Isolating avian paramyxovirus-1
in New Zealand poultry.
Research Project Veterinary Medicine Utrecht University
Author: Drs. C. Reijne
Student number: 3155943
Date: … January 2011
Project supervisors
- Massey University:
-
University of Utrecht:
Dr. N. Cave
Dr. M. Dunowska
Dr. H.F. Egberink
Foreword
In the final two years of Veterinary medicine at University Utrecht all students have to do a
three months during research project . This paper is the final report of my research on avian
paramyxoviruses in New Zealand.
When I went to New Zealand the original plan was to do a research on feline
immunodeficiency virus (FIV). The purpose of this research was to isolate and quantify FIV
derived from the plasma and PBMC's of naturally infected cells. The plan was to infect MYA1 cells in vitro, using PBMC and plasma from cats that were naturally infected with FIV.
Then the cell supernatant was going to be assayed at defined time points, to quantify the level
of virus produced by the cells. For quantification a real time PCR or p24 ELISA would be
used. With this research the optimal time from infection to harvest of the supernatant would
be determined, this information could be used to produce viral stock from a range of strains of
FIV from New Zealand.
Before me and my student colleague arrived in New Zealand, our supervisor already started
with trying to grow the MYA-1 cells. The goal was to grow the MYA-1 cells before we came
to New Zealand so we could start with our research when we arrived. Unfortunately the cells
were not growing as fast as expected and we had to wait until the ordered IL-2, that was
needed for the cells to grow would arrive. So when we arrived we first did a literature study
on FIV and started with learning the techniques using healthy PBMC's instead of MYA-1
cells. Also we started on a other little study to determine why CPT tubes don't work on cat
blood. The IL-2 that was needed for the growth of the MYA-1 cells wasn’t delivered and the
MYA-1 cells all died. Since there would not be enough time to order new MYA-1 cells and
grow them, the research projects had to be changed and it wouldn’t be possible to both do a
research on FIV. For this reason I switched to a research on avian paramyxovirus-1, and
started with a second literature study and a new research as described in this paper.
Even though things didn’t really go as I expected and it was sometimes hard to accept that
research takes a lot of time, I really liked my time at Massey University. I have learned many
techniques (flow cytometry, PCR, cell culture, RNA/DNA extractions, real time RT-PCR and
many more) and eventually finished a nice research project.
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
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Table of contents
Abstract
Page 4
Introduction
- Etiology
- Epidemiology
- Clinical signs
- Different virulent APMV-1 isolates
- Diagnosis
- Vaccination
- Avian paramyxoviruses in New Zealand
Page 5
Page 5
Page 6
Page 6
Page 7
Page 7
Page 8
Page 9
Materials and methods
- Sources and collection of samples
o Blood samples
o Cloacal and tracheal swabs
- RNA extraction
o Method 1: TRIzol ® LS Reagent
o Method 2: TRIzol ® LS Reagent combined with
Qiagen RNeasy columns
o Method 3: Qiagen RNeasy columns
- Quantification of the RNA
- RNA clean up
- Real time reverse transcriptase Polymerase Chain Reaction
- Agarose gel electrophoresis
Page 10
Page 10
Page 10
Page 10
Page 11
Page 11
Results
- Serology
- Quantification of the RNA
- Determining the positive control for real time RT-PCR
- Real time RT-PCR on the samples from the backyard poultry
o M-gene assay
o L-TET assay
- Real time RT-PCR on the samples from the chickens at the
poultry farm
- Agarose gel electrophoresis
Page 15
Page 15
Page 15
Page 16
Page 17
Page 17
Page 19
Discussion
- Method for RNA extraction
- Real time RT-PCR
Page 23
Page 23
Page 24
Acknowledgements
Page 25
References
Page 26
Attachments
- Attachment 1: Results from the spectrophotometer of the
cloacal samples
- Attachment 2: Results from the spectrophotometer of the
tracheal samples
Page 28
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
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Page 12
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Abstract
Avian paramyxoviruses (APMVs) cause a significant economic impact on the poultry
industry worldwide. There are nine different serotypes of APMVs, which can infect a wide
range of avian species. Newcastle disease (ND) is caused by avian paramyxovirus type 1
(APMV-1) with a ICPI above 0,7. Clinical signs seen in birds infected with APMV-1 vary
depending on the pathogenicity of the isolate. As signs of disease caused by APMV-1 in
different hosts vary significantly, the diagnosis of ND cannot be made based on clinical signs
alone. So far no ND outbreak has occurred in New Zealand and all New Zealand isolates of
APMV-1 to date have been apathogenic, so New Zealand is regarded free of the virulent form
of NDV. However, seroconversion to APMV-1 is occasionally recognized in commercial
poultry flocks during the lay period, and APMV-1 have been isolated from wild waterfowl in
New Zealand. A limited number of studies have been undertaken to characterize such NZ
isolates of APMV-1. For this reason this research had a look at which APMV-1 isolates are
circulating in New Zealand. To do so paired cloacal and tracheal swabs were taken from seronegative layer birds that were introduced into a commercial poultry flock with known
exposure to APMV-1. In total 100 birds were sampled: 50 on the first sampling (2 weeks after
introduction to the farm), then 25 on the second sampling (a week later) and 25 on the third
sampling (another 2 weeks later). In total 22 blood samples were collected for serology at the
second and third time point. From the cloacal and tracheal samples RNA was extracted using
Qiagen RNeasy columns and the 45 samples taken at the last time point were tested in a real
time RT-PCR using a M-gene assay and a L-TET assay. Also 162 samples from chickens and
other domestic birds kept on small backyard farms that were at high risk of getting a APMV-1
infection from wild waterfowl, were tested by this real time RT-PCRs. All samples that were
tested with the M-gene assay were negative, only the positive controls were positive. The Lprobe tested all the samples negative and there was no positive control sample for this test so
no conclusions can be drawn from these results. Since there were no positive samples it was
not possible to characterize any isolates. From the results of this research it can be concluded
that APMV-1 is not common in New Zealand. More research to characterize the APMV-1
isolates that are circulating in New Zealand should be done, to assure that all circulating
APMVs continue to belong to lentogenic and apathogenic types.
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
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Isolating avian paramyxovirus-1 in New Zealand poultry.
Introduction
In 1926 a highly contagious viral infection of poultry was first discovered on a poultry farm
near Newcastle UK and in Java, Indonesia. Shortly after the reported disease that was named
Newcastle disease, two further outbreaks occurred in the UK, in Somerset and in
Staffordshire. After the recognition of the disease it rapidly spread throughout the world to
Indonesia and South east Asia. At first Newcastle disease was recognized as a highly
pathogenic disease with up to 100% mortality. In the mid-1930s a relatively mild respiratory
disease with a low mortality, called pneumoencefalitis was observed in California. Later it
showed that this was caused by a virus which was immunologically indistinguishable from
Newcastle disease virus (NDV). After the observation that NDV was not always highly
pathological numerous reports of other strains with low virulence followed (1).
Etiology
Avian paramyxoviruses (APMVs) can be classified into nine serotypes, which can infect a
wide range of avian species as shown in table 1 (2). Within these subtypes there are many
isolates, which differ in their pathogenicity. In vivo an assessment of the pathogenicity of the
virus can be performed by the intracerebral pathogenicity index (ICPI). This index is the mean
score (0= normal, 1= sick, 2= dead) per bird per 24 hour observation over 8 days, after
intracerebrally injected diluted virus. Values approaching the maximum score of 2 represent
the most virulent viruses and low virulent viruses give values around 0. Newcastle disease is
caused by a high virulent avian paramyxovirus serotype-1 that has an ICPI of 0,7 or higher.
(2,3,4,5).
Avian paramyxovirus serotype 1(APMV-1) is a single stranded, non segmented RNA virus
with an envelope. The genome consist out of six genes that encode for the six structural
proteins: nucleoprotein (NP), phosphoprotein (P), matrix (M), fusion (F), hemagglutininneuraminidase (HN) and RNA polymerase (L) as shown in figure 1 (6,7). APMV-1 can be
divided into class I and class II based on phylogenetic grouping of the F-gene (8).
Prototype virus strain
APMV-1 (Newcastle disease)
Usual natural hosts
Numerous
APMV-2/chicken/California/ Yucaipa/56
Turkeys, passerines
APMV-3A/turkey/ Wisconsin/68
Turkeys
APMV-3A/parakeet/
Netherlands/449/75
APMV-4/duck/Hong Kong/D3/75
APMV-5/budgerigar/Japan/ Kunitachi/74
APMV-6/duck/Hong Kong/199/77
Psittacines,
passerines
Ducks
Budgerigars, lorikeets
Ducks
APMV-7/dove/Tennessee/4/75
Pigeons, dove
APMV-8/goose/Delaware1053/76
APMV-9/duck/New York/22/78
Ducks and geese
Ducks
Disease produces in naturally infected poultry
Very common worldwide, varies from very severe to
inapparent disease depending on strain and host infected
Common, probably worldwide, mild respiratory disease
or egg production problems; severe if exacerbating
occurs
Turkeys only, in North America and Europe mild
respiratory disease but severe egg production problems
worsened by exacerbating organisms or environment
No infection of poultry reported
None known
No infection of poultry reported
Mild respiratory disease and slightly elevated mortality
in turkeys; none in ducks or geese
Natural infections of turkeys with respiratory disease and
ostriches have been reported
No infection of poultry reported
None known
Table 1: Different types of paramyxoviruses and the host species they infect (2).
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
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Figure 1: Newcastle disease virus and genomic
organization of its structural proteins (3)
RNA viruses have a high mutation rate caused by the low fidelity and processivity of their
polymerase. Because the virus replicates very quick and the generation time is short there are
a lot of evolutionary rates. For this reason there are many different isolates of APMV-1 that
circulate in different parts of the world (6,8).
Epidemiology
APMV-1 is transmitted in poultry by direct contact with secretions of infected birds, for
instance through infected water or food (fecal/oral route) or by inhalation. Transmission from
an infected flock to an uninfected flock is possible through respiratory secretions, feces,
carcasses or by wild birds and waterfowl which may act as an reservoir (3).
Nowadays APMV-1 has a worldwide distribution and Newcastle disease is endemic in many
countries most of the enzootic strains are mesogenic or lentogenic (2,3). The disease has been
reported in temperate and tropical areas and it occurs in all seasons. Since there is a
widespread vaccination with mesogenic and lentogenic strains this is likely to mask the
presence of virulent strains. Newcastle disease is endemic in the commercial poultry industry
of southeast Asia (9). Newcastle disease is a serious problem in many countries in Africa,
were most of the poultry are held at the village and it is hard to achieve efficient vaccination.
In the United States and South America velogenic strains are reported. Luxembourg and
Norway are the only two European countries that never recorded outbreaks of Newcastle
disease. In Australia and New Zealand antibodies against APMV-1 are present but no disease
is evident, which indicates that there are avirulent strains present in the poultry population.
Because of the wide variation in the severity of the disease caused by different strains it is
likely that many outbreaks in which the birds are infected with low pathogenicity strains go
unreported (1).
Clinical signs
Newcastle disease has a wide host range with at least 241 species form 27 of the 50 orders of
birds reported to be capable of supporting replication of the virus, but the disease usually
occurs in chickens or in turkeys (9,4). The susceptibility in birds varies among species and
waterbirds seem to be the most resistant (4). Clinical signs associated with APMV-1 infection
in birds can be variable and depend on factors such as the virus isolate, host species, age and
immune status of the infected animal, the presence of other organisms and environmental
stress (2).
APMV-1 can be classified into different pathotypes based on the clinical signs seen in
infected chickens and the histopathological findings.
1. Viscerotropic velogenic: this is a highly pathogenic form, it causes an acute disease
with high mortality. Often haemorrhagic lesions in the intestines are seen.
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
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2. Neurotropic velogenic: this form causes respiratory and nervous signs followed by a
high mortality.
3. Mesogenic: this form has a low mortality and causes acute respiratory disease and
occasionally neurological signs in some birds.
4. Lentogenic: this AMPV-1 type may cause mild respiratory signs and decreased egg
production in some birds but can also be subclinical.
5. Asymptomatic enteric: usually consists of a subclinical enteric infection. (2,3,6,9)
Different virulent APMV-1 isolates
APMV-1 consists out of one serotype, nonetheless there are a lot of different strains which
have a wide difference in the pathogenicity, which ranges from causing asymptomatic disease
to killing the bird within a few days (5). Velogenic and mesogenic strains are virulent, while
lentogenic strains often have a low virulence (6). How virulent a APMV-1 strain is depends
on multiple genes, a critical site that is responsible for differences in virulence is the fusion
protein cleavage site (10,11). It has been shown that low virulent APMV-1 strains have fewer
basic amino acids at their fusion protein cleavage site and have a leucine at position 117
instead of a phenylalanine (12). The viral fusion protein is necessary for entry of the virus into
the cell. To get into the active form the fusion protein (F0) has to be cleaved to form F1 or F2.
It has been demonstrated that the presence of multiple basic amino acids at the fusion protein
cleavage site allows a wide range of proteases to cleave, which is characteristic for velogenic
and mesogenic APMV-1 isolates. These viruses cause severe diseases because they are able to
infect many tissues. On the other side the fusion cleavage site of apathogenic and lentogenic
isolates is recognized only by trypsin-like proteases in the respiratory and gastro-intestinal
tract. Because of this, these viruses can only cause mild localized infections (2,11). Highly
virulent NDV isolates (with an intracerebral pathogenicity index (ICPI) of 0.7 or higher) are
List A pathogens and have to be reported to the Office of International Epizooties. (4).
Diagnosis
As the symptoms of NDV infection vary widely, the diagnosis can’t be made based on the
clinical symptoms alone. When a bird is suspected to be infected with NDV the preferred
diagnostic methods are serological testing and virus isolation (3,5).
Serological tests measure the NDV-antibody titer in the blood, which can be done by a
haemagglutination (HI) test or an ELISA. The viral envelope has the ability to agglutinate red
blood cells, the HI test is based on the inhibition of this agglutination by specific antibodies.
When a blood sample does not contain any antibody against the virus haemagglutination
occurs, but when it does the antibodies will inhibit the agglutination and the red blood cells
will appear as a pellet at the bottom of the well. The immune status of the bird can be
determined by the haemagglutination titer, since a high antibody titer is indicative of a recent
infection. To determine whether the titer is increasing or decreasing it is necessary to take
multiple samples at different time-points. The ELISA test is based on anti-NDV antibodies,
which attach to viral antigen antibodies and causes a change of color, which can be seen by a
spectrophotometer. Identification of NDV through the presence of specific antibodies against
NDV only indicates that the bird has ever been infected or vaccinated and it does not
necessarily means that the bird was infected at the time the sample was taken (5).
By virus isolation the presence of the virus itself will be demonstrated. Virus isolation can be
performed on recently dead birds or moribund birds that have been killed humanely and must
be done on both oronasal swabs and samples collected from lung, kidneys, intestine, spleen,
brain, liver and heart tissue. Tracheal and cloacal swabs can be used for virus isolation in live
animals. Because of the variation in virulence and the use of live vaccines, identification of an
isolate APMV-1 does not confirm the diagnose of NCD. The definitive diagnosis can be done
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
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by an assessment of the pathogenicity of the virus, which in vivo can be performed by
determining the intracerebral pathogenicity index (ICPI). (3,5)
Vaccination
Avian paramyxoviruses cause a significant economic impact on the poultry industry
worldwide, which includes the direct costs of the disease itself, the costs of maintaining
preventive vaccination strategies and limitations on export of livestock (2). Since there is no
treatment for Newcastle disease, prevention of the disease is very important (3). For this
reason inactivated and live-virus vaccines for the control of NDV in poultry have been
developed (4). Three types of vaccines are developed as preventive measures to protect
against Newcastle disease: inactivated, live lentogenic and live mesogenic vaccines (5).
In 1946 inactivated vaccines were made commercially available to the poultry industry (4).
These type of vaccines contain inactivated viruses which are no longer capable of replication
or spreading in the flock, for this reason it has to be injected individually into every bird
needing vaccination. Live vaccines contain viruses that can replicate in the host, so it is not
necessary to inject the vaccine into every bird since the vaccine virus can spread through the
flock. A problem with live vaccines is that they can cause clinical signs, whether this occurs
depends on the vaccine strain and the presence of other organisms (5). Previous researches
indicated that both inactivated and live vaccines give protection against morbidity and
mortality and significantly lower shedding of the virus, but virus shedding is not prevented
and infection can still occur (4,8,13,14).
Different NDV isolates are used in vaccines. Most of the live lentogenic vaccines are derived
from field isolates that have a low pathogenicity but can produce an adequate immune
response (5). Vaccines that contain lentogenic isolates usually produce weak and short during
immunity, to maintain immunity frequent revaccination is needed. Vaccines that are prepared
from more pathogenic (mesogenic) isolates produce longer and stronger immunity but can
cause mortality in vaccinated chickens (15).
Most of the vaccines that are used are live vaccines. These vaccines have the potential to
effect the evolutionary genetics of the avian paramyxovirus because they can be transmitted
amongst birds. It is possible that recombination of the vaccine strains between wild and
domestic birds takes place, this was suggested when vaccinated birds were found protected
against the disease but not against infections with other strains of avian paramyxovirus-1. It is
also found that, although since 1950 vaccination occurs the population size of the mostly
used vaccine strain has steadily enlarged while contemporary genotypes, made in 1960, were
found to have decreased in population size in 1998. This can be explained by a change in
either poultry farming practices or disease. So although modified live viruses are of great
value for the control of NCD, it is important to keep in mind that the vaccination strategies
can change viral diversity and population dynamics (8).
Despite the vaccination of poultry flocks in many countries, outbreaks of Newcastle disease
have been reported in vaccinated populations as for example in The Netherlands in 1992 and
1993 and the USA in 2002 (12,13). These outbreaks suggest that the virus can spread in
partially vaccinated populations. Either because the vaccination coverage level is to low or
that vaccination does not provide full immunity. Herd immunity in poultry can only be
achieved when more than 85% of the birds have a high antibody titer, because of the high
transmissibility in poultry with low antibody titers (13).
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
8
The vaccines that are used at the moment are not perfect and new NDV vaccines and
strategies should be developed, so these can be used during outbreak situations to protect
chickens from disease and infection and to reduce virus shedding (14).
In New Zealand it is prohibited to vaccinate poultry or wild birds against Newcastle disease.
Other disease control measures such as screening are being used (16).
Avian paramyxoviruses in New Zealand
So far no Newcastle disease outbreaks have occurred in New Zealand (17). Avian
paramyxoviruses can be introduced into New Zealand by migration of birds from other
countries, import of birds for commercial purpose or by the import of poultry or products that
originate from poultry. Waterfowl birds are key vectors for the introduction of APMV but
other bird species like godwits, plovers, terns and knots are also able to carry APMV.
Although non-pathogenic strains of APMV-1 and APMV-4 have been found in poultry and
wild ducks in New Zealand and antibodies against NCD are occasionally found in commercial
poultry flocks during the lay period with serological testing, a disease outbreak associated
primarily with APMV has not yet been confirmed (18). This might be explained by the fact
that in general species of waterfowl bird don’t migrate to New Zealand and that there are
strict quarantine protocols for the other species. Also the geographical isolation of New
Zealand, the strict biosecurity protocols and the low migration rate of birds helps prevent the
entry of APMV (15,19).
Some research has been done on APMVs in New
Zealand. To determine the presence of APMV type 1, 2
and 3 in caged birds and poultry in New Zealand a
research was done by Stanislawek et al. In this research
several blood samples from caged and wild bird at
different New Zealand regions, as shown in figure 2,
were investigated by haemagglutination inhibition test.
They concluded that APMV-1 is present in wild birds,
caged birds and poultry in New Zealand, however they
found that there is no evidence of the presence of
APMV-3 and that APMV-2 can’t be excluded (19).
To find out more about the APMV status in New
Zealand Stanislawek et al. did another research in which
they sampled 321 live mallard ducks and used several
methods to determine the presence and pathogenicity of
AMPV. Phylogenetic analysis of the isolated APMVs
showed that there is a close relationship between the
New Zealand’s APMV-1 isolate and the Australian
APMV-1 isolate, which indicates that introduction of
the virus does sometimes occurs trough migration or by
vagrant birds from Australia. A reservoir of the Figure 2: Map of New Zealand showing locations of
pathogenic APMV-1 was not isolated, but mutation may sampling sites and number of caged (C) and/or wild (W)
play a role in producing this virus. The position where birds sampled from December 1997 to April 1999 (19)
the mutation occurs and the way this works is not clear,
it is suggested that it is more reasonable that virulent strains develop in poultry after
introduction from wild birds. Whether or not these mutations occur depends on the genetic
pool of the virus and the management and health status of poultry. Luckily the presence of
APMV-1 in New Zealand’s poultry is low, which suggest that the opportunity for the virus to
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
9
enter the poultry and mutate is limited. Nevertheless, this study showed the presence of
APMV-1 in the mallard duck population that have a close relationship with viruses thought to
have mutated to virulence. This shows why it is important for New Zealand to develop a
safety biosecurity on poultry farms and keep monitoring the presence and type of APMV in
wild and domestic birds (9). Still, to precisely predict the APMV status in the New Zealand
bird population, more number of birds from a wider scale of species is necessary (6).
All APMV-1 that have been isolated in New Zealand so far were apathogenic and thus New
Zealand is regarded as free of the virulent form of NDV. However antibodies against NCD
are occasionally found in commercial poultry flocks during the lay period with serological
testing and APMV have been isolated from wild waterfowl in New Zealand (7). The New
Zealand poultry industry has an on-going interest in continuous monitoring of avian
paramyxoviruses present in New Zealand in order to assure that all circulating APMVs
continue to belong to lentogenic/apathogenic types.
In addition to the above, Massey University wants to do more research on apathogenic
APMV-1 that are currently circulating in New Zealand. During some random serological
testing on a poultry farm antibodies against NCD were found. This research tried to determine
whether there is a apathogenic APMV-1 subtype circulating at this poultry farm. This study
also had a look at whether there are apathogenic strains amongst backyard poultry in New
Zealand. The objective of this research is to characterize avian paramyxoviruses isolates that
are currently circulating in New Zealand.
Materials and methods
Sources and collection of samples
Blood samples
Blood samples were collected from birds at the poultry farm a vet through venipuncture of the
brachial vein to monitor the birds’ exposure to APMV-1 serologically. On the second
sampling (3 weeks after introduction) 10 blood samples were taken and at the third sampling
(5 weeks after introduction) 12 blood samples were collected for serological testing.
Cloacal and tracheal swabs
To isolate and further characterize the APMVs present in New Zealand two sources of
samples were used.
1. Samples collected from a commercial poultry flock with known exposure to APMV-1.
On this farm there were no clinical signs of disease but the farm was selected as it has
been serologically positive for APMV-1 in the past. Since there is no vaccination
against Newcastle disease in New Zealand, there may be some APMV-1 viruses
circulating on this farm. The samples used in this research were taken from a group of
seronegative layer birds at different time points after their introduction into this
APMV-seropositive farm. A total of 100 birds were sampled: 50 on the first sampling
(2 weeks after introduction to the farm), then 25 on the second sampling a week later
and 25 on the third sampling another 2 weeks later. From these birds paired tracheal
and cloacal swabs were collected by a vet for real-time reverse transcriptase PCR. The
samples were taken by inserting a cotton-covered stick into the cloaca or trachea.
These swabs were collected into a commercial viral transport media (Copan) and
transported to the laboratory on ice within 24 hours after collection. An aliquot of each
sample was used for RNA isolation for real time RT-PCR. The samples were frozen at
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
10
-70 ºC until the RNA extraction was done. In total 100 cloacal samples and 95 tracheal
samples were collected (5).
2. Samples from chickens and other domestic birds (e.g. ducks, turkeys, geese, pigeons
etc.) kept on small backyard farms (not more than 50 chickens). For this part of the
research samples were used that were stored after a research done by T. Zheng et al,
2010 (20) to investigate the presence of avian influenza in these flocks in New
Zealand and their potential transmission pathways. Backyard poultry on 24 farms in
the Bay of Plenty and regions of the North Island of New Zealand were identified
from the AgriBase database. The birds all lived in regions near major water bodies
because this are areas with high potential for transmission of viruses from wild
waterfowl and areas selected based on major migration routes. From these birds
esophageal, tracheal and cloacal swabs were collected and put in separate vials
containing 1.5 ml of virus transport medium (Eagle’s minimum essential medium).
The samples were kept on ice for transport and stored at -70°C. On this samples a
RNA extraction was already done and this was stored in the freezer until they are used
for real time RT-PCR to test them on APMV, in total 162 samples were tested (20).
RNA extraction
In total 95 cloacal and 94 tracheal samples were collected from the chickens at the poultry
farm, on these samples a RNA extraction was performed. Before starting with the samples
that were taken from the chickens, the most suitable method for RNA extraction had to be
determined. For this other viral samples that were stored in a freezer were used to test three
different methods for RNA extraction: 1.TRIzol ® LS Reagent, 2. TRIzol ® LS Reagent
combined with Qiagen RNeasy columns and 3. Qiagen RNeasy columns.
Method 1: TRIzol ® LS Reagent
For this RNA extraction method 0,25 ml of sample was added to 0,75 ml of TRIzol LS
Reagent. This was mixed by pipetting the sample up and down and then left at room
temperature for five minutes for homogenization. TRIzol LS Reagent is a mono-phasic
solution of phenol and guanidine isothiocyanate. This causes lysis of the cells and dissolves
cell components while the integrity of the RNA remains the same. After five minutes 0,2 ml
of chloroform was added and the sample was shaken for fifteen seconds followed by
incubation at room temperature for ten minutes. Then the samples were centrifuged at 12000
G for fifteen minutes at 4 ºC. After centrifugation the solution was separated into an upper
aqueous phase, an interphase and an organic phase, which contains the phenol as shown in
figure 3. The RNA is in the aqueous phase, while DNA and proteins are in the interphase. The
aqueous phase was then transferred into a new tube without disturbing the interphase. The
aqueous phase was mixed with 0,5 ml isopropylalcohol and incubated at room temperature for
ten minutes. Then the sample was centrifuged at 12000 G for ten minutes at 4 ºC. After
centrifuging the RNA forms a gel-like pellet on one side of the bottom of the tube as seen in
figure 4. Before collecting, the RNA needs to be washed. To do this the supernatant was
removed by one smooth movement without losing the pellet. This was followed by the
addition of 1 ml 75% ethanol and then mixed by vortexing. The tube was centrifuged at 7500
G for five minutes at 4 ºC. After the centrifuging the supernatant was removed and the pellet
was dried by the air for approximately fifteen minutes. When the pellet was dry the RNA was
dissolved in RNase-free water at incubated at 55 ºC for ten minutes (a,15).
This method takes a bit longer than column-based methods but it has a higher capacity and
can yield more RNA. In this method chloroform and phenol are used, since these reagents are
both hazardous the previous described steps were all performed in a airflow-hood.
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
11
Method 2: TRIzol ® LS Reagent combined with Qiagen RNeasy columns
For this RNA extraction method 0,75 ml of TRIzol LS Reagent was added to 0,25 ml of
sample (mixed by pipetting up and down) and then left at room temperature for five minutes.
Then 0,2 ml of chloroform was added and the sample is shaken vigorously for fifteen seconds
followed by incubation at room temperature for ten minutes. After this the samples were
centrifuged at 12000 G for fifteen minutes at 4 ºC this causes the separation of the solution
into an upper aqueous phase, an interphase and an organic phase, which contains the phenol
(shown in figure 3). 450 µl of the aqueous phase, which contains the RNA, was transferred to
a clean 1,5 ml tube and mixed with 450 µl 70% ethanol. Of this liquid 450 µl was transferred
to a RNeasy column and centrifuged at 8000 G for 15 seconds, the flow-through was
discarded and this was repeated with the other half of the liquid. Followed by the addition of
700 µl buffer RW1, and centrifuging for 15 seconds at 8000 G, the flow-through was
discarded. After this step 500 µl of the buffer RPE was added in the column and the sample
was centrifuged at 8000 G for 15 seconds, then the flow-through was discarded. Another 500
µl of buffer RPE was put in the column and then centrifuged at 8000 G for 2 minutes. To get
rid of the remaining buffer the column was placed in a new 2 ml tube and centrifuged for 1
minute at full speed. Then the column was placed into a new 1,5 ml tube and 30 µl RNasefree water was added, this was centrifuged for 1 minute at 8000 G. After this centrifugation
the RNA was in the liquid at the bottom of the 1,5 ml tube and the column was discarded
(a,b).
Figure 3: Phase separation (21)
Figure 4: Isopropanol precipitation (21)
Method 3:Qiagen RNeasy columns
In the last method 875 µl buffer RLT was added to 250 µl of sample and vortexed for one
minute. This causes disruption of the cells to release all the RNA in the sample.
Homogenization is necessary to reduce the viscosity of the lysates otherwise RNA binds
inefficiently to the column and yielded RNA is reduced. After vortexing, 625 µl 100% ethanol
was added to promote selective binding of RNA to the membrane of the column, then 600 µl
of the sample was transferred to a RNeasy column and centrifuged at 8000 G for 15 seconds.
After centrifugation the flow-through was discarded and this step was repeated two more
times. Then RNA washing was performed to wash away any contaminants, first 700 µl buffer
RW1 was added, and the columns were centrifuged for 15 seconds at 8000 G, the flowthrough was discarded. After this step 500 µl of the buffer RPE was added in the column and
this is centrifuged at 8000 G for 15 seconds and the flow-through was discarded. Another 500
µl of buffer RPE was put in the column and then centrifuged at 8000 G for 2 minutes. To get
rid of the remaining buffer the column was placed in a new 2 ml tube and centrifuged for 1
minute at full speed. Then the column was placed into a new 1,5 ml tube and 30 µl RNasefree water was added and this was centrifuged for 1 minute at 8000 G. After this
centrifugation the RNA is in the liquid at the bottom of the 1,5 ml tube and the column was
discarded (b).
Isolating APMV-1 in New Zealand poultry
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Quantification of the RNA
After the RNA extraction the samples were measured by a Nanodrop spectrophotometer. This
machine measures the absorbance of the samples and determines the concentration of RNA
(260), protein (230) and contamination (280) and the purity of the sample (260/280). To make
a quantification 1,5 μl of sample was loaded onto the optical pedestal. Then the
spectrophotometer is closed and the sample is measured, when the measurement was
completed the surface was wiped and this was repeated for the other samples.
RNA clean up
The samples that had a 260/280 below 1,7 were cleaned. For this the sample was adjusted
with RNase free water to a volume of 100 µl and 350 µl buffer RLT was added. This was
followed by the addition of 250 µl 100% ethanol. This was transferred to a spin column and
centrifuged for 15 seconds at 8000 G, the flow-through was discarded. Followed by the
addition of 500 µl buffer RPE and centrifuging for 15 seconds at 8000 G to wash the spin
column membrane, the flow-through was discarded. Then again 250 µl buffer RPE was added
and this was centrifuged for 2 minutes at 8000 G this dries the spin column membrane which
ensures that there was no carryover of ethanol. The spin column was placed in a new
collection tube and centrifuged at full speed for one minute, then the column was placed in a
new 1,5 ml tube and 30 µl RNase free water was added, this was centrifuged for 1 minute at
8000 G. After this the samples were measured by a spectrophotometer and then stored at -70
ºC until real time RT-PCR was performed (b).
Real time reverse transcriptase Polymerase Chain Reaction
For the detection of the APMV-1 a real time RT-PCR was performed. Before the samples that
were taken from the chickens were tested, a real time RT-PCR was done with a negative
control and two positive control samples of APMV-1 that were collected earlier in New
Zealand, APMV1/Mallard/NZ/1/97 and APMV1/Mallard/NZ/7/97. This was done to
determine whether the PCR cycle was right and if these isolates could be used as a positive
control. After this the poultry samples were tested for APMV-1.
In the performed real time RT-PCR two sets of primers and probes were used. Primers/probes
for the L gene (table 2) and the M-gene (table 3) were combined to get a multiplex assay to be
able to detect a broader range of APMV-1 isolates. The M-gene assay had a sensitivity of 103
copies detected and the L-TET assay had a higher sensitivity as this detected 102 copies of the
target gene (22).Real time RT PCR was performed with 2,0 μL of template RNA, 2,5 μl one
step forward mix, 0,2 μl of L+ 8738 primer, L-8847 primer, M+ 4100 primer and M- 4220
primer, 0,12 μl of both the L+8762 probe and the M+ 4169 probe, 0,5 μl of RT enzyme and
3,96 μl of H2O (c). In each run two positive (APMV1/Mallard/NZ/1/97 and APMV1/Mallard/
NZ/7/97) and two negative (H2O) controls were included.
Polymerase
Position AY626266
Sequence
L-TET Probe
L+8762
5’[TET]TGCCTGGTCACACAAGATCCGCCG[3BHQ_1] 3’
Sense primer
L+8738
TGTTGAAAAGAAGCTGCTAGGC
Antisense primer
L-8847
TGGACCATGAAGAGTGGAACC
Table 2: Primer and probe sequences for the L-TET assay (22)
Polymerase
Position AY626266
Sequence
M Probe
M+4169
5’[FAM]TTCTCTAGCAGTGGGACAGCCTGC[BHQ_1] 3’
Sense primer
M+4100
AGTGATGTGCTCGGACCTTC
Antisense primer
M-4200
CCTGAGGAGAGGCATTTGCTA
Table 3: Primer and probe sequences for the M-gene assay (22,23)
Isolating APMV-1 in New Zealand poultry
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To perform the PCR the RNA must first be reverse transcribed into cDNA. The real time RTPCR used in this research is a one-step RT-PCR, in which both reverse transcriptase and real
time PCR take place in the same tube. The real time RT-PCRs were performed using a real
time PCR cycler (Rotor gene Q). The reactions started with 8 minutes at 50 °C in which
cDNA synthesis takes place, followed by initial denaturation at 95 °C for 30 seconds. This
was followed by 45 cycles of: denaturation for 1 second at 95 °C, primer annealing at 56 °C
for 20 seconds and primer extension at 72°C for 1 second. Then there was a final extension at
40 °C for 30 seconds.
The probes that were used in this reaction were hydrolysis probes. The sequence specific
probes are labeled with a quencher dye on the 5’ end and a reporter dye on the 3’ end. When
the probe is intact the quencher reduces the fluorescence intensity of the reporter dye by
fluorescence resonance energy transfer. When the probe binds to the right target DNA it will
get degraded by the DNA polymerase during the PCR’s extension step. Due to the
degradation of the probe the reporter gets separated from the quencher dye which causes a
increase in the fluorescence emission as shown in figure 5. This increase of fluorescence
emission is detected by the real time PCR cycler (24).
Figure 5: Hydrolisis probe
principe (24)
Figure 6: Curve of a positive sample in a real time
RT-PCR (24)
If a sample is positive for APMV-1 it shows a curve that similar to the curve that is shown in
figure 6. This curve consists out of four major phases. The first phase is the linear ground
phase in which the amount of fluorescence is not higher than the background. The second
phase is the early exponential phase where the fluorescence emission is above the
background. Then there is a log-linear phase in which the product is doubling after every
cycle. The last phase is the plateau phase, it depends on the number of cycles whether this is
shown (24).
Agarose gel electrophoresis
Samples that were not clearly defined positive or negative after real time RT-PCR were
checked by running them at a gel electrophoresis. The gel was prepared from 40 ml 0,5 TBE
solution and 0,8 gram agarose LE powder which were heated for 1 minute in a microwave.
Followed by the addition of 2 μl ethyl-bromide. In the first well a DNA ladder (O’GeneRuler
TM
DNA Ladder mix) was loaded. Of each tested sample 5 μl was mixed with 1 μl dye (6x
Orange DNA Loading Dye) and this was loaded in a well in the gel. A positive control
(APMV1 isolate) and a negative control (H2O) were also included. Then the gel was run
trough electrophoresis for 30 min at 100 V. After this the products were photographed during
UV transillumination.
Isolating APMV-1 in New Zealand poultry
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Results
Serology
To monitor the birds’ exposure to APMV-1 serologically, blood samples were collected from
the chickens at the poultry farm. At the second time point 10 blood samples were collected
and at the third time point 12 blood samples were collected. These 22 samples were tested by
the poultry vet services and came out all negative for antibodies against APMV-1.
Quantification of the RNA
To determine the best method for RNA extraction three different methods were tried before
extraction of the poultry samples was done. These methods were compared by measuring
them in a spectrophotometer, especially the RNA purity (260/280 ratio) was important and
this should ideally be between 1,8 and 2,2.
The results from the spectrophotometer of the samples extracted by the first method (TRIzol
® LS Reagent) are shown in table 3. As shown in this table all samples had a 260/280
between 1,8 and 2,2 but the 260/230 of these samples were all low (ideally between 1,8-2,2).
Sample number Concentration (ng/ul) A 260
A 280
260/280 260/230
PI 3 (1)
88,96
2,224
1,189
1,87
0,52
PI 3 (2)
50,46
1,262
0,657
1,92
0,66
Coronavirus
27,26
0,681
0,384
1,77
0,09
CDV
42,24
1,056
0,570
1,85
0,29
Table 3: Results from the samples that were extracted using method 1
Table 4 shows the results of the samples extracted by the second method (TRIzol ® LS
Reagent combined with Qiagen RNeasy columns). With this method again there were low
260/230 ratio’s, the 260/280 ratio’s higher then with the first method but the 260/280 of
ERBV(1) was too high.
Sample number Concentration (ng/ul) A 260
A 280
260/280 260/230
ERAV
16,34
0,409
0,201
2,04
0,35
ERBV (1)
5,98
0,150
0,040
3,73
0,01
ERBV (2)
6,97
0,174
0,073
2,37
0,02
Table 4: Results from the samples that were extracted using method 2
The results measured by the spectrophotometer of the samples extracted by the third method
(Qiagen RNeasy columns) are shown in table 5. The samples extracted by this method also
had a low 260/230, but the 260/280 were fine.
Sample number Concentration (ng/ul) A 260
A 280
260/280 260/230
CDV (1)
10,90
0,272
0,127
1,99
0,06
CDV (2)
44,50
1,112
0,590
1,89
0,29
Table 5: Results from the samples that were extracted using method 3
Isolating APMV-1 in New Zealand poultry
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After RNA extraction of the cloacal and tracheal samples that were taken from the chickens at
the poultry farm, these samples were also measured by a spectrophotometer. These samples (n
= 198) had a mean 260/230 of 0,45 (0,01-4,14) and a mean 260/280 of 2,16 (1,49-4,01). The
full results of the measurements by the spectrophotometer are shown in attachment 1 and 2.
Determining the positive control for real time RT-PCR
The samples taken from the chickens at the farm and the backyard poultry were examined by
real time RT-PCR. Before this was done, a positive control had to be found. For this two
isolates that were isolated in New Zealand by Stanislawek et al. were tested with the M-gene
assay and the L-TET assay. The results of the real time RT-PCR with the M probe are shown
in figure 7 with on the X-axis the number of cycles and on the Y-axis the amount of
fluorescence. This figure shows that the two isolates that were isolated in New Zealand earlier
(APMV1/Mallard/NZ/1/97 and APMV1/Mallard/ NZ/7/97) were both tested positive with the
M-gene assay. The curve of the two positive samples clearly shows a early exponential and a
log-linear phase the negative control that was included didn’t show these two phases and was
tested negative. The two isolates were also tested with the L probe real time RT-PCR, the
results of this test are shown in figure 8. In this figure there is no clear distinction between the
two positive samples and the negative sample. These two samples were tested again with the
L-TET assay and the results were similar.
Isolating APMV-1 in New Zealand poultry
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Real time RT-PCR on the samples from the backyard poultry
In total 63 tracheal, 50 esophageal and 49 cloacal samples were tested using a M-probe and a
L-probe real time PCR. The two isolates described earlier were used as positive controls.
M-gene assay results
In the first real time RT-PCR that was performed 59 tracheal samples that were collected form
the backyard poultry were tested. The raw results of the M-gene assay are shown in figure 9.
In this figure the red and the green line both rise more than the other samples, and these two
samples show a early exponential and a log-linear phase. These two samples represented the
positive controls, the red line is APMV1/Mallard/1/97 and the green line is
APMV1/Mallard/7/97. The end point data of this real time RT-PCR cycle are shown in figure
10, this shows that only the two positive control samples were tested positive with this Mgene assay.
Figure 10: End data results of tracheal samples collected from the backyard poultry
1 and 2 are the two positive controls
3 and 63 are the two negative controls
4-28: Tracheal 1 – Tracheal 25
29- 62: Tracheal 38 – Tracheal 71
Isolating APMV-1 in New Zealand poultry
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The second real-time RT-PCR cycle was performed with 4 tracheal and 47 esophageal
samples collected from the backyard poultry. The raw results of the M-gene assay are shown
in figure 11, is this figure only the red and the green line rise. These two samples were the
positive controls. In figure 12 the end point data are drawn, here only the first (APMV1/
Mallard/NZ/1/97) and the second sample (APMV1/Mallard/NZ/7/97) are above the threshold
(red line) and so only these two samples are tested positive.
Figure 12: End data results of M-gene assay of tracheal and esophageal samples collected from the backyard poultry
1 and 2 are the two positive controls
3 and 55 are the two negative controls
4-7: Tracheal 72 – Tracheal 75
8- 54: Esophageal bp 1 – Esophageal bp 47
The raw results of the M-gene assay performed on 3 esophageal and 49 cloacal samples taken
from the backyard poultry are shown in figure 13. In this figure again only the red and the
green line rise. Figure 14 shows the end point data of these samples, again only the first two
samples are drawn above the threshold and so these were tested positive. These samples were
the two positive controls. All the tracheal, esophageal and cloacal samples that were taken
from the backyard poultry were tested negative with the M-gene assay.
Isolating APMV-1 in New Zealand poultry
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Figure 14: End data results of M-gene assay of esophageal and cloacal samples collected from the backyard poultry
1 and 2 are the two positive controls
3 and 57 are the two negative controls
4-6: Esophageal bp 48 – Esophageal bp 50
7- 56: Cloacal bp 1 – Cloacal bp 49
L-TET assay results
The results of the real time RT-PCR with the L-probe/primers are shown in figures 15,16 and
17. All the measured samples show a small increase in the amount of fluorescence but there is
not a clear early exponential phase followed by a log-linear phase. Also the two NZ’s isolates
that were included as positive controls don’t show this curve. It was tried to make a end point
data figure, but this graph could not be displayed because the negative controls were either at
the same level or above the negative control. All samples tested with the L-probe, including
the positive controls, were tested negative.
Isolating APMV-1 in New Zealand poultry
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Isolating APMV-1 in New Zealand poultry
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Real time RT-PCR on the samples from the chickens at poultry farm
The samples that were taken from the chickens at the poultry farm at the third time point (five
weeks after introduction to the farm) were tested with the M-gene assay and the L-TET assay.
Figure 18 shows the raw results of the real time RT-PCR with the M-probe, this shows that
only the two positive control samples (red and green) show a early exponential and a loglinear phase, the other tested samples do not show this. In figure 19 the end point data are
drawn, this shows that all samples taken from the chickens were tested negative and that the
two positive controls were tested positive.
Isolating APMV-1 in New Zealand poultry
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Figure 19: End data results of M-gene assay of cloacal and tracheal samples collected from chickens at the poultry
farm 5 weeks after introduction to the farm
1 and 2 are the two positive controls
3 and 49 are the two negative controls
4-29: Cloacal 76 – Cloacal 100
7- 56: Tracheal 76 – Tracheal 95
The results of the real time RT-PCR with the L probe are shown in figure 15, in this figure all
samples show a slow linear increase in the amount of fluorescence on this scale, but none of
the samples shows a clear early exponential and a log-linear phase. In this figure there is also
no distinction between the positive and negative controls, all samples were tested negative.
Again it was not possible to make a end point graph.
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
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Agarose gel electrophoresis
After the first real time RT-PCR there were a view samples that were not clearly defined
positive or negative. These samples were run in a gel electrophoresis to see if they were
positive on APMV-1. Figure 21 shows that all of these samples have a band, which is formed
by the primers but only the two positive control samples had a second band. In this gel only
the two positive controls were positive and the other samples were negative. Since this first
real time RT-PCR round was preformed with 1 µl of template, all the samples were measured
again (these results are shown in figures 8 and 11) and the results were all negative (the figure
of the first PCR that was preformed with 1 µl of template is not included in this paper).
Figure 21: results of the gel
electrophoresis
A: DNA ladder
1: Tracheal 21
2: Tracheal 22
3: Tracheal 23
4: Tracheal 45
5: Tracheal 46
6: Tracheal 47
7: Tracheal 48
8: Tracheal 49
9: Tracheal 50
10: Tracheal 51
11: Tracheal 52
12: APMV1/Mallard/NZ/1/97
13: APMV1/Mallard/NZ/7/97
14: Negative control (H2O)
Discussion
So far there has not been an outbreak of Newcastle Disease in New Zealand. For this reason
there has not been a lot of research done on APMV-1 isolates that circulate in New Zealand.
The aim of this study was to isolate and characterize the New Zealand isolates of AMPV-1.
The study was designed to maximize the chance of detecting APMV-1, if the birds get
infected shortly after introduction to the farm. The power analysis was not relevant for the
study, as birds with different clinical outcomes were not compared, but only used to obtain
viral isolates for further characterization.
Method for RNA extraction
To determine the best method for the extraction of RNA the measurements from the
spectrophotometer of the samples were compared. As shown in tables 3, 4 and 5 the RNA
concentrations of the different samples differ a lot but since the extractions were all done on
different starting samples these were not compared in this research. The 260/280 of all
samples from all three of the different methods were above 1,8, except one sample extracted
by the first method with a 260/280 of 1,77. The tables also show low 260/230 ratio in all three
of the methods, the 260/230 were also low in the poultry farm samples as shown in
attachment 1 and 2. The reason for the low 260/230 wasn’t determined, the most reasonable
explanation is that the low 230/260 is caused by contamination, even though it is not so likely
that this occurred in so many samples (180 of 189 tracheal and cloacal poultry samples had a
230/260 below 1,7) and in all three methods. Possibly the 260/230 was low because of the
low RNA concentration of the samples, since it seemed that some samples with a higher RNA
concentration also had a higher 260/230. But this is not so likely since there were samples
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
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with a high concentration and a low 260/230 and also samples with a low RNA concentration
and a relatively high 260/230. A real time RT-PCR was done at a positive control sample that
also had a low 260/230 to see if this affected the outcome, the low 260/230 did not affect the
results of this PCR. For this reason it was decided that a low 260/230 was no problem for the
following real time RT-PCR. Based on the tests done in this research a good comparison
between the three methods can’t be made. To do so a lot more samples should be tested and
they should all have the same starting samples with the same amount of RNA and the same
virus to compare them. It is also important to add a control sample, of which the RNA
concentration is known, to determine the efficiency of the RNA extraction. In this research
this was not the main subject, all three methods were tried to see what would be the best
method to do the RNA extraction on the poultry samples. Since the 260/280 and 260/230 ratio
did not differ a lot between the three tested methods, in this research the fastest and easiest
method was chosen. For this reason the RNA extraction on the samples collected from the
chickens was performed using the Qiagen RNeasy columns (method 3).
Real time RT-PCR
The original plan was to measure all the cloacal and tracheal samples that were taken from the
chickens at the poultry farm. But since the serology of the blood samples that were taken from
22 chickens at two different time points (3 and 5 weeks after introduction of the chickens on
the farm) were all negative for antibodies against APMV-1, it is reasonable to assume that the
tracheal and cloacal samples taken at the first and second time points were negative. For this
reason it was decided not to measure the cloacal and tracheal samples that were taken at the
first two time points. The samples that were taken from the backyard poultry (n = 162) and
the tracheal and cloacal samples that were taken at the third time point from the chickens at
the poultry farm (n = 45) were tested by real time RT-PCR.
M-gene assay
Before the samples could be tested by real time RT-PCR, a positive control had to be found.
To do so APMV1/Mallard/NZ/1/97 and APMV1/Mallard/NZ/7/97 isolates that were isolated
in New Zealand earlier were tested. Both isolates were tested positive on the M-probe real
time RT-PCR (see figure 7) and so these were suitable as a positive control for this test.
L-TET assay
The two positive isolates were also tested with the L-TET assay, this is shown in figure 8. In
this figure it is not possible to distinguish between the negative control and the two APMV-1
isolates. Because the two isolates were not tested positive these can’t be used as a positive
control for the L-TET assay. Because there are not many APMV-1 isolated in New Zealand it
was not possible to test more APMV-1 isolates. Since the M-gene assay and the L-TET assay
could be performed at the same time in the same tube it was decided to also do the L-probe,
even though there were no positive controls. If a sample would be tested positive with the LTET assay this sample might be used as a positive control for this test. All the poultry samples
that were tested were negative, this can be explained in three ways. The first explanation is
that all tested samples really were negative, this is possible since the samples were all tested
negative with the M-probe real time RT-PCR as well. On the other hand it is also possible that
the L-probe did not work, this can be caused by a difference in sequence between the probe
and/or primers and the isolate sequence wherefore the increase in fluorescence doesn’t take
place. This is hard to say because there were no positive controls for this test, since the two
APMV-1 isolates were tested negative by this reaction this is also a likely explanation. It is
also possible that the RT-PCR cycle wasn’t right, and that this has to be adjusted. To
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
24
determine why the results from the L-probe real time RT-PCR were all negative more
different APMV-1 isolates should be tested.
Samples that were taken from the backyard poultry and the poultry farm
All the poultry samples that were collected from the backyard poultry and the chickens of the
poultry farm were tested negative with both the M-probe and the L-probe real time RT-PCR.
Because the positive controls were tested positive in all the M-gene assays it is likely that the
tested poultry samples really were negative on APMV-1. Off course it is still possible that
some of the samples were false negative, since previous researches have shown that the Mgene assay did not detect all low virulent isolates. In previous researches it was shown that the
M-gene assay detects 103 copies of the target gene. Samples that contained less than 103
copies of the virus were tested (false) negative in this assay. To see if the samples really were
negative a consensus PCR could be performed which detects a wider range of
paramyxoviruses. The L-TET assay has a higher sensitivity because it can detect 102 copies of
the target gene. All samples that were tested with the L-TET assay were tested negative. Since
there were no positive controls and it was not sure if the L-TET assay worked no conclusions
can be drawn from the results of the L-TET assay.
In this research 162 samples were tested that were collected from backyard poultry that had a
high risk for infection with APMV-1. These birds were selected in 24 areas near major bodies
across New Zealand were they could easily be in contact with wild waterfowls, which can
function as a reservoir for APMV1. These areas were also selected based on the major
migrating routes of wild waterfowl. From this study it can be concluded that APMV-1 is not
very common in New Zealand.
Since there were no positive samples it was not possible to characterize any isolates. The
continuous monitoring of avian paramyxoviruses present in New Zealand is very important, in
order to assure that all circulating APMVs continue to belong to lentogenic/apathogenic types.
For this reason more research to characterize the APMV-1 isolates that are circulating in New
Zealand should be done. In following research it is important to also take samples at later time
points after the introduction of the chickens to the farm, to see if there still is a apathogenic
strain circulating at the poultry farm blood samples from the introduced chickens should be
taken for serology after a view months. To see if the chickens have antibodies against APMV1, if they do it is likely that there still is a apathogenic strain at this poultry farm and more
research should be done. In following researches it is important to get a working positive
control for the L-TET assay, to be able to detect a broader range of APMV-1 viruses.
Acknowledgement
The author wishes to thank Dr. Nick Cave for all his time and effort to provide a great
research opportunity at Massey University and all his enthusiasm in providing this research.
Many thanks to Dr Herman F. Egberink for all his support and great advise, and also for
having a critical review on the manuscript.
Also many thanks to Dr. Magda Dunowska for all her help with the RNA extractions and real
time RT-PCR. Also for all her time, patience and great advise about this research.
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
25
Thanks also to Drs. Alison Stickney for showing us around on Massey University and making
us feel at home. Also thanks for all her patience in explaining an teaching the many research
methods we learned.
A special thank to Drs. Babette A. Ravensbergen, for helping me with my research, reading
my manuscript and all her support.
I also would like to thank Dr. Tao Zheng for giving me his stored samples that were taken
from the backyard poultry. And to Dr. Neil Christnsen for collecting all the samples from the
chickens at the poultry farm.
Last of all I would like to thank Massey University for giving me the opportunity to do
research at this university.
Sources and manufacturers
a. TRIzol ® LS Reagent, Invitrogen Corp., Carlsbad CA.
b. RNeasy ® Mini Kit, Qiagen Inc. Valencia, CA
c. 1-Step Fast qRT-PCR Kit, Quanta Biosience Inc.
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paramyxoviruses. In: L. Dufour-Zavala (Ed) A laboratory manual for the isolation,
identification, and characterization of avian pathogens (fifth edition). The American
Association of Avian Pathologists.
3. Organization of International Epizooties (2004). Chapter 2.1.15. Newcastle Disease
In: Manual of Diagnostic Test and Vaccines for Terrestrial Animals (mammals, birds
and bees) (fifth edition) World Organization for Animal Health
4. B.S. Seal, D.J. King and H.S. Sellers (2000). The avian response to Newcastle
disease virus. Developmental Comparative Immunology 24, pp 257-268
5. D.J. Alexander, J.G. Bell and R.G. Alders (2004). A technology review: Newcastle
disease, with special emphasis on its effect on village chickens. Food and
agriculture organization of the united nations (FAO), pp 1-22
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The American Association of Avian Pathologists. pp 55-59
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Drs. C. Reijne
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10. O.S. de Leeuw, G. Koch, L. Hartog, N. Ravenshorst and B.P. Peeters (2005).
Virulence of Newcastle disease virus is determined by the cleavage site of the
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haemagglutinin-neuraminidase protein. Journal of General Virology 86, pp17591769
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Quantitative Basic Residue Requirements in the Cleavage-Activation Site of the
Fusion Glycoprotein as a determinant of virulence for Newcastle disease virus.
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paramyxoviruses, and pneumovirus infections. In: Y.M. Saif, A.M. Fadly, J.R.
Glisson, L.R. McDougald, L.K. Nolan and D.E. Swayne (Ed) Diseases of Poultry.
Iowa State University pp75-116
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Herd immunity to Newcastle disease virus in poultry by vaccination. Avian
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14. D.R. Kapczynski and D.J. King (2005). Protection of chickens against overt clinical
disease and determination of viral shedding following vaccination with
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highly virulent virus from the California 2002 exotic Newcastle disease outbreak.
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Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
27
Attachment 1: Results from the spectrophotometer of the cloacal samples
Sample number Concentration (ng/ul) A 260
A 280
260/280 260/230
APMV C1
8,68
0,217
0,111
1,95
0,34
APMV C2
49,64
1,241
0,598
2,07
0,56
APMV C3
52,21
1,305
0,61
2,14
0,41
APMV C4
10,22
0,256
0,136
1,88
0,13
APMV C5
89,44
2,236
1,063
2,1
0,25
APMV C6/11
13,04
0,326
0,169
1,93
0,003
APMV C7/12
99,5
2,487
1,201
2,07
0,43
APMV C8/13
33,8
0,845
0,382
2,21
0,22
APMV C9/14
13,03
0,326
0,153
2,13
0,4
APMV C10/15
38,02
0,95
0,463
2,05
1,45
APMV C16
25,71
0,643
0,333
1,93
0,35
APMVC 17
123,81
3,095
1,484
2,09
1,09
APMV C18
49,29
1,232
0,583
2,12
0,24
APMV C19
32,633
0,816
0,401
2,03
0,3
APMV C20
51,52
1,288
0,634
2,03
1,05
APMV C21
34,82
0,871
0,384
2,26
0,08
APMV C22
44,97
1,124
0,59
1,9
0,14
APMV C23
34,37
0,859
0,408
2,1
0,91
APMV C24
236,37
5,909
2,845
2,08
2,18
APMV C25
36,43
0,911
0,439
2,07
0,26
APMV C26
114,28
2,857
1,382
2,07
1,74
APMV C27
57,5
1,437
0,682
2,11
0,58
APMV C28
43,22
1,08
0,527
2,05
0,88
APMV C29
326,76
8,169
3,902
2,09
1,02
APMV C30
61,84
1,546
0,747
2,07
2,32
APMV C31
71,8
1,795
0,862
2,08
2,13
APMV C32
7,32
0,183
0,123
1,49
0,03
APMV C33
15,63
0,391
0,184
2,12
1,1
APMV C34
144,53
3,613
1,77
2,04
2,17
APMV C35
23,68
0,592
0,286
2,07
0,07
APMV C36
21,25
0,531
0,252
2,11
0,2
APMV C37
9,33
0,233
0,126
1,86
0,17
APMV C38
363,38
9,084
4,366
2,08
2,25
APMV C39
14,48
0,362
0,179
2,03
1,2
APMV C40
74,56
1,864
0,891
2,09
2,1
APMV C41
12,39
0,31
0,155
1,99
0,53
APMV C42
145,3
3,633
1,742
2,09
0,95
APMV C43
30,49
0,762
0,371
2,06
0,08
AOMV C44
15,03
0,376
0,199
1,89
0,66
APMV C45
9,44
0,236
0,097
2,44
0,55
APMV C46
19,98
0,499
0,204
2,45
0,04
APMV C47
2,99
0,075
0,019
4,01
0,04
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
28
APMV C48
APMV C49
APMV C50
APMV C51
APMV C52
APMV C53
APMV C54
APMV C55
APMV C56
APMV C57
APMV C58
APMV C59
APMV C60
APMV C61
APMV C62
APMV C63
APMV C64
APMV C65
APMV C66
APMV C67
APMV C68
APMV C69
APMV C70
APMV C71
APMV C72
APMV C73
APMV C74
APMV C75
APMV C76
APMV C77
APMV C78
APMV C79
APMV C80
APMV C81
APMV C82
APMV C83
APMV C84
APMV C85
APMV C86
APMV C87
APMV C88
APMV C89
APMV C90
APMV C91
APMV C92
27,18
71,04
54,41
5,93
16,91
11,79
13,55
5,09
15,78
33,9
4,61
26
13,03
13,68
9,16
5,53
6,27
28,58
5,19
10,27
42,5
5,65
3,94
20,35
11,57
6,71
9,75
3,82
7,28
4,45
4,65
19,13
14,46
15,5
5,49
6,93
9,31
12,57
12,53
10,97
5,55
10,76
11,7
14,68
4,85
0,679
1,776
1,36
0,148
0,423
0,295
0,339
0,127
0,395
0,848
0,115
0,65
0,326
0,342
0,229
0,138
0,157
0,715
0,13
0,257
1,062
0,141
0,098
0,509
0,289
0,168
0,244
0,095
0,182
0,111
0,116
0,478
0,361
0,388
0,137
0,173
0,233
0,314
0,313
0,274
0,139
0,269
0,292
0,367
0,121
0,43
0,838
0,625
0,052
0,187
0,135
0,138
0,058
0,156
0,386
0,054
0,297
0,129
0,169
0,101
0,057
0,079
0,331
0,073
0,123
0,508
0,063
0,061
0,234
0,133
0,066
0,088
0,051
0,082
0,044
0,045
0,227
0,172
0,161
0,062
0,083
0,124
0,147
0,149
0,14
0,069
0,122
0,132
0,177
0,061
1,58
2,12
2,18
2,84
2,27
2,18
2,45
2,21
2,53
2,2
2,14
2,19
2,53
2,03
2,26
2,44
1,98
2,16
1,77
2,09
2,09
2,23
1,61
2,17
2,18
2,53
2,78
1,88
2,22
2,55
2,57
2,11
2,11
2,4
2,32
2,1
1,87
2,14
2,1
1,96
2,01
2,2
2,21
2,07
2
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
0,42
0,2
0,27
0,01
1,05
0,16
0,03
1,32
0,05
0,19
0,05
1,59
0,11
0,51
1,07
0,22
2,41
0,68
1,71
4,14
0,73
0,42
0,06
1,61
2,17
0,08
0,02
0,03
0,12
0,07
0,01
1,36
0,85
0,03
0,01
0,04
1,46
0,42
0,17
0,32
0,02
0,43
0,04
1,02
0,21
29
APMV C93
APMV C94
APMV C95
APMV C96
APMV C97
APMV C98
APMV C99
APMV C100
47,76
17,58
9,96
23,52
39,77
7,56
7,15
18,9
1,194
0,44
0,242
0,588
0,994
0,189
0,179
0,473
0,775
0,234
0,13
0,298
0,591
0,097
0,105
0,259
1,54
1,88
1,86
1,97
1,68
1,94
1,71
1,83
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
0,5
0,13
1,05
0,6
0,43
0,04
0,58
0,42
30
Attachment 2: Results from the spectrophotometer of the tracheal samples
Sample number Concentration ng/ul A 260
A 280
260/280 260/230
APMV T1
55,19
1,38
0,791
1,75
0,18
APMV T2
7,36
0,184
0,093
1,97
0,02
APMV T3
6,67
0,167
0,08
2,1
0,03
APMV T4
6,15
0,154
0,089
1,73
0,12
APMV T5
5,71
0,143
0,072
1,98
0,32
APMV T6
13,28
0,332
0,163
2,03
0,83
APMV T7
4,49
0,112
0,065
1,73
0,02
APMV T8
3,72
0,093
0,037
2,53
0,02
APMV T9
35,28
0,882
0,568
1,55
0,41
APMV T10
5,73
0,143
0,078
1,83
1,1
APMV T11
1,74
0,044
0,023
1,9
0,06
APMV T12
8,42
0,21
0,208
1,96
0,63
APMV T13
5,13
0,128
0,061
2,1
0,01
APMV T14
5,4
0,135
0,068
1,97
0,03
APMV T15/16
6,53
0,163
0,065
2,5
0,01
APMV T 17
5,1
0,128
0,06
2,11
0,06
APMV T 18
5,34
0,134
0,054
2,46
0,12
APMV T19
7,88
0,197
0,093
2,11
0,19
APMV T20
3,33
0,083
0,048
1,75
0,41
APMV T21
2,74
0,068
0,031
2,24
0,04
APMV T22
22,81
0,57
0,358
1,59
0,23
APMV T23
3,99
0,1
0,046
2,17
0,46
APMV T24
4,41
0,11
0,06
1,82
0,08
APMV T25
5,24
0,131
0,04
3,29
0,01
APMV T26
2,47
0,062
0,02
3,13
0,01
APMV T27
5,19
0,13
0,054
2,4
0,02
APMV T28
4,41
0,11
0,061
1,81
0,4
APMV T29
5,47
0,137
0,075
1,82
0,51
APMV T30
1,1
0,028
0,013
2,05
0,01
APMV T31
4,93
0,123
0,063
1,96
0,41
AOMV T32
7,94
0,198
0,117
3,67
0,02
AOMV T33
7,94
0,198
0,117
1,7
0,48
AOMV T34
9,28
0,232
0,114
2,03
0,64
APMV T35
7,12
0,178
0,098
1,81
0,28
APMV T36
7,93
0,198
0,117
1,7
0,85
APMV T37
12,18
0,304
0,145
2,1
0,17
APMV T38
8,17
0,204
0,117
1,74
1,17
APMV T39
3,69
0,092
0,054
1,7
0,07
APMV T40
1,72
0,043
0,015
2,9
0,01
APMV T41
4,09
0,102
0,055
1,87
0,33
APMV T42
3,74
0,094
0,054
1,75
0,09
APMV T43
4,05
0,101
0,041
2,44
0,45
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
31
APMV T44
APMV T45
APMV T46
APMV T47
APMV T48
APMV T49
APMV T50
APMV T51
APMV T52
APMV T53
APMV T54
APMV T55
APMV T56
APMV T57
APMV T58
APMV T59
APMV T60
APMV T61
APMV T62
APMV T63
APMV T64
APMV T65
APMV T66
APMV T67
APMV T68
APMV T69
APMV T70
APMV T71
APMV T72
APMV T73
APMV T74
APMV T75
APMV T76
APMV T77
APMV T78
APMV T79
APMV T80
APMV T81
APMV T82
APMV T83
APMV T84
APMV T85
APMV T86
APMV T87
APMV T88
9,44
5,13
6,53
1,43
14,08
10,5
6,88
9,51
7,03
5,45
8,72
3,52
10,46
27,47
30,95
5,7
4,62
8,02
3,78
3,58
5,02
5,88
5,18
7,92
6,26
4,35
5,07
8,55
5,64
5,1
4,09
7,5
6,51
5,1
3,23
8,8
6,71
9,43
4,27
3,29
7,8
10,93
8,33
5,04
6,64
0,236
0,128
0,163
0,036
0,352
0,263
0,172
0,238
0,176
0,136
0,218
0,088
0,262
0,687
0,774
0,142
0,115
0,2
0,095
0,089
0,126
0,147
0,129
0,198
0,157
0,109
0,127
0,214
0,141
0,127
0,102
0,188
0,163
0,127
0,081
0,22
0,168
0,236
0,107
0,082
0,195
0,273
0,208
0,126
0,166
0,118
0,058
0,091
0,011
0,22
0,099
0,053
0,132
0,094
0,062
0,116
0,048
0,131
0,427
0,484
0,066
0,04
0,115
0,036
0,024
0,062
0,069
0,047
0,093
0,083
0,052
0,065
0,089
0,065
0,04
0,055
0,075
0,088
0,069
0,047
0,085
0,087
0,086
0,041
0,04
0,108
0,151
0,11
0,043
0,063
2
2,22
1,79
3,14
1,6
2,66
3,26
1,8
1,87
2,2
1,88
1,84
2
1,61
1,6
2,17
2,89
1,75
2,66
3,68
2,04
2,12
2,76
2,14
1,88
2,11
1,96
2,41
2,16
3,17
1,87
2,49
1,85
1,85
1,71
2,59
1,92
2,74
2,61
2,04
1,81
1,81
1,9
2,91
2,62
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
0,05
0,01
0,15
0,01
0,32
0,03
0,02
0,02
0,17
0,23
0,41
0,35
0,26
0,42
0,43
0,02
0,01
0,35
0,06
0,02
0,08
0,51
0,35
0,86
0,56
0,03
0,06
0,05
0,01
0,02
0,33
0,32
0,2
0,36
0,04
0,03
0,31
0,02
0,39
0,01
0,18
0,83
0,48
0,01
0,09
32
APMV T89
APMV T90
APMV T91
APMV T92
APMV T93
APMV T94
APMV T95
9,05
5,88
3,6
11,68
14,27
8,44
6,6
0,226
0,147
0,09
0,292
0,357
0,211
0,165
0,102
0,048
0,044
0,148
0,215
0,081
0,065
2,22
3,09
2,02
1,98
1,66
2,6
2,41
Isolating APMV-1 in New Zealand poultry
Drs. C. Reijne
0,19
0,01
0,04
0,47
0,39
0,02
0,01
33