الفهرس 
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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.