Expression of NDV HN protein in rice and development of a semi-quantitative rapid method for detection of antibodies / 生物工程学报

The aim of this study was to develop a semi-quantitative immunochromatographic method for rapid detection of Newcastle disease virus (NDV) antibodies by expressing HN protein in rice endosperm bioreactor. The recombinant plasmid pUC57-HN was digested by MlyⅠ and XhoⅠ to retrieve the HN gene, while the intermediate vector pMP3 containing promoter, signal peptide and terminator was digested by NaeⅠ and XhoⅠ. The HN gene and the linearized pMP3 were purified and ligated to form a recombinant plasmid pMP3-HN1. Subsequently, pMP3-HN1 and plant vector pCAMBIA1300 were digested by EcoRⅠ and Hind Ⅲ, and the HN1 gene was cloned into pCAMBIA1300. The recombinant plasmid pCAMBIA1300-HN1 was introduced into Agrobacterium tumefaciens EHA105 by electrotransformation, and the pCAMBIA1300-HN1 was transferred into rice callus by agrobacterium-mediated method. After dark culture, callus screening, differentiation, rooting and transplanting, transgenic rice seeds were obtained 4 months later. PCR identified that the HN gene has been inserted into the rice genome. SDS-PAGE and Western blotting indicated that the HN protein was successfully expressed in the positive rice endosperm. The purity of the HN protein was more than 90% by SP cation exchange chromatography and gel filtration chromatography. According to the national standards for the diagnostic techniques of Newcastle disease HI test (HI≥4log2, positive antibody reaction), a colloidal gold labeled purified HN protein was used to prepare a semi-quantitative test strip by double-antibody sandwich method for rapid detection of NDV antibody. The results showed that the test strip did not cross-react with positive sera against other viruses, and the sensitivity of the test strip reached 1:102 400 for standard positive sera of Newcastle disease. Testing of a total of 308 clinical sera showed that the compliance rate of the test strip with HI test was 97.08%, and the Kappa value was 0.942. In conclusion, high purity recombinant HN protein was obtained from rice endosperm, and a simple, rapid, highly sensitive and highly specific semi-quantitative immunochromatographic strip was developed. The test strip could be used for immune evaluation of the Newcastle disease vaccine.

Immunomodulatory effects of Pimpinella anisum L. (Aniseed) in Broiler Chicks against Newcastle disease and infectious bursal disease viruses / Efecto inmunomodulador de Pimpinella anisum L. (anís) en pollos de engorde contra la enfermedad de Newcastley la enfermedad viral de la bursitis infecciosa

Pimpinella anisum L. (Aniseed) is mostly used as an immune stimulant, growth promoter, antifungal, antibacterial in many countries for centuries. The aim of this study was to determine the immunomodulatory effect of aniseed against Newcastle Disease (ND) and infectious bursal disease (IBD) viruses. The immunomodulatory effect of aniseed against ND and IBD viruses were determined by modifying splenic cell migration inhibition assay and differential leukocyte count for cellular immunity. Haemagglutination inhibition and indirect haemagglutination were used for measurement of humoral immune response against ND and IBD viruses, respectively. The present study suggests that the aniseed addition to basal diet at the rate of 0.5 g/kg and 1 g/kg of feed had best immunomodulatory activity both for humoral and cellular immune responses. However, at higher doses aniseed had adverse effects. Aniseed possesses significant immunomodulatory activity when it is added at lower doses i.e., 0.5 g/kg and 1 g/kg.

Pimpinella anisum L. (Anís) se utiliza principalmente como un estimulante inmunológico, promotor del crecimiento, antifúngico, y antibacteriano, en muchos países durante siglos. El objetivo de este estudio fue determinar el efecto inmunomodulador de anís contra la enfermedad de Newcastle (ND) y la enfermedad de la bursitis infecciosa (IBD). El efecto inmunomodulador de anís contra los virus ND y e IBD se determinaron mediante la modificación del ensayo de inhibición de la migración de células del bazo y recuento diferencial de leucocitos de la inmunidad celular. La inhibición de la hemaglutinación y hemaglutinación indirecta se utilizaron para la medición de la respuesta inmune humoral contra el virus de ND e IBD, respectivamente. El presente estudio sugiere que la adición de anís a la dieta basal a la tasa de 0,5 g/kg y 1 g/kg de alimentación tuvo una mejor actividad inmunomoduladora tanto para las respuestas inmunes humorales como celulares. Sin embargo, a dosis más altas de anís tuvo efectos adversos. El anís posee una importante actividad inmunomoduladora cuando se añade en dosis más bajas, es decir, 0,5 g/kg y 1 g/kg.

comparison of the effects of commercial prebiotic [safmannan, biomos and fermacto] on performance, egg quality and antibody titer of Avian Influenza and Newcastle disease in laying hens

Effects of different sources of prebiotic on performance and immune response in laying hens were evaluated in this study. The aim of this experiment was to compare the effects of commercial prebiotics on performance, egg quality and antibody titer against Newcastle disease [ND] and Avian Influenza [AI] in laying hens. The experiment was performed for 11 weeks [from 33 to 44 weeks of age] on 240 laying hens [the LSL strain], in a completely randomized design with 4 treatments, and 5 replicate for each treatment and 12 birds for each replicate. Treatments were: 1] control; 2] control +Safmannan; 3] control +Bio-Mos and 4] control +Fermacto. The results showed that feed intake, feed conversion ratio and egg production percentage were not affected by different sources of prebiotic. However, egg weight and egg mass were significantly increased [p<0.05] by adding prebiotic, in comparison with control diet. Egg specific gravity and shell thickness were increased [p<0.05] by prebiotic addition. The results of experiment showed that antibody titers against AI and ND were significantly increased [p<0.05] by prebiotics compared with control group. The results showed that the effects of different sources of prebiotic on performance and immune response of laying hen are similar. However, inclusion of prebiotic in the diet results in improvement of the performance and antibody titer against Influenza and Newcastle

Estudo da eficácia de espuma para a depopulação de avespara situações de emergência sanitária / Experimental evaluation of foam-based poultry depopulation for emergencies

São inúmeras as dificuldades observadas na depopulação de elevado número de aves e é importante proceder-se àdepopulação no interior do próprio aviário por questões de biossegurança do homem em casos de emergência sanitáriacausada por doenças de aves de elevada transmissibilidade, como a influenza aviária e a doença de Newcastle. Construiuseo equipamento gerador de espuma para provocar a anóxia física em aves. Verificou-se a eficácia da espuma geradaatravés do uso de líquido detergente de alta expansão sobre o sacrifício de galinhas, avaliando-se os sinais sensoriais,tempo de morte, letalidade e lesões macro e microscópicas. Duzentas galinhas de postura com 78 semanas de idadeforam divididas em dez grupos experimentais e submetidas a diferentes períodos de exposição à espuma, variando de5min30s a 8min15s . A partir de seis minutos de exposição à espuma 100% das aves foram mortas. A presença de bolhasde espuma na cavidade oral e traqueia foram constatadas em todos os períodos de exposição à espuma, indicando aocorrência de asfixia física. Congestão nas mucosas da cavidade oral e traqueia foram também observadas em todos osperíodos de exposição. No exame histopatológico dos pulmões de todas as aves, observou-se a presença de congestãoe hemorragia difusa de moderada intensidade. Nenhuma alteração foi observada na traqueia das aves. A eficácia daespuma como método de depopulação de aves foi constatada neste trabalho, podendo ser recomendada diante danecessidade de atendimento a emergências sanitárias.

There are several difficulties in the control of quick spreading and highly contagious poultry diseases such as avianinfluenza and Newcastle disease, which require large numbers of poultry to be rapidly killed inside the poultry housesin order to minimize human exposure and disease spreading. We have built and tested an equipment that uses a waterbasedfoam from high expansion fire detergent that induces poultry anoxia measured by sensorial signs, time to death,lethality and macro and microscopically lesions. Two hundred 78-weeks old hens were divided into ten groups andexposed to different times of death according to exposure to water-foam varying from 5min30s to 8min15s. All poultrydied after six minutes of water-foam cover. Foam bubbles were observed in oral cavity and trachea of all poultry ateach time of exposure to foam, revealing physical asphyxia. Macroscopically, congestion in oral cavity and in tracheamucosa was observed. Histopathology revealed diffuse and moderate hemorrhage in lungs and no lesion in trachea.The efficacy of water-foam procedure for poultry depopulation was demonstrated validating its recommendation incase of emergency.

[Effect of Echinacea purpurea dried extract effect on producing antibody from newcastle disease vaccine in broilers by HI test]

Newcastle disease is one of the most important causes of economic losses in the poultry production and can he resulted in high mortality. Antibody detection is also an important tool for assessment of the immunity against the disease. In the present study a trial was conducted to evaluate the effect of an immune stimulator[Echinacea purpurea] on antibody production against Newcastle disease vaccine. 450 one day old broiler chicks were divide into five groups of three repeat each. For three weeks from day one various doses of Echinacea purpurea extract was prescribed to four treatment groups and to the fifth group placebo in water was prescribed. All groups were vaccinated on days:11, 19, 38. Subsequently. serum samples were collected at days 10. 25, 34.52 of post vaccination from 21 chicks of each group [4 samples of each repeat] and were tested for Newcastle antibody titers by HI test. This experiment showed that the use of Echinacea purpurea extract with the rate of 29, 75Mg per kilo body weight per day had better effects on antibody titers and significantly increased between control group arid treatment groups [p<0.01]. It is also revealed that the use of Echinacea purpurea induces FCR improvement and mortality rate was decreased significantly [p<0.01]

Locally produced mucosal IgG in chickens immunized with conventional vaccines for Newcastle disease virus

Newcastle disease virus (NDV) is the causative agent of an economically important disease, which affects all species of birds worldwide. Current vaccination programs for NDV include the use of either low-virulent live-virus vaccines or inactivated vaccines to induce protective immunity while producing minimal adverse effects in birds. In order to further characterize the immune response elicited by live virus and inactivated NDV conventional vaccines in chickens, we evaluated the presence of specific antibodies in different secretions and in tissue culture supernatants of immunized birds. To this end, we analyzed all the samples by ELISA, using an indirect assay set up in the laboratory. Specific anti-NDV IgG antibodies were detected in tracheal and cloacal swabs and tracheal and intestinal washes of immunized animals. We also found specific anti-NDV IgG antibodies in tracheal and intestinal tissue culture supernatants, indicating that the IgG found in swabs and washes was not transudated from serum or, at least, was not all transudated from serum. Knowledge about the mechanisms involved in the immune response of chickens to different NDV vaccines should increase our understanding of the mucosal response against the virus and, eventually, provide new useful information for the development and evaluation of synthetic vaccines.

The use of scaning electron microscopy in postvaccinal evaluation of tracheal epithelium of Coturnix coturnix japonica

Este experimento foi realizado para avaliar o emprego da microscopia eletrônica de varredura no estudo da reaçäo respiratória pós-vacinal em epitélio traqueal de codornas (Coturnix coturnix japonica) imunizadas contra a doença de Newcastle. Foram utilizadas 36 codornas que foram distribuídas em quatro grupos, sendo: T1 - grupo de aves controle (näo vacinado), T2 - grupo de aves vacinadas com a estirpe Ulster 2C, T3 - grupo vacinado com a estirpe B, T4 - grupo de aves vacinadas com a estirpe LaSota. Independente do grupo experimental, as aves näo apresentaram sinais clínicos detectáveis de reaçäo respiratória pós-vacinal. Entretanto, na análise de fragmentos traqueais, ao microscópio eletrônico de varredura, observou-se que as codornas vacinadas com as estirpes B e LaSota desenvolveram descamaçäo epitelial da traquéia, enquanto as aves vacinadas com a estirpe Ulster 2C näo desenvolveram tal alteraçäo, mostrando um epitélio traqueal íntegro, semelhante ao grupo controle.

Studies on danofloxacin in chicks vaccinated with newcastle disease virus vaccine

In Hubbard chicks, oral administration of therapeutic and twofold dose of danofloxacin for three successive days post-vaccination with Newcastle disease virus vaccine induced a slight immunostimulant effect manifested by an increase in haemagglutination inhibition titres in vaccinated treated groups. Moreover, the drug at therapeutic doses, resulted in a significant [P < 0.05] increase of body gain in treated chicks compared with non treated ones. On the other hand, therapeutic doses of danofloxacin had no adverse effects on liver and kidney functions. Double therapeutic doses of danofloxacin elicited a significant [P < 0,05] increase in serum uric acid, creatinine, urea, alkaline phosphatase, aspartate amino transferase and a decrease in serum albumin. In addition, degenerative changes in liver and kidney, and lymphocytic depletion in the thymus, spleen and bursa of fabricius were detected. It could be concluded that therapeutic doses of danofloxacin Induce a slight immunostimulant effect in chicks immunized with Newcastle disease virus vaccine, coupled with no adverse effects on body gain, liver Nrand kidney functions

effect of feeding and lighting regimens on some behavioral patterns and performance of broiler chicks

This study was carried out to investigate the effect of feeding and lighting regimens on behavior and performance of broiler chicks. A sample of 150 Arbor Acres were used and divided into 3 equal groups, each of 50 chicks. The 1st group [G.1] received a continuous lighting and ad. libitum feeding acts as control, the 2nd group [G.2] permitted ad. libitum feeding with only day light, while the 3rd group [G.3] received a continuous lighting with intermittent feeding [three time daily, each of 2 hours]. The results obtained indicated that there was a significant [p <0.05] increase in the food-searching behaviors as feeding frequency, feed bout, and ground pecking by birds with intermittent feeding [G.3], while there was no effect of light regimen on these patterns. There was no significant difference in the final body weight gain between groups although birds in groups 2 and 3 consumed significantly [p <0.05] less food [3629.6, 3405 and 3420 g] and convert their feeding more efficiently [0.412, 0.423 and 0.421] than those in control group. Regarding the rest and comfort behaviors, there was no significant effect of feeding or lighting regimens except that the resting and ground scratch% were significantly [p <0.05] higher in group 3 than other groups. There was no significant effect of both treatments on agonistic behaviors except on fight and feather pecking patterns as their occurrences were higher between birds in group 3 than in the other two groups [1 and 2]

electrophoretic pattern of serum proteins in chickens vaccinated with newcastle [different strains and gumboro vaccines

The present work was planned to study the effect of vaccination with different strains of Newcastle disease virus vaccine [NDVV] as well as vaccination with Gumboro vaccine on the electrophoretic pattern of serum proteins in our local breed [Baladi]. One hundred and fifty three one day old chicks were used. Our results revealed that there was a significant increase in the levels of total serum proteins, albumin, total globulins and gamma-globulin in the sera of chickens vaccinated with NDVV whereas there was no significant difference between Gumboro vaccinated chickens and control group in the total serum proteins and its fractions. The chickens vaccinated with komarov strain of NDVV showed the highest mean values of total serum proteins, albumin, total globulins and gamma-globulin whereas the lowest values were observed in the chickens vaccinated with Gumboro and Lasota strain of NDVV. Concerning the type of vaccine and weeks post-vaccination. The mean values of total serum proteins, albumin, total globulins and beta-globulins were significantly increased in chickens vaccinated with NDVV either with komarov or Lasota strains at the 1st week post-vaccination. In the 2nd week post-vaccination, the mean values of beta-globulin fraction in group vaccinated with NDVV and chickens vaccinated with NDVV [without last dose] and Gumboro vaccine was significantly decreased. Furthermore, the total serum proteins were significantly decreased in chickens vaccinated with Gumboro and NDVV [K]. In the 3rd week post-vaccinated with Gumboro [L]. It was concluded that the vaccination of chicks with Gumboro vaccine at 14 day of age delay the response of immune system for vaccination with Newcastle disease virus vaccine [NDVV]