Download Effect of lentogenic Newcastle disease virus (Lasota) on low

Journal of Avian Research
RESEARCH ARTICLE
open access
Effect of lentogenic Newcastle disease virus (Lasota) on low pathogenic avian
influenza virus (H9N2) infection in fayoumi chicken
1
Sajid Umar , Tamoor Azeem2*, Salman Ahmed Abid2, Aqsa Mushtaq3, Kiran Aqil3, Muhammad Rizwan Qayyum3, Abdul
Rehman4
1
National Veterinary school Toulouse, France, 2 Department of Pathology, University of Veterinary and Animal Sciences, Lahore, Pakistan. 3 Veterinary
Research Institute, Lahore, Pakistan. 4Friedrich-Loeffler-Institute of Epidemiology Berlin, Germany
Abstract: Low pathogenicity avian influenza virus (LPAIV) and lentogenic Newcastle disease virus (lNDV) are are two of the most
economically important viruses affecting poultry worldwide. Co-infections usually occur but cannot be easily diagnosed due to
confusing similar clinical signs. Fayoumi is indigenous chicken of Pakistan on which the impact of co-infections is still unknown. The
objective of this study was to investigate the effect of lNDV on the infectivity and excretion of LPAIV in fayoumi chicken. Four week
old fayoumi chicks were inoculated intranasally with 106 median embryo infectious of lNDV vaccine strain (LaSota) and a H9N2
LPAIV (A/Chicken/Pakistan/UDL/08 H9N2) simultaneously. No clinical signs were observed in chickens infected with the lNDV. All
chicken showed mild to moderate respiratory distress with LPAIV alone or in combination with lNDV. Clinical and necropsy findings
revealed non synergistic behaviour of two viruses for the development of clinical signs and lesions. However, the pattern of virus
shed was different with co-infected chickens, which excreted lower titres of lNDV and LPAIV at first three days post inoculation (dpi)
as compared to singly inoculated chicken but after 3 dpi co-infection resulted in significantly higher number of oropharangeal and
cloacal swabs detected positive for LPAIV and lower number for lNDV. The knowledge obtained from the study serves the dual
purpose of shedding light on the different replication behaviours of LPAIV in early days of experiment which may be due to
competition for receptor binding with lNDV, as well as the more pathogenic behaviour of LPAIV (H9N2) in fayoumi chickens of
Pakistan.
Key words: Poultry, LPAIV, NDV, co-infection
Citation: Umar S, Azeem T, Abid AS, Mushtaq A, Kiran A, Qayyum MR, Rehman A. Effect of lentogenic Newcastle disease virus (Lasota) on low pathogenic
avian influenza virus (H9N2) infection in fayoumi chicken. J Avian Res, 2015, 1(1):1-4
Received: Jan 01, 2015; Revised: Feb 28, 2015; Accepted: Mar 11, 2015
Copyright: 2015 Umar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Competing Interests: The authors have declared that no competing interests exist.
*Corresponding author: Tamoor Azeem; Email: [email protected]
Introduction
Newcastle disease virus (NDV) and Avian influenza virus (AIV)
have been great threat to poultry industry and have caused great
economical losses by decreasing egg and meat production. These
viruses originated from wild birds and then transmitted to domestic
poultry birds producing mild to severe clinical infections of
respiratory tract. Both of these are negative-sense single stranded,
RNA viruses.
NDV belong to Paramyxoviridae family and also known as avian
Paramyxovirus. Based on virulance and pattern of sequences
present near protease cleavage site of fusion (F) protein NDV have
been catogaorized into different pathotypes producing diseases of
different types and severity in chicken, those are viscerotropic
velogenic, neurotropic velogenic, mesogenic, lentogenic or
asymptomatic [1].
AIV are member of Orthomyxoviridae family and also known as
orthomyxovirus. Avian influenza viruses are classified as low
pathogenic (LP) and high pathogenic (HP) viruses depending upon
the presence of multiple basic amino acids at cleavage site of the
HA precursor protein and virulence in chicken [2]. Diseases
produced by virulent strains of NDV (velogenic and mesogenic)
and highly pathogenic avian influenza (HPAI) viruses are notifiable
to the World Organization for Animal Health [3]. Lentogenic
strains of NDV are mostly used as live vaccines for the protection
virulent Newcastle disease virus (vNDV) infections in poultry.
These lentogenic or vaccine strains of NDV are known to cause
little or mild respiratory distress in specific pathogen free (SPF)
chickens under experimental conditions [1]. However, during field
outbreaks they have been reported to cause severe respiratory
distress leading to decrease production of eggs and meat especially
when joined by other respiratory pathogens or immunosuppressive
agents of the environment.
Lasota and B1 are the most commonly used strains of NDV which
are used in live and killed lNDV vaccines for the prevention of
vNDV outbreaks in developed and developing countries [1].
Similarly, LPAIVs produce very mild respiratory infections in SPF
experimental chickens but co-infections with other pathogens
including viruses can exacerbate disease outcomes under field
conditions leading to severe respiratory problem. [4]
LPAI infection is an emerging threat to most of the countries
especially Middle East and Asian countries [5]. Low pathogenic
avian influenza virus (H9N2), circulating in Pakistan has been
declared to have novel genotype. However, vaccines for the
prevention of LPAI are not used routinely and only inactivated and
vectored vaccines are allowed to be used against LPAI. Coinfections LPAIV and lNDV commonly occur in field and present a
complicated clinical picture of mixed clinical signs and lesions
which often leads to misdiagnosis of these viruses [6,7].To date
very little is known about the co-infections of these two viruses
especially in indigenous chicken. Virus and bacteria co-infections
have been reported with increased clinical signs and lesions as
compared to single infection in chicken [8,9]
Conversely, infection of a host with one virus may affect infection
by a second virus, a phenomenon explained by the occurrence of
viral interference, in which cells infected by a virus do not permit
multiplication of a second virus [10]. Measurable differences may
include changes in tissue permissiveness or tropism, viral
replication, patterns of virus progeny production and release,
latency, pathology including immunopathology, and immunological
responses [11]. In addition, viral interference may be detrimental to
detecting viruses in co-infected flocks since lower or undetectable
virus titers might fail to give a complete diagnosis [12]. Exposure to
lNDV, either as live vaccines or field strains, is nearly unavoidable
for commercial and non-commercial poultry worldwide, and co-
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J Avian Res, 2015, 1(1):1-5
infections with LPAIV are likely to occur. Both viruses replicate in
epithelial cells of the respiratory and intestinal tracts, where there
are trypsin-like enzymes, likely competing for target cells or
replicating in adjacent cells. Whether co-infections with LPAIV and
lNDV will exacerbate clinical signs of disease in infected birds or
produce viral interference, masking infections by one or other virus,
is unknown. In this study we examined the effect of co-infections of
fayoumi chickens with LaSota lNDV vaccine strain and a LPAIV
(A/Chicken/Pakistan/UDL/08 H9N2) by inoculating the viruses
simultaneously and determining differences in pathogenesis
(clinical signs, lesions), presence of the viruses in tissues, duration
and titer of virus shedding and Seroconversion to both viruses. Such
a study design replicates field situations in countries free of virulent
NDV, but with active NDV vaccination programs and where LPAI
outbreaks periodically occur [1, 5].
Materials & methods
Virus
The UDL-01/08 H9N2 virus was a field isolate obtained from
Quality operation laboratory, University of Veterinary and Animal
Sciences, Lahore, Pakistan. Viral stocks were prepared and titrated
in 9-day-old to 10-day-old specific pathogen free embryonated
chicken eggs [13]; the median embryo infectious dose (EID50) was
calculated using previously reported methods [14]. The viral stocks
were diluted in sterile brain–heart infusion medium containing
antimicrobials to yield a final titre of 106 EID50/0.1 ml [15].
Birds
Experimental study protocol was approved by the Animal care and
research committee of the University of Veterinary and Animals
Sciences, Lahore and experimentation was carried out according to
the guidelines of committee. Three-week-old fayoumi chicks
(indigenous layer hens), were acquired from a local supplier. Blood
and buccal swab samples were taken from all birds and analysed by
haemagglutination inhibition (HI) and virus isolation (VI) in eggs
using standard methods [16] to ensure that the birds were
serologically naïve and free from influenza virus and Newcastle
disease virus infection prior to the start of the experiment. Each
group of birds was housed separately in cages in separate rooms
(Table 1).The birds were acclimatized for 7 days prior to
inoculation. Feed and water were provided ad libitum. All treatment
groups contained 10 birds and were inoculated by the intranasal
routes with a dose of 106 EID50 of each virus or sham inoculum.
The viruses were given alone and in combination simultaneously on
the same day.
The birds were monitored three times daily for clinical signs
(reluctance to move, anorexia, congestion of eyes, respiratory signs
mainly sneezing, swollen head, haemorrhage on shanks Blood
samples for serology were taken before virus inoculation (day 0)
and 14 days post inoculation (dpi). Oropharyngeal (OP) and cloacal
(CL) swabs were collected from all birds from 1 to 7 dpi to assess
virus shedding. Three birds from each group were euthanized at 3
dpi for necropsy findings and tissues were collected in 10% neutral
buffered formalin to evaluate microscopic lesions and the extent of
virus replication in tissues as described previously [15, 18]. At 14
dpi remaining birds were bled for serology and euthanized by the
intravenous (IV) administration of sodium pentobarbital (100
mg/kg body weight).
RNA extraction and Real-Time PCR
Oropharyngeal and cloacal swabs were collected in 2 mL of BHI
broth with a final concentration of gentamicin (200 μg/mL),
penicillin G (2000 units/mL), and amphotericin B (4 μg/mL) and
kept frozen at −70 °C until processed. RNA was extracted using the
QIAamp viral RNA isolation kit. Quantitative real time RT-PCR
(qRT-PCR) for AIV and Newcastle disease virus (NDV) detection
was performed as previously described [19] with modifications.
qRT-PCR reactions targeting the influenza virus M gene [20] and
NDV M gene [21] were conducted using Quanti-fast SYBR green
RT-PCR one step RT-PCR Kit (Qiagen) and the Light cycler 480,
Real-Time PCR system (Roch Life sciences Switzerland). The RT
step conditions for both primer sets were 10 min at 45 °C and 95 °C
for 10 min. The cycling conditions for AIV were 45 cycles of 15 s,
95 ° 45 s, 60 °C; and for NDV were 40 cycles of 10 s, 94 °C; 30 s,
56 °C; 10 s, 72 °C. The calculated qRT-PCR lower detection limit
was for AIV was 100.5RNA copies (log10)/mL and 100.6 RNA copies
(log10)/mL for NDV. A standard curve for virus quantification was
established with RNA extracted from dilutions of the same titrated
stock of the challenge virus, and results also reported as Log10 RNA
copies/mL equivalents
Serology: Hemagglutination inhibition (HI) assays were performed
to quantify antibody responses to virus infection as previously
described [3] with serum collected from birds at 14 dpi. Titers were
calculated as the highest reciprocal serum dilution providing
complete haemagglutination inhibition. Serum titers of 1:8 [20] or
lower were considered negative for antibodies against AIV or NDV
Statistical analysis
Data were analyzed using Prism v.5.01 software (GraphPad
Software Inc. La Jolla, CA, USA) and values are expressed as the
mean ± standard deviation of the mean (SDM). One-way ANOVA
with Tukey post-test was used to analyze HI titers. The number of
birds shedding virus were tested for statistical significance using
Fisher’s exact test. Two-way ANOVA with Bonferroni multiple
comparison analysis was used to evaluate virus titers in swabs. For
statistical purposes, all qRT-PCR negative oropharyngeal and
cloacal swabs were given a numeric value of 100.5RNA copies
(log10)/mL for LAIV and 100.6 RNA copies (log10)/mL for lNDV.
All HI-negative serum was given a value of 3 log2. These values
represent the lowest detectable level of virus and antibodies in these
samples based on the methods used. Statistical significance was set
at p < 0.05 unless otherwise stated.
Results
Clinical signs
No clinical signs were observed in chicken exposed to lNDV alone.
However, all chicken exposed to LPAIV, regardless of lNDV
exposure, presented mild to moderate clinical signs consisting of
periocular edema, conjunctivitis, sinusitis, ruffled feathers, and
lethargy. These clinical signs were first observed at 3 dpi and lasted
until 7 dpi.
No differences in the severity of the clinical signs were observed
between the groups inoculated with both LPAIV and lNDV and the
group inoculated only with LPAIV.
Virus shedding
All birds were negative for H9N2 AIV by serology and virus
isolation in eggs prior to inoculation. All birds in inoculated groups
became infected with H9N2 AIV as determined by detection of the
H9N2 AIV matrix gene in OP and CL swabs. The total number of
positive swabs and the duration of virus shedding varied among
different groups (Table 3). LPAIV viral shedding was mainly
through the oropharyngeal (OP) route (p<0.05). At early time
points (1–3 dpi), co-infected birds presented lower amounts of viral
shedding than birds receiving lNDV or H9N2 virus alone. These
differences were significant at 1 and 3 dpi when chickens coinfected with LPAIV and lNDV showed significantly lower viral
titers compared to single lNDV or H9N2 virus infected birds
(p<0.05). The peak H9N2 AIV shedding in OP swabs varied
between groups as determined by RT PCR results (Fig. 1 & 2).
Birds in the lNDV + H9N2 group had peaks for AIV OP shedding
at 5dpi. Similarly, peak of lNDV OP shedding was at 5dpi in lNDV
group. Moreover, virus shedding period was longer in birds of the
lNDV+H9N2 group than those in the H9N2 group. Lower virus
titer of lNDV was observed in co-infected group than lNDV
infected group.
Necropsy and histopathological findings
No gross lesions were observed in any of the birds necropsied at 3
dpi, except for the chicken inoculated with LPAIV alone or in
combination with lNDV, which had mild conjunctivitis, sinusitis,
enteritis and moderate tracheitis and airsaculitis. The microscopic
lesions observed were consistent with gross lesions of LPAIV and
lNDV infection (Table 2). Lesions present in tissues from LPAIVinfected chicken included mild rhinitis, sinusitis and enteritis
characterized by mild accumulation of inflammatory cells and
exudate while moderate airsaculitis and tracheitis having moderate
accumulation inflammatory cells and exudate. No significant
difference in the severity of lesions was found between chicken
infected only with LPAIV and chicken co-infected with LPAIV and
lNDV.
Serology
HI assays were used to test for antibodies against LPAIV and lNDV
(Table 4). All chickens seroconverted to both LPAIV and lNDV,
among the inoculated groups. However, a clear difference was
found in lNDV and LPAIV titers in chicken exposed to alone than
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J Avian Res, 2015, 1(1):1-5
Table. 1: Experimental design
Groups
Age
(days)
Number of
birds
A
B
C
28
28
28
10
10
10
D
28
10
Route of
inoculation
Viral strain
Dose per bird
Negative control (non inoculated)
Lasota vaccine strain
(A/Pakistan/chicken/UDL-01/08)
(A/Pakistan/chicken/UDL-01/08)+
Lasota vaccine strain
0.5ml normal saline
106 EID50/0.5 ml lNDV Lasota strain
106 EID50/0.5 ml LPAIV
106 EID50/0.5 ml LPAIV
106 EID50/0.5 ml lNDV
IN
IN
IN
IN
Table. 2: Microscopic lesions at day 3 PI
Groups
Rhinitis/sinusitis
tracheitis
bronchitis
A
_
_
_
B
+
C
+
++
+
D
+
++
+
Notes: Whereas, -no lesions, + mild , ++ moderate , +++ severe
pnuomonitis
_
+
+
Air sacculitis
_
++
++
enteritis
_
+
+
Table. 3: Infection and viral sheddinga (Viral sheddinga of LPAIV detected in OP and CL swabs after single infection or co-infection with
lNDV and H9N2 LPAIV in Fayoumi chicken).
Swabs
OP
Groups
B
C
D
CL
B
C
D
1
(lNDV)
(LPAIV)
LPAIV
lNDV
(lNDV)
(LPAIV)
LPAIV
lNDV
d
4/7
5/7
2/7
3/7
2/7d
3/7
2/7
4/7
2
3
Days post-inoculation
4
5
6
7
8
9
10
Totalb
meanc
2/7
3/7
3/7
4/7
3/7
4/7
3/7
6/7
6/7
7/7
5/7
7/7
4/7
5/7
2/7
7/7
7/7
7/7
7/7
5/7
7/7
5/7
6/7
3/7
0/7
0/7
0/7
0/7
0/7
1/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
25
35
27
23
20
30
26
24
3.57
5
3.85
3.2
2.8
4.2
3.7
3.4
4/7
7/7
7/7
3/7
3/7
7/7
7/7
3/7
2/7
4/7
2/7
1/7
1/7
3/7
4/7
1/7
0/7
2/7
1/7
0/7
0/7
2/7
2/7
0/7
a
Determined by rRT-PCR.
b
Total number of positive swabs.
c
Mean number of viral shedding days.
d
Number of positive birds/total number of birds (n = 7 indicates the number of birds that were sampled during the entire study period,
excluding the birds that were euthanized in the first 3 days post single infection or co-infection
combined infections, indicating that the presence of LPAIV might
be interfering with the production of antibodies against lNDV.
These results suggest that infection with a heterologous virus may
result in temporary competition for cell receptors or competent cells
for replication, most likely interferon-mediated, which decreases
with time.
Discussion
Co-infections of different viruses and bacteria especially lNDV and
LPAI are expected to occur and have been reported in poultry
[22,23], but the impact of such co-infections on several host
responses including clinical outcome, viral shedding dynamics,
seroconversion, and sites of virus replication in fayoumi chickens is
unknown. Co-infections of AIV and NDV have been studied in
vitro using cell cultures or chicken embryos, and interference
between these viruses has been reported, with one virus inhibiting
the growth of the other [7,24, 25] In contrast to in vitro or in ovo
studies, in vivo experiments examine the overall effect of coinfections by incorporating the complexity of the whole organism,
including different target cells and immune responses. In our coinfection studies, all chickens became infected with lNDV and
LPAIV as shown by virus shedding results, and a significant
reduction in virus replication was observed for first 3 days when
birds were co-infected versus single virus infected. In spite of the
differences in virus replication, co-infection of LPAIV and lNDV
had no effect on the severity of clinical signs. Typically, chickens
lack clinical signs in experimental infection with LPAIV or lNDV,
which was corroborated by mild microscopic lesions.
Lack of clinical signs was also observed in chicken infected with
lNDV alone. However, all chicken infected with LPAIV, coinfected or not, showed mild transient upper respiratory signs and
moderate inflammation and necrosis in the epithelia of nasal trachea
and air sacs accompanied by mild exudate. These results are
consistent with previous reports mentioning similar findings [26,
27].
In addition, this H9N2 LPAIV strain is known to be chicken
adapted, with a very low mean bird infectious dose required to
produce infection [28]. Therefore, the host species is a factor that
can influence the severity of clinical signs and amount of virus
replication in such virus co-infections. In this study, an effect in the
pattern of viral shedding was also found in the fayoumi chickens,
indicating that virus interference can occur, but to a lesser degree,
as long as there is viral replication. We do expect similar results
with other viruses. In this study, viral shedding patterns were
clearly affected in chickens exposed to lNDV and LPAIV
simultaneously. Although LPAIV OP shedding was significantly
less when measured at 3 dpi but peak virus shedding time was same
for all groups. Likewise, co-infection of chickens with LPAIV also
affected lNDV OP virus shedding, with initial lower virus
replication in co-infected birds than birds receiving only LPAIV,
but higher and more prolonged virus shedding at later time points.
The presence of high virus titers was associated with clinical signs
and microscopic lesions in respiratory tissues of all chicken infected
with LPAIV. lNDV OP virus shedding was clearly affected by
LPAIV replication, with fewer co-infected birds shedding lNDV
and with significantly lower lNDV titers than chicken infected with
lNDV alone. However, lNDV replication increased slowly in birds
that received LPAIV probably because by then the effect of LPAIV
replication had diminished. This suggests that infection with a
heterologous virus may result in temporary competition for cell
receptors or susceptible cells, resulting in decreased initial
replication of the second virus; but as replication of the first virus
declines, the second virus increases to fill the gap. Viral
interference is a phenomenon in which a cell infected by a virus
does not permit multiplication of a second homologous or
heterologous superinfectant virus [10]. Viral interference can be
explained by different mechanisms including: competing by
attachment interference therefore reducing or blocking of receptor
sites for the super-infecting virus; competing intracellularly for
replication host machinery; and virus-induced interferon
interference [29]. lNDV and LPAIV replicate in cells where there
are trypsin-like enzymes such as in the upper respiratory and
intestinal epithelia [4,30] and might compete for the same target
cells or replicate in adjacent cells. The LaSota virus, as a lNDV,
1
J Avian Res, 2015, 1(1):1-5
binds through the HN glycoprotein to sialic acid-containing
receptors on cell surface, as well as the HA glycoprotein does for
LPAIV [31]. Replication of one virus might also be affected by
previous replication in the same site of another virus that has
already activated antiviral immune responses including
Table. 4: Serological status for LPAIVand lNDV, as determined
by HI test, of fayoumi chicken at before inoculation ( day 0) and
after inoculation (14 days) in single infection and co-infection
groups.
HI titre results a
Day 0
Day 14
B
7/7b (HI<8)
7/7 (256)
C
7/7 (HI<8)
7/7 (512)
lNDV
7/7 (HI<8)
7/7 (128)
D
LPAIV
7/7 (HI<8)
7/7(512)
a
Number of positive birds/total (HI titre).
b
Number of positive birds/total number of birds (n = 7 indicates the
number of birds that were sampled during the entire study period,
excluding the birds that were euthanized in the first 3 days post
single infection or co-infection)
Groups
immunomodulators or recruitment of immune cells. Although the
LaSota lNDV strain is known to be a weak interferon inducer as
part of their low virulent phenotype profile [32] local interferon
production might still be able to interfere with LPAIV replication.
In fact, previous studies in embryonating eggs showed that LaSota
lNDV could suppress growth of a H9N2 LPAIV’s, if given prior to
the LPAIV [24]. Influenza viruses also induce interferon [33,34],
which could have been one mechanism by which the high LPAIV
replication in the turkeys inhibited lNDV replication. Viral
interference has also been suggested in other studies with influenza
virus such as the pandemic H1N1 when it was shown that an
increase in the proportion and number of rhinovirus diagnoses in
humans occurred in parallel with the decrease of influenza
diagnoses, suggesting that rhinoviruses inversely affected the
spread of the pandemic H1N1 virus [35, 36].
Fig. 1: Mean virus titre values (log10 RNA copies/ml) of lNDV
detected in OP and CL swabs/day post inoculation in group B
and D
Fig. 2: Mean Virus titre values (log10 RNA copies/ml ) of LPAI
detected in OP and CL swabs/day post inoculation in group C
and D
Experimental in vivo co-infections of NDV and LPAIV are scarce
in chickens. França et al. [37] performed co-infection in wild ducks
with LPAIV and lentogenic NDV wild bird strains and observed
differences in the pattern of virus shedding depending on the time
of co-infection. The authors suggest that competition for replication
sites and/or differences in fitness for replication may explain the
effects of co-infection with lentogentic lNDV and LPAIV. Other
studies have examined co-infection of LPAIV and lNDV with other
respiratory viruses of poultry. Research has shown that infectious
bronchitis virus (IBV) interfered with the replication of lNDV [38,
39] However, IBV live vaccine increased the severity of H9N2
LPAIV infections [11, 12], and it was suggested that IBV was a
supplier of trypsin-like proteases therefore enhancing the reach of
systemic sites by the virus. In our case, such an exacerbation would
not occur, since neither the LaSota lNDV strain, nor the LPAIV can
provide extra enzymatic activity to each other. In other studies, coinfection of turkeys with lNDV and another respiratory virus, avian
pneumovirus (APV), induced more severe disease compared to
turkeys infected with APV or NDV [40-42], and dual vaccination of
turkeys with lNDV and hemorrhagic enteritis virus (HEV) live
vaccines enhanced the pathologic response of the host [16]. In
chickens, although the co-infection with LPAIV and lNDV
interfered with viral replication as seen in the viral shedding
patterns, the reduction in the humoral immune response was not
observed, since all chickens seroconverted with similar antibody
titers to LPAIV and NDV, regardless if they were co-infected or
not. This is similar to what has been reported with experimental coinfections with IBV and live lNDV and some other diseases
vaccines in broilers [39]. Although no significant effect of coinfection was observed on HI titers with these particular viruses, an
effect might be seen in chickens infected with more virulent strains
of NDV and AIV. [22]. Our co-infection study was performed
under controlled conditions in order to examine the specific
interactions between the two viruses when given at high challenge
doses. This might not be representative of what happens under field
conditions where poultry are exposed to many viruses and other
infectious and non-infectious disease agents. However, the results
obtained underline the importance of co-infections which can either
exacerbate clinical disease, or, like in our study, affect virus
replication by lowering viral titers to under the levels of detection
and affecting serological results, and in some cases increasing the
time virus was shed which could favor prolonged transmission. In
addition, exposure to lower challenge doses of these viruses in the
field could also affect the results of co-infection. The effects of
virus co-infection will most likely vary depending on how well
adapted the viruses are to a specific bird species, on the virulence of
the viruses involved, on the timing of co-infections, and on other
concomitant infectious and environmental factors. Evaluating the
infectious status in birds might be necessary when developing
vaccination protocols using live attenuated vaccines. The role of
viral interference in the spread of AIV and NDV needs further
examination as also the role of co-infections in terms of altering the
severity of clinical signs and lesions. The identification of factors
that influence co-infection interference or elements that favor a
delay in infection of one virus at expense of another virus will
provide new insights in the pathogenesis of these viruses, allowing
better development of new diagnostic and vaccine technologies for
prevention and control of these infections.
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