Detection and Isolation of Exotic Newcastle Disease Virus
from Field-Collected Flies
DANIEL J. KING,
CAROL J. CARDONA,
DOUGLAS R. KUNEY,
AND ALEC C. GERRY
J. Med. Entomol. 44(5): 840Ð844 (2007)
ABSTRACT Flies were collected by sweep net from the vicinity of two small groups of “backyard”
poultry (10Ð20 chickens per group) that had been identiÞed as infected with exotic Newcastle disease
virus (family Paramyxoviridae, genus avulavirus, ENDV) in Los Angeles County, CA, during the
2002Ð2003 END outbreak. Collected ßies were subdivided into pools and homogenized in brain-heart
infusion broth with antibiotics. The separated supernatant was tested for the presence of ENDV by
inoculation into embryonated chicken eggs. Exotic Newcastle disease virus was isolated from pools
of Phaenicia cuprina (Wiedemann), Fannia canicularis (L.), and Musca domestica L., and it was
identiÞed by hemagglutination inhibition with Newcastle disease virus antiserum. Viral concentration
in positive pools was low (⬍1 egg infectious dose
per ßy). Isolated virus demonstrated identical
monoclonal antibody binding proÞles as well as 99% sequence homology in the 635-bp fusion gene
sequence compared with ENDV recovered from infected commercial egg layer poultry during the
KEY WORDS ßies, Newcastle disease virus, poultry, chickens, mechanical vector
Exotic Newcastle disease (END) is a contagious and
fatal viral disease affecting the respiratory, nervous,
and digestive systems, of poultry and other birds. END
is so virulent that many birds may die without ever
showing clinical signs of illness. There is a near 100%
mortality in unvaccinated poultry, and even poultry
vaccinated against the endemic low-virulence New-
castle disease virus strains are not adequately pro-
tected against END virus (family Paramyxoviridae,
genus avulavirus, ENDV) (USDAÐAPHIS 2006).
Newcastle disease control in poultry has been on-
going in the United States since the Þrst infections
were identiÞed in the early 1940s. Introductions of the
more virulent strains of Newcastle disease virus, the
cause of END, have occurred, and in most cases, the vi-
rus has been quickly eradicated (Utterback and
Schwartz 1973). During two END outbreaks, virus was
not quickly eradicated. The Þrst occurred during
1971Ð1973, and this outbreak resulted in the quaran-
tine of eight California counties, the destruction of
11.9 million birds, and eradication costs of $56 million
(USDAÐAPHIS 1978). The second occurred during
2002Ð2003, and it resulted in the quarantine of 18,345
premises, the destruction of 3.2 million birds, and
eradication efforts cost of $170 million (Breitmeyer et
ENDV is primarily spread by direct contact be-
tween infected and healthy birds. However, it also can
be transmitted indirectly via contaminated equipment
and persons (Utterback and Schwartz 1973, Burridge
et al. 1975). During the 1971Ð1973 California END
outbreak, Rogoff et al. (1975) isolated virus from pools
of Fannia spp. collected at commercial poultry oper-
ations with ENDV infected birds, implicating this spe-
cies as a possible vector of ENDV.
Many insects, especially ßies, are commonly asso-
ciated with poultry operations, and they are known to
disperse into surrounding areas (Lysyk and Axtell
1986, Axtell 1999, Sawabe et al. 2006). Wet manure is
an excellent breeding environment for several ßy spe-
cies, and it also likely to support the survival of ENDV
(Kinde et al. 2004). Flies are capable of harboring a
diverse range of animal and human pathogens (Cali-
beo-Hayes et al. 2003, Graczyk et al. 2005, Sawabe et
al. 2006), and they may be involved in transmission of
these pathogens to otherwise healthy hosts (Shane et
al. 1985, Calibeo-Hayes et al. 2003, Ahmad et al. 2007).
However, the extent to which insects may be involved
in the dispersal and transmission of ENDV to unin-
fected birds remains unknown.
In this study, ßy species collected at two private
homes with ENDV-infected poultry in Los Angeles
County, CA, were examined for the presence of
ENDV. Isolated ENDV was characterized by mono-
clonal antibody binding proÞle and sequence analysis
of the fusion gene.
Department of Entomology, University of California, Riverside,
USDAÐARSÐSEPRL, Athens, GA 30605.
Veterinary Medicine Extension, University of California, Davis,
University of California Cooperative Extension, Riverside, CA
Corresponding author, e-mail: firstname.lastname@example.org.
Materials and Methods
Flies were captured by sweep net from the imme-
diate vicinity of two private homes in Los Angeles Co.,
southern California. Both locations, one location in
the city of Compton (home A), and the other location
in Inglewood (home B), contained chicken ßocks
(⬍20 birds) that had been identiÞed by the California
Department of Food and Agriculture (CDFA) as hav-
ing one or more ENDV-infected birds within the pre-
ceding 5 d. Flies were collected before poultry were
euthanized and removed from the private homes by
the END Task Force (a joint United States Depart-
ment of Agriculture and CDFA organization) respon-
sible for efforts to eradicate END in California. Fly
collectors followed all biosecurity precautions recom-
mended by the CDFA while sampling at these homes.
Multiple 5-min sweep collections were made at each
home, taking care not to touch poultry cages or the
ground with the net while sweeping. Sweep net col-
lections continued at each home until ßies were no
longer readily captured. Captured insects were killed
on dry ice, placed into labeled vials by using sterilized
forceps, and held on dry ice until they could be re-
turned to the laboratory to be identiÞed and counted.
Sweep nets and forceps were sterilized between each
5-min collection by submergence in 10% Lysol (Rec-
kitt Benckiser Inc., Wayne, NJ) after which nets were
allowed to dry before reuse.
Flies were kept frozen on dry ice during identiÞ-
cation in a biological safety cabinet, and then they
were pooled into groups of Þve or fewer ßies of the
same species from the same home. Fly pools were
stored at ⫺80⬚C until shipment on dry ice to the USDA
Southeast Poultry Research Laboratory (SEPRL), in
Athens, GA, where they were again placed at ⫺80⬚C
until tested for the presence of ENDV. Pools were
homogenized in 1.5 ml of brain-heart infusion (BHI)
broth with antibiotics (200
g of gentamicin/ml, 2000
U of penicillin/ml, and 4
g of amphotericin B/ml,
Sigma-Aldrich, St. Louis, MO) by using a tissue grinder
with sterile plastic pestles in microfuge tubes and
centrifuged at 16,000 ⫻ g for 10 min. Virus isolation
was performed by inoculating 100
l of the superna-
tant into the allantois of each of three 9- or 10-d-old
embryonated chicken eggs (ECE). Eggs were incu-
bated at 37⬚C in a standard humidiÞed incubator. The
embryonated chicken eggs were obtained from the
SEPRL speciÞc-pathogen-free White Leghorn ßock.
Eggs were candled to determine embryo death each
24 h through 7 d postinoculation. Embryos that died
within the Þrst 24 h were discarded. Embryos that died
between 24 h and7daswell as all survivors at 7 d were
chilled at 4⬚C. Amnio-allantoic ßuid (AAF) harvested
from chilled eggs was tested for hemagglutination
(HA) activity to detect ENDV. Virus presence in HA-
positive samples was conÞrmed by hemagglutination-
inhibition (HI) with Newcastle disease virus (family
Paramyxoviridae, genus Avulavirus, NDV)-speciÞc
antiserum (King 1996a). Amnio-allantoic ßuids from
HA negative dead embryos and embryos alive at 7 d
postinoculation were subjected to a second serial pas-
sage by inoculation of 100
l of the AAF into each of
three additional embryonated chicken eggs. Eggs
were candled, and killed embryos were handled as
before. If by day 7 postinoculation there was no HA
activity in the AAF of the second passage dead or
surviving embryos, the specimen was regarded as neg-
ative for ENDV.
A monoclonal antibody proÞle was determined for
each isolated ENDV using nine monoclonal antibodies
(mAbs) with different Newcastle disease virus HI
speciÞcities by using previously described methods
(King 1996a, Kommers et al. 2001). The mAbs in-
cluded 15C4, AVS, B79, 161/167, P11C9, P3A11,
10D11, P15D7, and P10B8 (Kommers et al. 2003).
RNA was extracted from AAF by using TRIzol LS
according to manufacturerÕs instructions (Invitrogen,
Carlsbad, CA); 750
l of TRIzol LS reagent was added
l of allantoic ßuid. The ßuid was vortexed and
incubated at room temperature for 7 min. RNA was
separated into the aqueous phase with the addition of
l of chloroform, followed by precipitation with
isopropanol. After one wash with 70% ethanol, RNA
was dried and resuspended in RNase-free water. The
5⬘ end of the ENDV fusion gene was ampliÞed by
polymerase chain reaction (PCR) and sequenced us-
ing primers targeting a 635-bp fragment that included
the fusion protein cleavage site (forward primer, 5⬘-
GAG GTT ACC TCY ACY AAG CTR GAG A-3⬘;
reverse primer, 5⬘-TCA TTA ACA AAY TGC TGC
ATC TTC CCW AC-3⬘). These primers amplify the
NDV genomic region between positions 4317 and
5084. Standard 50
l reverse transcription (RT)-PCR
reactions were carried out using a kit (SuperScript III
One Step RT-PCR, Invitrogen) with annealing tem-
perature at 56⬚C. PCR-ampliÞed samples were sepa-
rated on a 1% agarose gel, and the bands were excised
and eluted using the QuickClean 5M gel extraction kit
(GenScript Corp., Piscataway, NJ). Once the PCR
products were cleaned, samples were quantiÞed using
a standard spectrophotomer and sequenced. All dou-
ble-stranded nucleotide-sequencing reactions were
performed with ßuorescent dideoxynucleotide termi-
nators in an automated sequencer (ABI 3700 auto-
mated sequencer, Applied Biosystems, Foster City,
CA). Nucleotide sequence editing and analysis were
conducted with the LaserGene sequence analysis soft-
ware package (LaserGene, version 5.07, DNAStar,
Inc., Madison, WI). The virus was sequenced and
compared by Blast analysis (Altschul et al. 1990) to an
END viral sequence [chicken/U.S.(AZ)/236498/03]
obtained from commercial poultry infected with
ENDV during the 2002 outbreak. It also was compared
with other available GenBank ENDV sequences.
Genomic sequences from viruses recovered from ßies
at each collection site were deposited in GenBank
(accession nos. EF424375 and EF424376).
Overall, 87 ßies composing nine species were col-
lected from the area surrounding the two private
homes housing ENDV-infected birds (Table 1). Most
September 2007 C
HAKRABARTI ET AL.: ENDV ISOLATED FROM FIELD-COLLECTED FLIES 841
of the collected ßies were Phaenicia cuprina (Wiede-
mann), Musca domestica L., or Fannia canicularis (L.).
There were 24 ßy pools representing groups of ⱕ5 ßies
of the same species from the same home. Five pools of
ßies contained ENDV; three pools of P. cuprina (two
pools from home A and the other pools from home B)
and one each of F. canicularis and M. domestica (both
from home B). The amount of virus recovered from
each of the ßy pools was very low (⬍1 egg infectious
per ßy) and required a second serial
passage in ECE for detection.
The mAb binding proÞle (Table 2) of the virus
isolated from each of the ßy pools was identical to an
ENDV strain obtained from infected commercial egg-
layer poultry during the 2002Ð2003 END outbreak
[chicken/U.S.(CA)/12430/02] and different than the
proÞle of the heterologous NDV B1 strain included as
a control. The NDV B1 strain is commonly used as a
live virus vaccine in commercial poultry. The partial
sequence of the fusion gene of the virus isolated from
the ßies showed 99% homology (634/635 bp) to a
2002Ð2003 ENDV isolate [chicken/U.S.(AZ)/236498/
03] and 87% homology (552/635 bp) to a 1971Ð1973
ENDV isolate [chicken/U.S.(CA)/1083(Fontana)/
72], both obtained from commercial egg-layer poultry
during ENDV outbreaks. At the amino acid level, the
partial sequence of the ßy isolate was 100% identical
to the 2002Ð2003 outbreak strain and 92% identical to
the 1971Ð1973 outbreak strain. Viruses recovered from
ßies at both collection sites were 100% identical to
Although all ßy species were collected in small
numbers, ENDV was isolated from the three most
abundant species collected during this study. This is
the Þrst report of ENDV isolated from Þeld-collected
P. cuprina and M. domestica; ENDV had previously
been isolated from F. canicularis and Fannia femoralis
(Stein) from a commercial poultry operation during
the 1971 END outbreak (Rogoff et al. 1975). Given the
small numbers of ßies collected, the prevalence of
ENDV-infected ßies was high with ⬇30% of the pools
containing the three species mentioned above having
one or more ßies per pool infected with virus. The
virus prevalence in Fannia spp. collected from com-
mercial poultry operations during the 1971 END out-
break was far lower with only two of 78 pools of Fannia
spp. (3,926 total ßies) collected before or within2dof
poultry removal containing ENDV, and no virus was
recovered from other common ßy species (Rogoff et
al. 1975). It is unclear to what extent Rogoff et al.
(1975) may have collected ßies from poultry facilities
where poultry or even manure had already been re-
moved from the site, perhaps resulting in the much
lower ENDV infection prevalence in ßies relative to
this study. The higher ENDV prevalence in this study
also may be due to differences between commercial
poultry and backyard poultry in animal housing, ma-
nure handling, or vaccination status of the poultry
against Newcastle disease virus. Backyard poultry are
often housed together on the ground or in wooden
boxes, giving them greater access to an infected bird
and its manure relative to commercial egg-layer poul-
try that are separated into cages suspended above the
ground where the manure is allowed to accumulate.
Additionally, backyard poultry are usually not vacci-
nated against Newcastle disease virus and infected
Table 1. Detection of ENDV from ﬂies collected at two homes
(A and B) in Los Angeles County with ENDV-infected poultry during
2002–2003 ENDV outbreak
ABAB A B
Calliphora spp. 1 1
Fannia canicularis 10 10 2 2 1 (n ⫽ 5)
Fannia femoralis 11
Musca domestica 8152 3 1(n ⫽ 5)
Muscina stabulans 2111
Phaenicia sericata 21
Phaenicia cuprina 14 21 3 5 2 (n ⫽ 10) 1 (n ⫽ 5)
Phormia regina 11
Stomoxys calcitrans 11
Table 2. Hemagglutination-inhibition test results of ENDV isolated from ﬁeld-collected ﬂies against NDV-speciﬁc mAbs
15C4 AVS B79 161/617 P11C9 P3A11 10D11 P15D7 P10B8
P. cuprina (A1) ⫹⫺⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹
P. cuprina (A2) ⫹⫺⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹
P. cuprina (B1) ⫹⫺⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹
F. canicularis (B1) ⫹⫺⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹
M. domestica (B1) ⫹⫺⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹
CK/CA/12430/02 (outbreak strain) ⫹⫺⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹
CK/US/B1/48 (vaccine strain) ⫹⫹⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺
Antibody-inhibited HA (⫹); no HA inhibition (⫺). 15C4 inhibits all NDV but pigeon PMV-1. AVS inhibits lentogenic strains like the B1
and LaSota ND vaccine virus. B79 inhibits most NDV including most pigeon PMV-1. 161/617 inhibits only pigeon PMV-1. P11C9, P3A11, P15D7,
and P10B8 inhibit various NDV strains. 10D11 inhibits neurotropic NDV mesogens and velogens such as virulent Texas GB. CK/CA/12430/02
is an ENDV strain recovered from commercial poultry during the 2002Ð2003 outbreak in California. CK/US/B1/48 is a strain widely used as
a Newcastle disease vaccine in the United States. The antibody inhibition proÞle of the viruses tested indicates the ENDV isolates from three
ßy species at premises A and B are antigenically similar as well as being similar to the CK/CA/12430/02 ENDV isolate from chickens but are
different than the vaccine strain B1.
842 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no. 5
birds would be expected to shed a greater concentra-
tion of ENDV in their manure relative to vaccinated
commercial poultry (Kapczynski and King 2005).
The mAb proÞle and sequence analysis of the virus
recovered from each of the ßy pools indicated that
these viruses were the same ENDV strain circulating
in commercial and backyard poultry during this same
period. Viruses recovered during 2002Ð2003 were ge-
notypically different from viruses recovered during
the earlier 1971Ð1973 ENDV outbreak (genotypes V
and VI, respectively) (Czegledi et al. 2006) with virus
isolates from the 2002Ð2003 ENDV outbreak being
most similar to ENDV isolated from chickens in Mex-
ico during 1998Ð2000 (Pedersen et al. 2004).
Flies are thus contacting infectious ENDV in the
environment and are capable of harboring at least low
levels of this virus. The ENDV infective dose for a
chicken is reported to range from ⬇10
(Alexander et al. 2006), far greater than
the ⬍1 EID
per ßy found in our Þeld-collected ßy
pools. However, given the small number of ßies col-
lected, it would be premature to assume ßies are not
capable of carrying substantially higher viral loads. It
is entirely possible that these ßies had contacted a
source of ENDV several days before our sampling and
the low viral load represented residual virus still as-
sociated with the ßies at the time of their capture.
The mechanism by which the ßies might be acquir-
ing virus has not been determined. It has been shown
that ENDV-infected poultry shed a considerable
amount of virus in their feces (Kapczynski and King
2005) and that virus can persist in the manure for up
to 16 d (Kinde et al. 2004). Wet manure provides an
excellent developmental environment for several ßy
species as well as a substrate on which adult ßies feed.
Manure also provides an excellent media for main-
taining and transmitting infectious agents to ßies as the
viscosity of the manure enhances manure attachment
to ßy tarsi and cuticular bristles (Tan et al. 1997). In
addition, ßies feeding on bacteria in the manure would
be expected to consume some amount of the infec-
tious agent. Further studies are needed to determine
whether ENDV is associated with these ßy species
simply as an external contaminant or if the virus is
protected or even ampliÞed within the gut or other
tissues of the ßy.
Shane et al. (1985) demonstrated the potential for
house ßies to transmit pathogens by placing ßies into
cages with Campylobacter jejuni-infected chickens
and subsequently moving the ßies to cages with patho-
gen-free chickens, resulting in C. jejuni infection in the
previously uninfected chickens. Similarly, turkey
poults housed in isolation units became infected with
turkey corona virus (family Coronaviridae, genus
Coronavirus, TCV) when house ßies orally inoculated
with TCV were released into the isolation units for
24 h (Calibeo-Hayes et al. 2003), and cattle held in
conÞned pens became infected with Escherichia coli
O157:H7 after house ßies orally inoculated with the
bacteria were released into the pens for 48 h (Ahmad
et al. 2007). However, the actual mechanism of bac-
terial transmission from the ßies to the chickens or
cattle in the studies mentioned above remains un-
Poultry are known to consume adult ßies in labo-
ratory studies (Calibeo-Hayes et al. 2003) as well as in
the Þeld (A.C.G., unpublished observation). Addi-
tionally, ßies may regurgitate and defecate virus while
feeding or resting on the surface of foods (Greenberg
1973). It is possible that chickens and other birds can
consume virus with recently infected ßies or contam-
inated ßy feces and regurgitated matter (Sawabe et al.
2006). The consumption of many adult ßies in a short
period might result in infection even if the viral load
of each individual ßy is lower than the required in-
Fly larvae developing in infected manure also might
serve as a source of infection when consumed by
poultry having access to the manure (typical of back-
yard poultry and some commercial poultry), although
this has not been evaluated. Additional studies are
required to evaluate the vector competence of ßies
and other poultry associated arthropods.
Dispersal of infectious virus by ßies moving from
premises with infected poultry to nearby premises
with uninfected poultry is an important biosecurity
concern for END and other avian diseases. Greenberg
(1973) reported that house ßies were capable of ßights
ranging from 2.3 to 11.8 km within 24 h. Although ßy
involvement in virus movement between commercial
poultry facilities is probably minor relative to other
means of virus movement (e.g., on personnel, shared
equipment, or manure hauling vehicles), ßy involve-
ment in virus movement among poultry houses at a
single commercial facility or among backyard poultry
may be signiÞcant given the short distances ßies would
need to move between infected and uninfected poul-
try. Backyard poultry are geographically clumped in
southern California with small numbers of poultry at
many adjacent homes and only a few meters often
separating poultry at one home from the next. During
the 2002Ð2003 END outbreak, homes with ENDV-
infected poultry were often very close to each other,
forming a cluster that was distinct within the some-
what larger geographic clumping of backyard poultry
(C.J.C., unpublished data). In addition to ßy dispersal,
movement of birds or personnel between these clus-
tered homes also may have occurred.
Although this study has not shown ßies to be com-
petent vectors of ENDV, a conservative approach
would indicate that biosecurity measures should in-
clude ßy control on and near premises with END-
infected poultry as also suggested by Bram et al.
(1974). Fly control measures would be especially im-
portant for a quarantined poultry facility (to include
backyard ßocks) before removal of birds and their
manure by a state and/or federal task force as part of
the disease eradication effort. Removal of birds and
manure would be expected to encourage ßy dispersal
into the surrounding area. It is recommended that
future END task force organizations consider the use
of insecticides providing rapid knockdown of adult
ßies immediately before initiating eradication efforts
at commercial and backyard poultry facilities and that
September 2007 C
HAKRABARTI ET AL.: ENDV ISOLATED FROM FIELD-COLLECTED FLIES 843
control measures continue throughout the period of
manure removal and site disinfection.
We thank Diane Zhang for ßy colony maintenance and
Timothy Olivier and Dawn Williams-Coplin for help in con-
ducting the experiments. This study was funded by USDAÐ
CSREES grant 2003-34439-13366 (“Exotic Pests and Dis-
eases”) managed by the UCANR Statewide IPM Program and
awarded to A.C.G. and C.J.C.; and USDAÐARS CRIS project
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Received 21 March 2007; accepted 3 June 2007.
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