الفهرس | Only 14 pages are availabe for public view |
Abstract Poultry breeding are considered an important industries, as it is characterized by the production cycle with a short period of time compared to the cycle of breeding and production of red meat production, but poultry like any living organism were infected with numerous diseases such as viral, bacterial, fungal or nutritional deficiencies and other diseases, viral diseases are most prevalent diseases in poultry breeding. The Office International des Epizooties (OIE), organize the viral diseases of poultry during the breeding period to a range of lists depending on how serious, two types of viruses namely Highly pathogenic avian influenza (HPAI) and Newcastle disease (ND which put in the list (A), depending on the extent of the seriousness of this disease and the speed of deployment and its impact on poultry. Our study has been focused on the Newcastle disease virus , and it was the first appearance of the NDV in 1926, Newcastle , England ,and is also called avian paramyxovirus-1, which is a very contagious virus that affects a wide range of birds , it is a spherical virus , polymorphic , and particle diameter ranged from 100 to 500 nanometers which is surrounded by a shell of lipids . Newcastle disease virus can be divided into 3 groups based on their virulence such as lentogenic, mesogenic and velogenic. The strains differ among themselves in the signs as well as the mortality rate in infected poultry flocks. The signs vary from respiratory signs, nervous or digestive disorders, the greenish diarrhea, surrounded by white secretions also infected poultry lost their ability to lay eggs, even if the production of eggs happened, the eggs be easy breakage and has a soft shell. The virus is transmitted to humans, but not through eating the meat of infected poultry, but by dealing with the infected poultry and the most common sign of infection in humans is conjunctivitis that develops within24 hours of NDV exposure to the eye. The aim of the present study was to isolate and define some strains of Newcastle disease virus and try to reduce the incidence of NDV and reduce the percentage of the mortality by the production of vaccine for the virus, and the use of certain natural substances as food additives to diets to raise the immune status of poultry flocks. - To reach to the desired goals the following work were done: Total number of 13 samples were collected from infected poultry flocks (10 infected poultry from each farm) likely presence of the virus by depending on external and anatomical signs, from different regions in Egypt: Giza , Fayoum , Qaliubiya , Gharbia, Qena , Beni Suef, the following data was recorded : type of poultry samples, age, number of the flock , the date of vaccination and signs. After extracting the virus from the collected samples and clarification it by using low speeds centrifugation, the supernatant from each sample cultivated into 9-11 days SPF embryonated chicken eggs in the allantoic cavity (five eggs per sample) and then injected eggs were incubated at 37 ° C in the presence of moisture and observed twice daily for 7 days and recording results. The total sample of 13 only 9 samples could cultivate in chicken embryos eggs with different times and the mortality ranged from 60 minutes to 102 hours. After that, the allantoic fluids that have been cultivated on chicken embryos eggs were detected for the presence of the virus by using serological tests and electron microscope examination. The results were obtained as follows:Detection of Newcastle disease by heamagglutination test gave positive with nine samples from a total of 13 which have been cultivated in embryos eggs, this test is no specific for the Newcastle virus only, so heamagglutination inhibition test using specific antiserum for Newcastle virus was done and gave positive with the nine samples. The agar gel precipitation test was conducted using specific antiserum for Newcastle virus. All isolates gave precipitation line with the specific antiserum. Heamagglutination inhibition test was conducted to distinguish between the Newcastle disease virus and the avian influenza virus using specific antiserum for avian influenza virus and one sample gave positive result with this test which shows that this sample is mixed infection of both Newcastle virus and avian influenza virus. Newcastle virus was also detected using a negative technique and examined by electron microscope. Samples that gave a positive result with the previous tests, observed that the virus particles was polymorphic, spherical with different diameters ranging from 100 to 500 nanometers . Identification of viral isolates depending on some of the properties mentioned depending on the International Committee on Taxonomy of viruses, these properties are: Biological properties, virion properties, antigenic properties and genome organization. One of biological properties that have been studied is the pathogenicity of viral isolates which were identified through three tests: injection in the brains of one day old chicks, injection in intravenous for 6 weeks chickens and estimate the mean death time in 9 to 11 days chicken emryonated eggs.The results which obtained were as follows: When injected nine isolates (that gave positive result with various detection tests) in the brains of one-day old chicks. (8 chicks for each isolate) and injection of a group with of phosphate saline solution as a comparison and chicks were observed for 8 days and recorded observations , the obtained values of the nine isolates were 1.77 , 1.83 , 1.3 , 1.96 , 1.5 , 0.33 , 0.17 and 0.43 respectively. Referring to the reference values for the Newcastle virus shows that the three isolates were velogenic strains, two isolates were mesogenic strains and four isolates were lentogenic strains. One velogenic, mesogenic and lentogenic isolates were selected based on the high concentration in the heamagglutination and heamagglutination inhibition tests to complete the rest of their practical experience. When the three isolates that selected were injected in intravenous of 6 weeks old chickens (10 chickens for each isolation) and observed for ten days the data revealed that the values that were obtained were 3.31, 0.11, 0.00 , respectively, and by referring to the reference values for the Newcastle virus shows that , the three isolates were velogenic, mesogenic and lentogenic , repectively. The clinical and postmortem signs were observed on the injected chickens like respiratory signs , greenish watery diarrhea, , paralysis, twisting the neck ,heammorhages in the intestines, proventicular and changes in the liver color. When the decimal dilutions of the three isolates injected in ten emryonated chicken eggs (9-11 days) and estimate the mean death time , the time which required for the deaths of embryos is 51.78 and 96 hours to three isolates respectively. By referring to the reference values for the Newcastle virus make sure we have velogenic, mesogenic and lentogenic isolates.Following these steps, make a propagation of the three isolates in eggs embryos (the age of 9 to 11 days) in the allantoic cavity and the allantoic fluids which containing the virus were collected. Purification of the three isolates was done using alternative centerfugation using a low speed and high speed and repeats this step again and then re-suspend the pellets in the volume of 300 microliter of phosphate buffer saline. The second of the biological properties to identify the viral isolates is studies the host range of the virus, where velogenic isolate was injected in pigeons, common quails, Turkish and ducks ( six birds as a replicates ), the clinical and postmortem signs ,the rate of mortality , and the responsiveness of each type to the virus infection were recorded It has concluded from this study that the pigeon is sensitive host, where the infection rate was 100% and the rate of mortality was 90%, while the Turkish is tolerant host which showed clinical signs, but the mortality are not registered any ratio while both ducks and quail are resistance host for virus infection. After the blood samples were collected from the pigeon as a sensitive host and Turkish as a tolerant host, it became clear from the results which were obtained that rise in total lipid, cholesterol levels , triglycerides , liver enzymes, urea, uric , creatinine and decrease in the percentage of glucose and total protein which will reflect the occurrence of accumulation of lipid and consumption of proteins as a source of energy and then a DROP in the weights with infected birds rates. As it reflected in the decline in the influence weights rates in infected birds compared to non- infected birds. The third biological properties that have been studied is studies the tropism of virus, to find this tissue, a series of decimal dilutions of the velogenic isolate in drinking water were done and the chickens allowed to drink from contaminated water with the virus after deprive the birds for a period of 4 hours prior to infection.The clinical and postmortem signs were recorded and mortality rate in each treatment as well as the control treatment, and blood samples were collected from two chickens randomly, starting from the second day of infection and the virus was obtained in the various tissues using heamagglutination test. The results obtained that, from the second day of infection the virus appeared with low concentrations in both the liver and proventicular, from the third day the virus concentration increased in these organs with the emergence of the virus with a low concentration in the spleen and the kidneys the results revealed that the liver, proventicular then spleen and kidneys are the organs and tissues where the virus has found in it with the highest concentration. In virion properties, which was used to identify of viral isolates is some of the physical and chemical factors and its effects on the virus. The effect of heat , radiation, the concentration of pH , formalin , sodium hypochlorite , chloroform and ethylether , it appeared from the results which obtained by the following: When exposing the three isolates of Newcastle disease virus to a temperature of 56 ° C, all isolates lost its ability to cause infection after exposure to this temperature for 45 minutes, but when temperatures rise to 60 ° C all isolates lost its ability to infection after exposure to this temperature for five minutes. To study the effect of pH degrees, the viral isolates were incubated with different degrees of pH (3, 7, 13). It was found that the viral isolates remained retains its ability to infection in the pH 7, and lost its ability to infection in the alkaline and acidic range with the exception of lentogenic isolate which remained retains its ability to infection in the acidic range. As for the impact of UV radiation on the infectivity of the viral isolates was clear from the results that, these rays do not have effectiveand tangible impact in influencing the activity of the different viral isolates. It was clear from the treatment of viral isolates with different concentrations of formalin 3, 5, 10 % that all isolates lost its ability to infection after 5 minutes of treatment with different concentrations. While when the viral isolates treated with sodium hypochlorite, it was observed that all isolates lost its activity after exposure to this substance for a period of 45 minutes. But when the effect of both chloroform and ethylether on viral isolates were studied, it was observed that all isolates lost its ability to infection after treatment of these solvents for 30 minutes. Among the properties that have been reliable in the definition of viral isolates it was viral genome properties. The fusion gene was chosen as it is responsible for identifying pathological capacity of Newcastle disease virus strains and it amplified by the polymerase chain reaction and the sequence of nucleotides was studied and the bioinformatics used to analyze the results of the sequence of nucleotides and build the phylogenetic tree. It was clear from the results of the sequence of nucleotides that two isolates of virus have high virulence and one isolate belong to the lentogenic strain. The antigenic properties were studied by inhibition of the velogenic isolate on the temperature 56 ° C for 30 minutes and then injected into the pure rabbits strains. The obtained antiserum was tested by the agar gel precipitation test. It became clear from the results of this test that serological relationships between different viral isolates were found because of all isolates belong to the same serotype (serotype -1). It is the principle that prevention is better than cure, two application experiments were done to raise the immune status of the flock and protect it against infection with Newcastle disease virus, the first experiment is aims to produce a vaccine from lentogenic strain after inhibited by formalin then infection with the velogenic strain was done. It appeared from the results that, despite all the birds were infected with velogenic strain but the strong importance of the vaccine appeared to prevent mortality among chickens and minimizing clinical and postmortem signs compared with untreated chickens . The second experiment done using of certain substances as food additives in chickens feed. One-day old broiler ere used and left two days in order to acclimatize to the new environment. Starting from the third day they were weighed and divided into three main groups who are: ginger group, cinnamon and yeast, each main group containing four subgroups. The chickens fed for a month on these additions individually, after two weeks of feeding, the infection with the velogenic strain was done. The clinical and postmortem signs, the mortality rate and the rate of weight per week were recorded. Our study revealed that the best of these additives in terms of improving weights rates were cinnamon, but the best of them in reducing mortality rate and severity of the signs yeast was followed by ginger and cinnamon. Recommendations that could be listed by the results of this study: 1. Ensure the quality and efficiency of the vaccine which used to protect poultry against the Newcastle virus disease and the emphasis on the use of vaccines made from local strains and move away from the use of imported vaccines. 2-Educate breeders for the risks that may occurs when used improper or excessive for these vaccines, where it is the vaccine itself may become the wrongful source to increase the infection, which leads to an imbalance in the diagnosis of these strains and the actual infection of the virus that leads to the re-division of the known genotypes at the level of the world and with the large number of fortifications, this may lead to mutation in the strains, which is given to the birds on farms and lead to an increase in ferocity. 3. Taking into account the health conditions and disinfecting and their contents sterilized properly and use more of the substance to increase the efficiency of sterilization. 4. When the virus cultivated in the eggs embryos, not relies conclusively on the results of the first passage, but make at least two passage in embryonated chicken eggs. 5. Do not rely on the clinical and postmortem signs in diagnosis of poultry diseases because of the similarity of many of diseases on the signs in infected poultry. 6. Use of certain natural substances as food additives in poultry feed, particularly substances which known its medical impact is useful and presented with diets or drinking water from the early days until the end of the production period. |