Avian inßuenza virus (AIV) is an enveloped RNA virus that belongs to the genus Influenzavirus A of the family Orthomyxoviridae. Inßuenza A viruses are classiÞed into subtypes by the combination of their two surface glycoproteins hemagglutinin and neuraminidase, of which 16 (H1ÐH16) and nine (N1ÐN9) antigens, respectively, have been recognized (reviewed in Pantin-Jackwood and Swayne 2009). AIV of H5 and H7 subtypes occurs as lowpathogenic (LPAIV) and high-pathogenic (HPAIV) strains, based on their virulence in chickens. The natural reservoir of avian inßuenza virus is wild aquatic birds within the orders Anseriformes and Charadriiformes (Stallknecht 2003) , and AIV circulates between wild birds and domestic poultry as well as within poultry ßocks through complex routes that are not fully understood (Alexander 2008) . In domestic poultry, most AIV infections are subclinical or give rise to mild clinical disease. Low-pathogenic isolates of the H5 and H7 subtypes may mutate into highly pathogenic (HP) variants after introduction into fowl, which induce severe, systemic disease with high mortality (Swayne and King 2003 , Swayne 2007 , Pantin-Jackwood and Swayne 2009 .
During outbreaks of HP avian inßuenza (AI), spread of AIV between poultry ßocks is assisted by human activity, but spread by contact with wild birds also may contribute to dissemination (Alexander 2008) . Although the role of insects in transmission of AIV has been considered, few studies have been published. AIV was detected in 25 of 72 pools of house ßies, Musca domestica L. (Diptera: Muscidae), collected within a 4-mo period from poultry ßocks in Pennsylvania and Maryland, infected with H5N2 AIV (Wilson et al. 1986 ). Also, HPAIV H5N1 has been isolated from two blow ßy species, Aldrichina grahami (Aldrich) and Calliphora nigribarbis Snellen van Vollenhoven (Diptera: Calliphoridae), collected in the vicinity of infected poultry at a farm in Kyoto, Japan (Sawabe et al. 2006) . The persistence of HPAIV H5N1 in house ßies and blow ßies as well as Newcastle disease virus (family Paramyxoviridae, genus avulavirus, NDV), turkey coronavirus (family Coronaviridae, genus Coronavirus, TCV), and reticuloendotheliosis virus (family Retroviridae, genus Gammaretrovirus, REV) has been studied in the laboratory (Calibeo-Hayes et al. 2003; Davidson and Braverman 2005; Chakrabarti et al. 2007 Chakrabarti et al. , 2008 Watson et al. 2007; Sawabe et al. 2009; Barin et al. 2010; Wanaratana et al. 2011) . In all studies, ßies fed with the viruses became transiently contaminated and the persistence of the virus in crop or alimentary canal declined with prolonged incubation period of the ßies. Importantly, a recent study by Wanaratana et al. (2011) demon-strated that H5N1 HPAIV can persist in the viscera of house ßies up to 72 h postexposure in the laboratory. Watson et al. (2007) and Calibeo-Hayes et al. (2003) further examined the transmission of NDV (Roakin strain) and TCV, respectively, to susceptible poultry by house ßies. Based on these studies, Watson et al. (2007) concluded the house ßy to be a poor vector of NDV (Roakin). In contrast, Calibeo-Hayes et al. (2003) successfully infected turkeys by placing them in contact with TCV-exposed ßies.
The current study investigated the persistence of LPAIV in the alimentary tract of house ßies that were fed various concentrations of subtypes H5N7 and H7N1 viruses and incubated at different temperatures for up to 24 h. Flies were subsequently tested for the persistence of infective virus. full volume of virus solution were discarded. Fed ßies were transferred to closed cages in groups of Þve for each combination of virus dilution, temperature, and incubation period. Fed ßies were stored at room temperature until all 60 ßies included in each experimental group were fed, after which the cages were placed in incubators and the incubation period of 6, 12, or 24 h was started. Importantly, the order with which ßies were fed with the different virus dilutions was consistent among all experiments. Flies had unlimited access to whole-fat, pasteurized and homogenized milk during incubation. Sham-fed ßies dined on equal volumes of sterile AF and were incubated as described above. Also, 0-h experiments were included, in which groups of Þve ßies were fed 1-l volume of either sterile AF or the three virus solutions for each virus strain. These ßies were subsequently kept at room temperature (24ЊC) for 10 min without access to milk before termination. Likewise, for each experimental round another Þve ßies were tested for virus as described below without any feeding or incubation.
Virus Isolation From Flies. All ßies were killed by freezing at Ϫ20ЊC for at least 30 min. Subsequently, body ßuids, crop and intestinal organs from the abdomen were immediately harvested aseptically as described below. Alternatively, ßies were kept at Ϫ80ЊC up to 48 h before harvest. While holding the ßy with a sterile forceps in a sterile petri dish, the area surrounding the ßyÕs cloaca was sterilized with a heated scalpel and punctured by a sterile needle. The content of the abdomen was harvested by a micropipette with a sterile disposable tip. The contents from each group of Þve ßies were pooled in 0.5 ml of medium [PBS (8 mM) with penicillin (8,100 U/ml), streptomycin (8,100 g/ml), gentamicin sulphate (200 g/ml), nystatin (8,100 U/ml), fetal bovine serum (4.5% vol:vol), and phenol red 2‰ (0.4% vol:vol)]. To detect the presence of infectious virus, undiluted 200-l aliquots of each pooled sample supernatant were inoculated into two SPF ECEs within 12 h of preparation by following a modiÞed version of the EU Directive 2005/ 94/EC (http://vla.defra.gov.uk/science/sci_ai_reßab_ man.htm). Samples were homogenized before inoculation. The individual groups of ßies were scored as virus-positive if any of the SPF ECEs were infected (dead embryo and hemagglutinin-positive allantoic ßuid) by primary inoculation or after one blind passage. Similarity between inoculated and isolated virus was conÞrmed by sequencing of reverse transcription-polymerase chain reaction (RT-PCR) amplicons. For each subtype of AIV, samples representing different combination of fed virus concentration, temperature, and incubation period were examined separately.
Persistence of Virus Dilutions. After each experiment, the three two-fold diluted aliquots of the AIV stocks were incubated at 15, 25, and 35ЊC for 12 and 24 h. After incubation, selected virus-dilutions (the 2 Ϫ4 dilution incubated at 35ЊC, the 2 Ϫ2 dilution incubated at 25ЊC, and the 2 0 dilution incubated at 15ЊC) were titrated. Titrations were performed by 10-fold dilutions in SPF ECEs.
Extraction of Viral RNA. Viral RNA was extracted from AF from selected eggs with virus isolates using the RNeasy mini kit (QIAGEN, Hilden, Germany) as described previously (Slomka et al. 2007 ). RNA was stored at Ϫ18ЊC.
RT-PCR and Sequencing. H5 and H7 subtype spe-ciÞc PCRs were performed by following the H5 conventional PCR protocol and H7 cleavage-site real-time PCR protocol described in Slomka et al. (2007) and the European Union protocol (http:// vla.defra.gov.uk/science/docs/sci_ai_vi536.pdf), respectively. Both strands of the amplicons were sequenced using the PCR primers at DNA Technology A/S (Risskov, Denmark).
Statistical Analysis. The statistical analysis was carried out by logistic regression, using the General Linear model (GLM) procedure in SPlus (Insightful Corp. 2002) . The dependent variable, AIV-positiveness of samples, was dichotomous and attained values of 0 or 1 only.
The probability p that an observation X describing virus status for an individual pool of Þve ßies takes the value one or 0 (i.e., the ßy pool is virus-positive [X ϭ 1] or the ßy pool is virus-negative [X ϭ 0]) is modeled as follows, where logit is the logistic transformation logit(p) ϭ log(p/(1 Ϫ p)):
so that logit(p) is regressed on temperature (T; room temperature [24ЊC], 15, 25, and 35ЊC), incubation period postfeeding of the ßies (I; 0, 6, 12, and 24 h), and AIV virus concentration (C; H5N7: 10 6.7 , 10 7.3 , and 10 7.9 ELD 50 /ml; H7N1: 10 7.3 , 10 7.9 , and 10 8.5 ELD 50 / ml); the fed virus concentrations were log 10 transformed to stabilize the variance. The constant ␣ was allowed to depend on virus strain (S), and interactions of up to third order between the four dependent variables T, I, C, and S was investigated using a stepdown procedure where the interaction terms were added simultaneously to the model 1 and subsequently deleted to reduce this full model. This was done as stepwise reduction of the full model, including interactions, excluding nonsigniÞcant parameters (P Ͼ 0.05) from the model, by using likelihood ratio tests for model reduction.
Because the analyzed pools of Þve ßies are independent samples, the probability p for containing a virus-positive ßy pool after a given incubation period may be translated as an estimate for the frequency of ßy pools to be positive for virus. Hence, with q denoting logit(p), the probability p is found from q as
Overall, only two ßies of the total 1,195 ßies died during the incubation period. Both of these represented H7N1-fed individuals kept at 35ЊC for 24 h. The ßiesÕ level of activity and foraging at the three incubation temperatures was strongly affected by their poikilothermic nature. As expected, the pools of Þve ßies selected randomly as negative controls before each experimental setup were all negative for virus, conÞrming the ßies be bred under virus-free conditions. Similarly, all groups of ßies fed with sterile allantoic ßuid were negative for virus (Table 1 ). The persistence of infectious virus of the H5N7 and H7N1 AIV subtypes in the ßy pools are given in Table 1 . Correspondence between fed virus subtype and harvested samples from the ßies was tested on randomly selected samples by RT-PCR and sequencing of the amplicons; identical similarity was conÞrmed in all cases. Virus persistence in the three virus dilutions for both virus strains was tested by incubating aliquots at 15, 25, and 35ЊC for 12 and 24 h. Subsequent titration of selected aliquots demonstrated the titers of the virus to be stable (data not shown).
As seen in Table 1 , the isolation of infectious virus from house ßies strongly depended on T, fed C, and I. The importance of the four covariates T, C, I, and S was statistically tested by the logistic regression presented in model 1. Stepwise reduction of the full model gave rise to the Þnal model described in model 3:
logit ͑p) ϭ ␣ S ϩ ␤ T *T ϩ ␤ I *I ϩ ␤ T:I *T*I ϩ ␤ S:C *C [3] which included the main effects of T and I and in addition the interactions between temperature and incubation period T:I and between virus strain and fed virus concentration S:C. The estimated parameters in the reduced model 3 are listed in Table 2 , and the constant terms ␣ S and the parameter ␤ S:C are virus strain speciÞc. The logistic regression transformation was based on the experimental replicates presented in Table 1 . The room temperature was Ϸ24ЊC, and the effect of this approximation on the statistical analysis was tested by a sensitivity analysis in which the room temperature was varied from 20 to 28ЊC. Stepwise reduction from model 1 including all interaction terms was repeated until no signiÞcant factors could be removed, and in all situations, the stepwise reduction gave rise to the reduced regression model 3, indicating the approximation did not affect the model. Due to the method used to feed the ßies, there was uncertainty about the determination of the time covariate T; however, a sensitivity analysis demonstrated that this did not affect the reduced regression model 3. Likewise, a sensitivity analysis conÞrmed that the inclusion of the 0 h experimental data did not alter model 3. Statistical analyses conÞrmed that the persistence of infective virus from virus-fed ßy pools decreased as a function of virus concentration, incubation period postfeeding, and temperature ( Table 2 ). The coefÞcients for T and I cannot be regarded on their own, because of the signiÞcant T:I interaction effect (Table 2) . Thus, for a temperature of 15ЊC, the effect of incubation period is given by the slope ␤ I ϩ 15␤ T:I , which Table 2 estimates as 0.206 Ϫ15⅐0.0165 ϭ Ϫ0.0415. Obviously, the slope is lower for higher temperatures. Similarly, the effect of temperature at an incubation period of 6 h is ␤ T ϩ 6␤ T:I , which is estimated as 0.0328 Ϫ 6⅐0.0165 ϭ Ϫ0.0662. In the same way, the coefÞcients for S and C must be combined with the corresponding interaction coefÞcient, to represent the effects of strain and virus concentration, respectively. As examples, the estimated persistence of virus-positive ßy pools for each employed AIV subtype fed with a virus concentration of 10 7.3 ELD 50 /ml are shown in Fig. 1 . The estimated incubation periods at which 50% of ßy pools fed with either of the virus The regression model was based on sample size n ϭ 171. Parameter abbreviations are explained in the main text under Statistical Analysis. Estimates denote the parameter values, and SE is the standard error of the estimates of the parameters. a P value includes removal of interaction term. b The effect of C is dependent on S and shown under S:C. 
Our results demonstrated the decline of virus at all concentrations and temperatures over time. In fact, only one group of ßies was found virus positive out of 36 tested (3%) after 24 h at 25ЊC and 35ЊC, indicating that the two LPAIV strains did not replicate in the ßyÕs alimentary tract. This indicates that the house ßy may be a potential passive carrier of LPAIV, in agreement with previous observations on the survival of HPAIV, NDV, TCV, and REV in house ßies and blow ßies (Calibeo-Hayes et al. 2003 , Davidson and Braverman 2005 , Watson et al. 2007 , Chakrabarti et al. 2008 , Sawabe et al. 2009 , Barin et al. 2010 , Wanaratana et al. 2011 .
The statistical analyses demonstrated the persistence of infectious LPAIV in the alimentary tract to be virus strain dependent. Although it cannot be excluded that the primary, secondary, and tertiary structural differences in the projecting surface molecules on the two LPAIV strains used may be correlated with different inactivation kinetics and excretion rate, this contribution was assumed to be questionable. The observed differences may reßect strain-related adaption of the two virus strains to cultivation in ECEs, strain-related pathogenicity and virus doses. The difference in persistence is especially prominent for the most diluted virus solutions. For example, one of the 0-h experiments with the most diluted virus solution of H5N7 was virus-negative, although the ßies were fed only 10 min before termination. However, the reason for this strain difference is at present unknown, and further experiments including a variety of different LPAIV and HPAIV strains are required to address this issue.
Our study was based on ßies individually fed known virus concentrations, and only those ßies that ingested all of the provided volume of virus solution were included in the study. Although this procedure was time-consuming and caused some time delay before incubation, it was considered essential to ensure that all virus-exposed ßies had ingested the required volume of virus. The house ßy frequently regurgitates and defecates during foraging (Greenberg 1973) , so the ingested doses of virus cannot be controlled by using ad libitum access to infective allantoic ßuid. The focus of the current study was on the persistence of AIV in the alimentary tract; therefore, virus particles present on the ßyÕs exoskeleton had to be excluded in the recovery procedures for infectious virus in the experiments. To obtain this, a method was developed for aseptic harvest of the content of the ßyÕs posterior abdomen that involved thermal inactivation of external virus particles. The house ßyÕs irregular integument surface may complicate external disinfection by solutions, a potential obstacle which we solved by thermal inactivation. This approach also made it possible to subsequently process the harvested pooled ßy samples in small volumes convenient for virus isolation. However, it cannot be excluded that the method may have led to some thermal inactivation of infective virions located within the alimentary canal.
Excretion of virus particles during regurgitation and defecation in addition to inactivation of virions in the alimentary tract due to exposure to digestive enzymes, low pH levels, and bacterial ßora probably contributed to the decrease in the number of virus positivepools over time (Sinha 1976 , Espinoza-Fuentes and Terra 1987 , Terra et al. 1988 , Nazni et al. 2005b . Table 2 .
Despite our using preoviposit females in the study, egg development was pronounced after 24 h of incubation, which occasionally decreased the volume of harvested samples; an observation that might have affected the detection of AIV in these samples upon cultivation in ECEs.
In the presented work, the use of LPAIVs was based on the common occurrence of these viruses in wild birds and domestic poultry and the presumed transfer between these groups of hosts. Also, LPAIV of H5 and H7 subtypes have the potential to mutate to HPAIV in poultry, causing devastating outbreaks of disease. Because of their high morbidity and mortality, HPAIV do not circulate between wild birds and domestic poultry as does the LP variants. Furthermore, LPAIVs were used in our study for safety reasons because it was not feasible to contain house ßies exposed to HPAIV in the laboratory. LPAIV may not represent a complete model for persistence of HPAIV in house ßies. HPAIV H5N1 was recently shown to persist in the viscera of house ßies up to 72-h postexposure in the laboratory (Wanaratana et al. 2011) , whereas only one of the 36 virus-fed ßy pools incubated at 25 or 35ЊC for 24 h was positive for virus in our study. Similarly, a velogenic exotic NDV strain was demonstrated to persist in house ßies for a longer period of time than the less virulent mesogenic NDV (Roakin) strain (Watson et al. 2007 , Chakrabarti et al. 2008 ). However, differences in the sensitivity of laboratory detection of viruses related to their pathogenicity may inßuence these results.
The synanthropic and omnivorous house ßy is a very common and abundant livestock farm pest, which is attracted by moist manure and other fermenting organic waste that serves as a food source as well as developing environment of the ßy (Axtell 1999 , Malik et al. 2007 , Hald et al. 2008 . Because chickens capture and eat ßying insects (Hald et al. 1998 , Hald et al. 2008 , ßies foraging on virus-containing feces represent a risk in the spread of disease. Chickens infected with AIV shed virus in secretions and excretions, and although HPAIV typically are found in both feces and respiratory secretions, the primary route of shedding of LPAIV depends on the virus strain (Spickler et al. 2008 ). Viral quantities of 10 3.2 Ð10 7.9 and 10 5.5 Ð10 8.1 EID 50 /ml have been reported in cloacal and fecal samples, respectively, from chickens infected in the laboratory with LPAIV H1, H4, and H6 subtypes (Morales et al. 2009 ). Shed titers up to 10 4.5 and 10 4.3 EID 50 /ml have been observed in cloacal swabs from chickens infected with LPAIV H5N1 and H7N2 subtypes, respectively (Swayne and Beck 2005, Spackman et al. 2007 ). In our experiments, high virus doses were used to be able to monitor the persistence over time. Each ßy ingested a minimum dose of 10 3.7 ELD 50 (H5N7) and the pool of Þve ßies a minimum total of 10 4.4 ELD 50 , which was sufÞcient to infect ECEs. Under natural conditions, the dose of virus that a house ßy may ingest when foraging on virus-containing feces will depend on the amount and persistence of shed virus, the ßyÕs hunger state as well as the size of the ßy. Large ßies are expected to uptake more food when foraging than smaller individuals and thus ingest more virus. A female house ßy can ingest a volume to 2Ð 4 l (Kobayashi et al. 1999) , and the frequency of foraging depends on factors such as temperature, level of stress and the food source. Although an individual ßy might not ingest a sufÞcient virus dose to infect a chicken, the uptake of several infected ßies may be critical and lead to infection. In this respect, Calibeo-Hayes et al. (2003) demonstrated that under controlled conditions the number of turkeys infected with TCV increased when placed in contact with higher numbers of TCVexposed ßies per bird.
In general, adult house ßies remain within a few kilometers of their breeding sites for their entire life (Nazni et al. 2005a , Boase 2007 , Stafford 2008 ). Therefore, house ßies represent potential carriers of AIV among chickens placed in the same building or farm or between poultry located on premises in proximity.
Our study demonstrated that infective low-pathogenic avian inßuenza virus of the H7N1 and H5N7 subtypes can be isolated from the alimentary tract of house ßies for at least 24 h postfeeding and that factors such as temperature, incubation period postfeeding, and load of ingested virus play an important role in the persistence of infective virus. Because house ßies commonly occur on premises with poultry production, the data indicate that house ßies can be a potential risk in the dissemination of avian inßuenza virus. However, studies that address whether ßies and in particular house ßies and blow ßies are able to cause infection after ingestion of AIV in poultry should have high priority. Similarly, studies on the possibilities of ßies to ingest chicken infective doses of AIV in poultry production environments and in nature are needed.