green pit viper Trimeresurus albolabris. Since antivenom produced from horses may cause numerous clinical side effects, the present study was conducted aiming to develop an alternative antivenom antibody (immunoglobulin Y - IgY) from leghorn chickens. Methods IgY in egg yolk from white leghorn chicken previously injected with T. albolabris venom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot, and its neutralizing assay was conducted on mice. Results Chickens injected multiple times with T. albolabris venom elicited strong antibody responses, and from their egg yolk IgY was isolated and purified, which exhibited a single protein band on SDS-PAGE and two bands (about 65 and 35 kDa, respectively) under reduced conditions. Immunoblot analysis revealed that these IgY are polyclonal antibodies since they bind with most venom components. In the neutralizing assay, all mice survived while the ratios of IgY/venom reached up to 3.79 (50.0 mg/13.2 mg). Conclusions IgY antibody response was successfully conducted in white leghorn chicken injected with T. albolabrisvenom. IgY against T. albolabris venom was obtained for the first time, and it exhibited strong neutralizing potency on mice. These results may lay a foundation for the development of IgY antivenom with clinical applications in the future.(AU)
Subject(s)
Animals , Immunoglobulins , Antivenins , Trimeresurus/immunology , Antibodies , Electrophoresis, Polyacrylamide GelABSTRACT
Snakebite incidence in southwestern China is mainly attributed to one of the several venomous snakes found in the country, the white-lipped green pit viper Trimeresurus albolabris. Since antivenom produced from horses may cause numerous clinical side effects, the present study was conducted aiming to develop an alternative antivenom antibody (immunoglobulin Y - IgY) from leghorn chickens. Methods IgY in egg yolk from white leghorn chicken previously injected with T. albolabris venom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot, and its neutralizing assay was conducted on mice. Results Chickens injected multiple times with T. albolabris venom elicited strong antibody responses, and from their egg yolk IgY was isolated and purified, which exhibited a single protein band on SDS-PAGE and two bands (about 65 and 35 kDa, respectively) under reduced conditions. Immunoblot analysis revealed that these IgY are polyclonal antibodies since they bind with most venom components. In the neutralizing assay, all mice survived while the ratios of IgY/venom reached up to 3.79 (50.0 mg/13.2 mg). Conclusions IgY antibody response was successfully conducted in white leghorn chicken injected with T. albolabrisvenom. IgY against T. albolabris venom was obtained for the first time, and it exhibited strong neutralizing potency on mice. These results may lay a foundation for the development of IgY antivenom with clinical applications in the future.
Subject(s)
Animals , Immunoglobulins/biosynthesis , Crotalid Venoms/analysis , Crotalid Venoms/isolation & purification , Crotalid Venoms/chemistry , Trimeresurus/immunologyABSTRACT
The production of antibodies in chickens and the extraction of specific antibodies from egg yolk (IgY antibodies) are increasingly attracting the interest of the scientific community, as demonstrated by the significant growth of the IgY literature. The objective of the study was to evaluate the oral acute toxicity of IgY anti HIV on Mice. In acute toxicity study, mice by administering once orally graded doses of the IgY anti HIV in the ranges of 0.9375 g to 15g /kg body weight and observed for 14 days and the number of dead mice was recorded and used in the calculation of the acute toxicity value (LD50). The mice were also observed for other signs of toxicity, such as convulsion, diarrhea, cornea reflex, dyspnea, righting reflex, straub. Oral administration of IgY anti HIV at dose of 0.9375; 1.875; 3.75; 7.5; 15 g/kg body weight showed there no mortalities or evidence of toxicity effects, suggesting that the LD50 value of IgY anti HIV was more than 15000 mg/kg body weight. Throughout 14 days of the treatment no changes in behavioural pattern, clinical sign of toxicity, vital organs weight (liver, lung, heart, spleen and kidney) and body weight of mice in both control and treatment groups. Also there were no any significant alterations in the biochemical analysis of the blood serum (SGPT, SGOT, BUN and Creatinine).The overall finding of this study indicates that the oral administration of IgY anti HIV did not produce any significant toxic effect and practically non toxic in mice. Hence, the IgY anti HIV can be utilized for immunotherapy on HIV patient.