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Susceptibility of Muscovy (Cairina Moschata) and mallard ducks (Anas Platyrhynchos) to experimental infections by different genotypes of H5N1 avian influenza viruses
Veterinary Microbiology 148 (2011) 168–174
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Research article
Susceptibility of Muscovy (Cairina Moschata) and mallard ducks (Anas Platyrhynchos) to experimental infections by different genotypes of H5N1 avian influenza viruses Do Quy Phuong a,b,c,*, Nguyen Tien Dung a, Poul Henrik Jørgensen d, Kurt Jensen Handberg d, Nguyen The Vinh a, Jens Peter Christensen b a
National Institute of Veterinary Research, 86 Truong Chinh, Dong Da, Hanoi, Viet Nam Department of Veterinary Pathobiology, Faculty of Life Sciences, University of Copenhagen, Stigbøjlen 4 - building 1-20 1870 - Frederiksberg C, Copenhagen, Denmark c Sub-department of Animal Health of Thai Binh province, 14 Quang Trung, Thai Binh city, Viet Nam d National Veterinary Institute, Danish Technical University, Hangøvej 2, DK-8200 A˚rhus N, Denmark b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 March 2009 Received in revised form 21 August 2010 Accepted 7 September 2010
It is a fact that in Viet Nam, Muscovy ducks are raised in large populations (approximately 8 million), usually kept in small flocks together with mallards and chickens. As a result, it is a great concern for epidemiologists to elucidate possible differences in relation to these species being exposed to infection with H5N1. To do this, an experimental study on infections with different genotypes of H5N1 in mallards and Muscovy ducks have been conducted, where it was found that the mortality of the inoculated Muscovy ducks was at least 80%, regardless of the virus strain employed. In contrast, the mortality of the mallards ranged from nil to 100%, which suggests that Muscovy ducks are more susceptible to HPAIV H5N1 infection in terms of disease development and mortality. It was also found that higher virus titers developed in vital organs of Muscovy ducks compared to mallards, particularly in the brain. Due to their high susceptibility, it is unlikely that Muscovy ducks act as a silent reservoir. The virus strains used in this study, to a certain degree, differed in their virulence properties to the bird species in question. ß 2010 Elsevier B.V. All rights reserved.
Keywords: H5N1 Avian influenza Ducks Muscovy ducks Mallard ducks Experimental infection
1. Introduction The highly pathogenic avian influenza (HPAI) virus H5N1 was, for the first time, isolated and recognized as the cause of avian influenza outbreaks (AI) in poultry in Viet Nam in late 2003 (Delquigny et al., 2004). At the start, the outbreak was already fast and massive. As a result, 57 out of the 64 provinces of the country were affected, where it was found that massive die-off and culling of poultry and
* Corresponding author at: Sub-department of Animal Health of Thai Binh province, 14 Quang Trung, Thai Binh city, Viet Nam. Tel.: +84 36 3831539. E-mail address: [email protected] (D.Q. Phuong). 0378-1135/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2010.09.007
other bird species took place. In addition, 27 human cases affection with H5N1 avian influenza were reported in the first 2 years of the AI crisis, of whom 20 were fatal (WHO, 2004). With regard to location, at the start, H5N1 AI outbreaks mainly occurred in the Red river and Mekong river deltas (Pfeiffer et al., 2007; WHO, 2007) and it was observed that chickens showed clinical disease and consequently died at a higher rate than ducks. Nevertheless, ducks were also affected in the early phase of the outbreak, accounting for 23% of the total 44 million birds destroyed in the 2003–2004 period (Anonymous, 2007Viet Nam DAH, 2007) and were considered as the natural carriers of the AI virus, i.e. ducks infected with AI viruses did not develop clinical diseases (Sturm-Ramirez et al., 2005; Saito et al., 2008).
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To respond to the outbreak, a national vaccination campaign was started in September 2005, after which it was found that the number of outbreaks in both chickens and ducks had a sharp decline and that AI cases were reported neither in humans nor in birds in 2006 (WHO, 2007). However, ducks still appeared to be infected and most H5N1 AI outbreaks reported in 2007 occurred in unvaccinated duck flocks being less than 3 months old. As an illustration, between May and July 2007, it was reported that 245,000 birds had been culled because of the AI crisis, in which 94% were waterfowl (Anonymous, 2007Viet Nam DAH, 2007). It had been thought that mallard and Muscovy ducks were both waterfowl by nature and therefore would be infected in the same manner. By contrast, observations have shown that the two species would react differently to the infection, when severe clinical diseases with a high rate of mortality were often seen in Muscovy ducks in Viet Nam (Nguyen, personal observation). Similar observations have also been reported from Italy (Capua and Mutinelli, 2001). As mallards and Muscovy ducks mingle all over the country, it is highly relevant to elucidate the differences in response to infection with H5N1 between the two species and the possible significance for the epidemiology of AI. For this reason, experimental infections with different genotypes of H5N1 virus in mallard and Muscovy ducks have been conducted. 2. Materials and methods 2.1. Experimental studies All the experiments were performed in a reference laboratory for influenza at the National Institute of Veterinary Research (NIVR), Viet Nam under BSL-3 conditions. Viruses: Four HPAI H5N1 viruses were used in this study: A/duck/Vietnam/40D/04 (Dk/vn/04), A/Duck/ Vietnam/1469/05 (Dk/vn/05), A/M.duck/Vietnam/1455/ 06 (M.dk/vn/06) and A/Duck/Vietnam/37/07 (Dk/vn/07). They originated from clinical outbreaks of HPAI and represented different genotypes of H5N1 (Table 1). Stock viruses were grown in 10-day-old embryonated chicken eggs for 36–48 h at 37 8C. The allantoic fluid was
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harvested, and aliquots were stored at 80 8C until use. Virus titer was determined according to Reed and Muench (1938). The isolate Dk/vn/04 was used to infect the birds as a reference study with a detailed characterization of the infection in mallard and Muscovy ducks. The other three isolates were used in more simple additional studies to supplement the former reference study. Inocula: Each inoculated bird was inoculated with 0.2 ml of virus stock diluted in phosphate buffered saline, equivalent to 106 EID50 by the intranasal route. Experimental animals: Mallard ducks, Muscovy ducks and chickens (Luong Phuong line) were raised on isolated farms. They were transferred to the NIVR facilities for experimental infections at the age of 4–6 weeks, and no vaccinations had been applied to the birds. Oropharyngeal, cloacal swabs and serum samples were collected from these birds before inoculation to confirm freedom of active infection with an AIV and serum antibodies specific to subtype H5 AIV. 2.2. Experimental design 2.2.1. Main infection study The main infection study was performed in two separate infection experiments, both employing A/duck/ Vietnam/40D/04. Fifteen mallard ducks aging 4–6 weeks old were inoculated and later introduced with contact birds of 2 mallards and 2 chickens at intervals of day 1, 5, 10, and 15 post-inoculation (p.i.), respectively. The contact birds shared a cage with the inoculated birds for 24 h, then were transferred to a separate cage for 1 more week of observation. In the second experiment, 10 Muscovy ducks were inoculated and the contacts birds (2 Muscovy ducks and 2 chickens) were only introduced on days 1, 5 and 10 following infection. Signs of disease were recorded on daily basis according to the following scoring system: low, medium and high. Low scoring was used when only unspecific clinical signs were observed; medium scoring was given if some disease signs and low to medium mortality rate were observed; and the high scoring was used if obvious clinical signs including CNS signs and a high mortality occurred. However, for welfare reasons, any bird showing difficulty in eating or drinking and/or severely impaired ability to
Table 1 Mean dead time, mortality and clinical signs in mallards, Muscovy ducks and chickens following infection with four different H5N1 avian influenza viruses. Virus strains
Genotype
Year
Sub-lineage
Species
MTD (in days)
No. dead/total
Inoculate
Contact
Inoculate
Contact
Clinical score Inoculate
Contact
Dk/vn/04
Z
2004
Clade 1
Mallard Muscovy Chicken
5.4 (5–6) 4.6 (4–6) ND
No dead 4.8 (2–5) 3.1 (2–5)
8/15 8/10 ND
0/18 4/6 10/15
Medium High ND
Low High High
Dk/vn/05
Z
2005
Clade 1
Mallard Muscovy
4.4 (3–6) 3.6 (3–5)
4.8 (3–6) 5.2 (3–7)
5/5 5/5
4/5 5/5
High High
High High
M.dk/vn/06
G
2006
Clade 2.3.2
Mallard Muscovy
No dead 5.3 (5–6)
No dead 5.5 (5–7)
0/5 4/5
0/5 2/5
Low High
Low Medium
Dk/vn/07
Z
2007
Clade 2.3.4
Mallard Muscovy
4.0 (3–4) 3.8 (2–5)
5.4 (4–5) 4.8 (3–4)
4/5 5/5
5/5 5/5
High High
High High
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move was killed and scored as dead. Oropharyngeal and cloacal swabs were collected in 1.5 ml transport medium from all birds every 2 days p.i. for virus detection. All the birds (both inoculated and contact birds) were subjected to post-mortem investigation, where tissue samples which were obtained from brains, lungs, spleens, intestines, trachea and muscles at necropsy, were stored at 80 8C until virus isolation and titration. Surviving inoculated birds and contact birds were tested from day 5 p.i. every 2 days for H5 antibodies in serum. The experiment continued until shedding of virus from inoculated birds could not be detected by real-time RT-PCR in cloacal and oropharyngeal swabs. Virus recovered at day 10 p.i. from the infected mallards was used in a further infection study aiming at determining any possible changes in virulence of the virus. For this purpose, 8 mallards and 3 chickens were inoculated as described above. 2.2.2. Additional infection study Mallards and Muscovy ducks in groups of five aged 5–6 weeks were inoculated with the following strains: Dk/vn/ 05, M.dk/vn/06 and Dk/vn/07, respectively. Then, five contact birds of the same species were introduced to the inoculated birds at day 1 post infection and were monitored/scored for 15 days. The birds were observed for clinical signs of disease every 24 h over a period of 10 days, and each bird was scored as described above. Tissue samples from the brain of all dead or euthanized birds were collected for virus isolation and titration. 2.3. RT-PCR and virus titration Every 2 g of tissue samples were homogenized in 4 ml of transport medium by the use of a porcelain mortar and pestle. RNA extraction and RT-PCR were performed as described (Munster et al., 2005). RNA from swabs and tissue suspensions was extracted by using QIAGEN RNeasy Mini Kit as recommended by the producer (QIAGEN GmbH, D40724 Hiden). Real-time RT-PCR assays were performed on a BioRad iQ5 Sequence Detection System machine by using QIAGEN One Step RT-PCR Kit Cat, no. 210212 (QIAGEN GmbH, D-40724 Hiden). The test used a hybridization probe (6-FAM-TCAGGCCCCCTCAAAGCCGA-TAMRA) and specified primers (forward: 50 -ARAT-GAGTCTTCTRACCGAGGTCG-30 and reverse: 50 -TGCAAAGACATCYTCAAGYYTCTG-30 ) to detect the matrix gene segment AIV as previously described (Runstadler et al., 2007). For each run, the samples were prepared and processed in parallel with several negative and positive control samples. Virus was quantified by inoculation of serial 10-fold dilutions of supernatant from the homogenized tissue samples and swabs in quadruplicate of MDCK cell cultures, as described by Rimmelzwaan et al. (1998). To determine possible modification of the inoculated virus strain in the HA gene during the infections in the reference infection experiment, 11 isolates were obtained throughout the study from the brain and the intestine. Samples were obtained from both dead and killed chickens and ducks until day 7. In addition, virus was isolated from contact ducks and chickens day 2–5 post contact.
2.4. Sequencing Sequencing was done according to previous reports (Slomka et al., 2007). The PCR primers KHA-1 and KHA-3 were used both as initial RT-PCR primers and as sequencing primers. The resulting sequences were approximately 350 bp long and including HA0 cleavage site. The sequences were assembled and aligned by the Invitrogen software Vector NTI version 10.3. 2.5. Serological test Pre- and post-infection serums were tested for H5 antibodies by using standard procedures (Thayer and Beard, 2008). The HI tests were performed by using a 0.5% suspension of chicken erythrocytes in phosphate-buffered saline. HI was assessed using 25 ml each of a series of serum dilutions 1:2, and 25 ml of HA antigen, standardized at 8 hemagglutination units (HAU) by hemagglutination titration. Serum was incubated for 30 min at 56 8C in a water bath to eliminate the nonspecific inhibitors, and the first dilution on the test plate was 1:8. All HI titers >8 were considered positive. None of the birds used in the infection studies were antibody positive to H5 prior to infections. 3. Results 3.1. Experimental infections Except for the strain M.dk/vn/06, all the strains used in the study caused clinical signs, mortality and gross lesions typical of HPAI following intranasal inoculation in both mallard ducks and Muscovy ducks. Although M.dk/vn/06 did not cause any mortality in mallards and only mild clinical signs, but it affected Muscovy ducks at a similar level with the other strains used. The clinical signs observed throughout the study varied from mild to severe (Table 1). Differences in mortality between mallard ducks and Muscovy ducks were observed following inoculation with Dk/vn/04 and M.dk/vn/06 and were also expressed in the level of mortality observed in the contact birds. Following infection with Dk/vn/04, no mortality was observed in mallards in contact with inoculated mallards while 66% of the Muscovy ducks in contact with inoculated Muscovy ducks died. M.dk/vn/06 showed low virulence to mallards and their contacts but showed full virulence for Muscovy ducks and their contacts, with 40% of the contact Muscovy ducks being fatal. It was also observed that the strains Dk/vn/04 and Dk/vn/07, both belong to genotype Z but to different antigenic clades differed in their virulent properties in mallards. Dk/vn/07 was in contrast to Dk/vn/ 04 in that the former is more capable of causing high mortality than the latter in contact mallards. In general observations, the mean death time (MDT) in Muscovy ducks is shorter than of those in mallards, regardless of virus strains. Mortality of the contact chickens was not observed after the second introduction of chickens to inoculated mallards; whereas the third group of chickens in contact with inoculated Muscovy ducks died (no further introduction of the fourth group) (Tables 2 and 3).
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Table 2 Reference infection study on susceptibilities of mallards to A/duck/Vietnam/40D/04. Group of birds
MDT (in days) Inoculate mallard
Inoculate group Contact group.1 (day 1 p.i.)
b
Contact mallard
Contact chicken
No dead
Inoculate mallard
Clinical score
Contact mallard
Contact chicken
4.5 (4–5)
Contact chicken
Contact mallard
Contact chicken
0/2
2/2
Low
High
3–6
NDa
No dead
2 (2)
0/2
2/2
Low
High
3–6
ND
No dead
No dead
0/2
0/2
Low
Low
NSb
NS
No dead
No dead
0/2
0/2
None
None
NS
NS
8/15
Inoculate mallard
Shedding time
Contact mallard
5.4 (5–6)
Contact group.2 (day 5 p.i.) Contact group.3 (day 10 p.i.) Contact group.4 (day 15 p.i.) a
No. dead/tatal
Medium
Inoculate mallard 7–13
ND: not done. NS: not shed.
As shown in Tables 2 and 3, none of the inoculated surviving mallards and Muscovy ducks shed virus for more than 13 and 14 days, respectively. The investigation into the pathogenicity of virus isolated late in the main infection study did not reveal any changes, compared to the initial inoculate.
found that the virus titers obtained from vital tissues of Muscovy ducks were higher than those of mallards at day 5 and 6. In mallards alone, it was observed that the titers were higher in intestines than in tracheas (day 5 and 6), whereas, titers obtained from the trachea of Muscovy ducks were always higher than those in the intestines.
3.2. Virus in tissues
3.3. Sequencing
The virus titers obtained from different tissues of all groups of birds that died at day 5 post infection (or contact) or killed day 5 due to severe clinical signs are shown in Fig. 1. The most obvious difference between the groups of birds was in relation to the titer obtained from the brain tissue, which is significantly higher in the inoculated Muscovy ducks than in any other group. Furthermore, the contact Muscovy ducks also developed high titers in the brain. The titers obtained from the inoculated mallards were more than 50% lower in Muscovy ducks, and no positive titers were recorded from the brain of the contact mallards (Fig. 1). A more detailed comparison of the titers obtained from different tissues of the infected mallards and Muscovy ducks is shown in Fig. 2. As no mallards died before day 5 post infection, no data were obtained during the first sampling points for this type of ducks. However, the data that were obtained from dead Muscovy ducks demonstrated high titers of the virus in lung and trachea, especially during courses of per-acute/ acute of infections. The brain and muscles also showed high titers. General speaking, when comparing data, it was
The partial sequencing of the HA gene of 11 isolates of Dk/vn/04 obtained from brain and intestinal samples showed no differences – all sequences were identical. 3.4. Serology In the survival cases, both the infected mallards together with their contacts and the infected Muscovy ducks together with their contacts developed a positive antibody titer (HI titer (log 2) >6–7) at day 7. No seropositive birds were observed before day 7. However, the surviving mallards and chickens in contact groups 3 and 4 did not develop a positive titer, but only the two of them in contact group 3 were positive to virus in tissues. 4. Discussions Few reports are available on HPAI and Muscovy ducks. However, Capua and Mutinelli (2001) reported that during the 413 outbreaks of HPAI (H7N1) occurring in Italy 1999– 2000, Muscovy ducks and geese were the only species of waterfowl being affected in spite of the fact that several of
Table 3 Reference infection study on susceptibilities of Muscovy to A/duck/Vietnam/40D/04. Group of birds
MDT (in days) Inoculate Muscovy
Inoculate group Contact ggroup.1 (day 1 p.i.) Contact ggroup.2 (day 5 p.i.) Contact ggroup.3 (day 10 p.i.) a
ND: not done.
No. dead/tatal
Contact Muscovy
Contact chicken
4
Clinical score
Contact Muscovy
Contact chicken
3–4
1/2
4
4
2–3
3
4.6 (4–6)
Inoculate Muscovy
Shedding time
Contact Muscovy
Contact chicken
2/2
High
1/2
2/2
2/2
2/2
8/10
Inoculate Muscovy
Contact Muscovy
Contact chicken
High
NDa
ND
High
High
ND
ND
High
High
ND
ND
High
Inoculate Muscovy 9–14
[(Fig._1)TD$IG]
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172 8.0
Virus titer (log10 TCID50/ml)
7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 Inoculated mallard group (3 birds)
Inoculated Muscovy group (4 birds)
contact mallard group (5 birds)*
contact Muscovy group (4 birds)**
Contact chicken group (3 birds)***
Groups of birds Brain
Lung
Spleen
Intestine
* Include contact duck group 1, 2 and 3 **Include contact Muscovy duck group 1 and 2 *** Include contact chicken group 1 and 2 Fig. 1. Virus titer in different tissues of the different groups of birds in the main study at day 5 post inoculation with A/duck/Vietnam/40D/04 (included dead and killed birds). * Include contact duck groups 1, 2 and 3. **Include contact Muscovy duck groups 1 and 2. *** Include contact chicken group 1 and 2.
the flocks also contained mallard ducks. Few histopathological lesions were observed in the dead Muscovy ducks, and if they were present, they were mild ones. The previous challenge of 6 weeks old Muscovy ducks with a strain of H5N1 resulted in 100% mortality within 1 week,
[(Fig._2)TD$IG]
Virus titer (log TCID50/ml)
a
but no further characterization of the infection was done (Steensels et al., 2007). In this study, however, the mortality of the inoculated Muscovy ducks was found to be at least 80% regardless of the virus strain employed. In contrast, the mortality of the
Virus titer in different tissues of dead mallard ducks 7 6 5 4 3 2 1 0
No mallard ducks died before day 5 p.i
Day 2
Day 3
Day 4
Day 5
Day 6
(0 birds)
(0 birds)
(0 birds)
(5 birds)
(3 birds)
Time after infection (day) Brain
Spleen
Intestine
Trachea
Muscular
Virus titer in different tissues of dead Muscovy ducks
b Virus titer (log TCID50/ml)
Lung
7 6 5 4 3 2 1 0 Day 2
Day 3
Day 4
Day 5
Day 6
(1 bird)
(1 bird)
(4 birds)
(4birds)
(2birds)
Time after infection (day) Brain
Lung
Spleen
Intestine
Trachea
Muscular
Fig. 2. Virus titer in different tissues of dead mallard and Muscovy ducks at period of time post inoculation with A/duck/Vietnam/40D/04 (included inoculated and contact birds).
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inoculated mallards ranged from nil to 100%, which suggests that Muscovy ducks are more susceptible to HPAI H5N1 virus infection with regard to the development of disease and mortality. A shorter MDT of the Muscovy ducks further substantiated this. The virulence of a particular virus following the infection of either Muscovy ducks or Mallards was, to some degree, reflected in the mortality that was observed among the contacts birds. In the main infection study, Dk/vn/04 was found to be moderately virulent for infected mallards but of low virulence for the contact mallards. The same isolate was found highly virulent for infected as well as contact Muscovy ducks. The mortality of chickens following contact to infected mallards and Muscovy ducks was 100% for the first 2 groups of contact chickens (introduced on day 1 and day 5). After that, no chickens in the third contact group died when brought into contact with the infected mallards at day 10 post infection. Only three contact groups were introduced to the infected Muscovy ducks and they all died. As it was demonstrated that the isolate shed from the mallards at day 10 was as virulent as the initial strain. The results also indicated that the level of excretion from the inoculated birds and the susceptibility of the contacts were the main reasons for the disease/ mortality pattern that was observed during the experiment rather than a reduced virulence of the initial strain following the challenge. Both the mallards and the Muscovy ducks shed virus for up to from 13 to 14 days, which is in accordance with the results from a previous study (Hulse-Post et al., 2005). Thus, the last introduced contacts (at days 10 and 15) were probably exposed to lower concentrations of the virus, as suggested by Hulsepost et al. (2005), and only the highly susceptible contacts (Muscovy ducks and chickens) contracted fatal infections. Our results show that higher titers develop in vital organs of Muscovy ducks in comparison with mallards, particularly in the brain. It is clear that the ability to produce severe diseases and mortality is associated with high virus replication titers in the host, especially in vital tissues such as brain and heart tissues (Lo¨ndt et al., 2008; Swayne, 2007; Pantin-Jackwood and Swayne, 2007). Muscovy ducks are the only domestic ducks that are not derived from Mallard stock. They are a species of South American origin and may genetically differ from mallards regarding susceptibility to infections. This has previously been documented for diseases such as parvovirus infection and duck virus hepatitis (Hoekstra et al., 1973; Hwang, 1974). The virus strains used in the study differ in their virulence properties. All the investigated strains demonstrated high virulence for Muscovy ducks, whereas a difference was observed between the strains for mallards. Even the strains belonging to the same genotype (Z) differed as e.g. Dk/vn/04 was moderately virulent for mallards whereas Dk/vn/05 and Dk/vn/07 were highly virulent. This suggests that only minor genetic differences may be responsible for the differences in virulence observed in the present study. Different strains of genotype Z have previously been shown to differ in virulence for ducks. Kim et al. (2008) also found that an isolate of clade 2.3.4 was of extreme virulence to Pekin ducks whereas a clade 1 virus was of lower virulence, which is in accordance
173
with our results concerning mallards. However, our study also demonstrates a difference in virulence between Dk/ vn/04 and Dk/vn/05 strains in clade 1 for mallards, which underlines that highly related strains may differ in virulence properties. It was also observed that a virus originally isolated from Muscovy ducks (genotype G) was of low virulence to mallards. Genotype G, which differs from genotype Z viruses in the source of the PB2 gene, has only been isolated since 2004 but no reports concerning virulence for this genotype seem to be available (Smith et al., 2006). In the present study, the strain Dk/vn/04 was stable in the HA gene segment being investigated following passage of the virus in four groups of contacts of the main infection study. Previous studies have shown that modification of the virus may happen quickly. Hulse-Post et al. (2005) demonstrated that HA amino acid differences may develop following repeated infections in ducks. Thus, Dk/vn/04 was shown to develop four amino acid differences in HA following 13 days of infection in one mallard. Modification was not observed with other virus strain infections in the same study, suggesting that modification is a quite unpredictable event. In conclusion, the present study shows that Muscovy ducks have a susceptibility similar to chickens in terms of disease development and mortality to different genotypes of H5N1. Consequently, there is no indication that this species acts as a ‘‘silent reservoir’’ for H5N1 in endemic areas. Once again, it was also demonstrated that mallards differ significantly concerning their susceptibility to different H5N1 viruses, underlining the risk that this species may pose a risk as a reservoir for some H5N1 viruses. The mechanisms behind the major differences observed concerning virulence properties of closely related viruses for mallards need further investigation. Acknowledgements This research was supported by the Danish Foreign Ministry (DANIDA) through the grant: ‘‘Epidemiology of avian influenza in Viet Nam – the role of ducks and survival of virus in water’’; and the MARD of Viet Nam. We would like to express our thanks to the staff members of the Virology Department from the NIVR (Hanoi – Viet Nam) for their technical assistance. References Anonymous, 2007. Vietnam Department of Animal Heath, Epidemiology Division. In: Seminar of Evaluating the Strategy of AI Prevention and Controlling, and Petition for Solution, Hanoi, 27 July. Capua, I., Mutinelli, F., 2001. Mortality in muscovy ducks (Cairina moschata) and domestic geese (Anser anser var. domestica) associated with natural infection with a highly pathogenic avian influenza virus of H7N1 subtype. Avian Pathology 30, 1979–1983. Delquigny, T., Edan, M., Nguyen, D.H., Pham, T.K., Gautier, P., 2004. Impact of Avian Influenza Epidemic and Description of the Avian Production in Vietnam. Food and Agriculture Organization, Rome, Italy, p. 119. Hoekstra, J., Smit, T., van Brakel, C., 1973. Observations on the host range and control of goose virus hepatitis. Avian Pathology 2, 169–178. Hulse-Post, D.J., Sturm-Ramirez, K.M., Humberd, J., Seiler, P., Govorkova, E.A., Krauss, S., Scholtissek, C., Puthavathana, P., Buranathai, C., Nguyen, T.D., Long, H.T., Naipospos, T.S.P., Chen, H., Ellis, T.M., Guan, Y., Peiris, J.S.M., Webster, R.G., 2005. Role of domestic ducks in the propagation and biological evolution of highly pathogenic H5N1
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Research article
Susceptibility of Muscovy (Cairina Moschata) and mallard ducks (Anas Platyrhynchos) to experimental infections by different genotypes of H5N1 avian influenza viruses Do Quy Phuong a,b,c,*, Nguyen Tien Dung a, Poul Henrik Jørgensen d, Kurt Jensen Handberg d, Nguyen The Vinh a, Jens Peter Christensen b a
National Institute of Veterinary Research, 86 Truong Chinh, Dong Da, Hanoi, Viet Nam Department of Veterinary Pathobiology, Faculty of Life Sciences, University of Copenhagen, Stigbøjlen 4 - building 1-20 1870 - Frederiksberg C, Copenhagen, Denmark c Sub-department of Animal Health of Thai Binh province, 14 Quang Trung, Thai Binh city, Viet Nam d National Veterinary Institute, Danish Technical University, Hangøvej 2, DK-8200 A˚rhus N, Denmark b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 March 2009 Received in revised form 21 August 2010 Accepted 7 September 2010
It is a fact that in Viet Nam, Muscovy ducks are raised in large populations (approximately 8 million), usually kept in small flocks together with mallards and chickens. As a result, it is a great concern for epidemiologists to elucidate possible differences in relation to these species being exposed to infection with H5N1. To do this, an experimental study on infections with different genotypes of H5N1 in mallards and Muscovy ducks have been conducted, where it was found that the mortality of the inoculated Muscovy ducks was at least 80%, regardless of the virus strain employed. In contrast, the mortality of the mallards ranged from nil to 100%, which suggests that Muscovy ducks are more susceptible to HPAIV H5N1 infection in terms of disease development and mortality. It was also found that higher virus titers developed in vital organs of Muscovy ducks compared to mallards, particularly in the brain. Due to their high susceptibility, it is unlikely that Muscovy ducks act as a silent reservoir. The virus strains used in this study, to a certain degree, differed in their virulence properties to the bird species in question. ß 2010 Elsevier B.V. All rights reserved.
Keywords: H5N1 Avian influenza Ducks Muscovy ducks Mallard ducks Experimental infection
1. Introduction The highly pathogenic avian influenza (HPAI) virus H5N1 was, for the first time, isolated and recognized as the cause of avian influenza outbreaks (AI) in poultry in Viet Nam in late 2003 (Delquigny et al., 2004). At the start, the outbreak was already fast and massive. As a result, 57 out of the 64 provinces of the country were affected, where it was found that massive die-off and culling of poultry and
* Corresponding author at: Sub-department of Animal Health of Thai Binh province, 14 Quang Trung, Thai Binh city, Viet Nam. Tel.: +84 36 3831539. E-mail address: [email protected] (D.Q. Phuong). 0378-1135/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2010.09.007
other bird species took place. In addition, 27 human cases affection with H5N1 avian influenza were reported in the first 2 years of the AI crisis, of whom 20 were fatal (WHO, 2004). With regard to location, at the start, H5N1 AI outbreaks mainly occurred in the Red river and Mekong river deltas (Pfeiffer et al., 2007; WHO, 2007) and it was observed that chickens showed clinical disease and consequently died at a higher rate than ducks. Nevertheless, ducks were also affected in the early phase of the outbreak, accounting for 23% of the total 44 million birds destroyed in the 2003–2004 period (Anonymous, 2007Viet Nam DAH, 2007) and were considered as the natural carriers of the AI virus, i.e. ducks infected with AI viruses did not develop clinical diseases (Sturm-Ramirez et al., 2005; Saito et al., 2008).
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To respond to the outbreak, a national vaccination campaign was started in September 2005, after which it was found that the number of outbreaks in both chickens and ducks had a sharp decline and that AI cases were reported neither in humans nor in birds in 2006 (WHO, 2007). However, ducks still appeared to be infected and most H5N1 AI outbreaks reported in 2007 occurred in unvaccinated duck flocks being less than 3 months old. As an illustration, between May and July 2007, it was reported that 245,000 birds had been culled because of the AI crisis, in which 94% were waterfowl (Anonymous, 2007Viet Nam DAH, 2007). It had been thought that mallard and Muscovy ducks were both waterfowl by nature and therefore would be infected in the same manner. By contrast, observations have shown that the two species would react differently to the infection, when severe clinical diseases with a high rate of mortality were often seen in Muscovy ducks in Viet Nam (Nguyen, personal observation). Similar observations have also been reported from Italy (Capua and Mutinelli, 2001). As mallards and Muscovy ducks mingle all over the country, it is highly relevant to elucidate the differences in response to infection with H5N1 between the two species and the possible significance for the epidemiology of AI. For this reason, experimental infections with different genotypes of H5N1 virus in mallard and Muscovy ducks have been conducted. 2. Materials and methods 2.1. Experimental studies All the experiments were performed in a reference laboratory for influenza at the National Institute of Veterinary Research (NIVR), Viet Nam under BSL-3 conditions. Viruses: Four HPAI H5N1 viruses were used in this study: A/duck/Vietnam/40D/04 (Dk/vn/04), A/Duck/ Vietnam/1469/05 (Dk/vn/05), A/M.duck/Vietnam/1455/ 06 (M.dk/vn/06) and A/Duck/Vietnam/37/07 (Dk/vn/07). They originated from clinical outbreaks of HPAI and represented different genotypes of H5N1 (Table 1). Stock viruses were grown in 10-day-old embryonated chicken eggs for 36–48 h at 37 8C. The allantoic fluid was
169
harvested, and aliquots were stored at 80 8C until use. Virus titer was determined according to Reed and Muench (1938). The isolate Dk/vn/04 was used to infect the birds as a reference study with a detailed characterization of the infection in mallard and Muscovy ducks. The other three isolates were used in more simple additional studies to supplement the former reference study. Inocula: Each inoculated bird was inoculated with 0.2 ml of virus stock diluted in phosphate buffered saline, equivalent to 106 EID50 by the intranasal route. Experimental animals: Mallard ducks, Muscovy ducks and chickens (Luong Phuong line) were raised on isolated farms. They were transferred to the NIVR facilities for experimental infections at the age of 4–6 weeks, and no vaccinations had been applied to the birds. Oropharyngeal, cloacal swabs and serum samples were collected from these birds before inoculation to confirm freedom of active infection with an AIV and serum antibodies specific to subtype H5 AIV. 2.2. Experimental design 2.2.1. Main infection study The main infection study was performed in two separate infection experiments, both employing A/duck/ Vietnam/40D/04. Fifteen mallard ducks aging 4–6 weeks old were inoculated and later introduced with contact birds of 2 mallards and 2 chickens at intervals of day 1, 5, 10, and 15 post-inoculation (p.i.), respectively. The contact birds shared a cage with the inoculated birds for 24 h, then were transferred to a separate cage for 1 more week of observation. In the second experiment, 10 Muscovy ducks were inoculated and the contacts birds (2 Muscovy ducks and 2 chickens) were only introduced on days 1, 5 and 10 following infection. Signs of disease were recorded on daily basis according to the following scoring system: low, medium and high. Low scoring was used when only unspecific clinical signs were observed; medium scoring was given if some disease signs and low to medium mortality rate were observed; and the high scoring was used if obvious clinical signs including CNS signs and a high mortality occurred. However, for welfare reasons, any bird showing difficulty in eating or drinking and/or severely impaired ability to
Table 1 Mean dead time, mortality and clinical signs in mallards, Muscovy ducks and chickens following infection with four different H5N1 avian influenza viruses. Virus strains
Genotype
Year
Sub-lineage
Species
MTD (in days)
No. dead/total
Inoculate
Contact
Inoculate
Contact
Clinical score Inoculate
Contact
Dk/vn/04
Z
2004
Clade 1
Mallard Muscovy Chicken
5.4 (5–6) 4.6 (4–6) ND
No dead 4.8 (2–5) 3.1 (2–5)
8/15 8/10 ND
0/18 4/6 10/15
Medium High ND
Low High High
Dk/vn/05
Z
2005
Clade 1
Mallard Muscovy
4.4 (3–6) 3.6 (3–5)
4.8 (3–6) 5.2 (3–7)
5/5 5/5
4/5 5/5
High High
High High
M.dk/vn/06
G
2006
Clade 2.3.2
Mallard Muscovy
No dead 5.3 (5–6)
No dead 5.5 (5–7)
0/5 4/5
0/5 2/5
Low High
Low Medium
Dk/vn/07
Z
2007
Clade 2.3.4
Mallard Muscovy
4.0 (3–4) 3.8 (2–5)
5.4 (4–5) 4.8 (3–4)
4/5 5/5
5/5 5/5
High High
High High
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move was killed and scored as dead. Oropharyngeal and cloacal swabs were collected in 1.5 ml transport medium from all birds every 2 days p.i. for virus detection. All the birds (both inoculated and contact birds) were subjected to post-mortem investigation, where tissue samples which were obtained from brains, lungs, spleens, intestines, trachea and muscles at necropsy, were stored at 80 8C until virus isolation and titration. Surviving inoculated birds and contact birds were tested from day 5 p.i. every 2 days for H5 antibodies in serum. The experiment continued until shedding of virus from inoculated birds could not be detected by real-time RT-PCR in cloacal and oropharyngeal swabs. Virus recovered at day 10 p.i. from the infected mallards was used in a further infection study aiming at determining any possible changes in virulence of the virus. For this purpose, 8 mallards and 3 chickens were inoculated as described above. 2.2.2. Additional infection study Mallards and Muscovy ducks in groups of five aged 5–6 weeks were inoculated with the following strains: Dk/vn/ 05, M.dk/vn/06 and Dk/vn/07, respectively. Then, five contact birds of the same species were introduced to the inoculated birds at day 1 post infection and were monitored/scored for 15 days. The birds were observed for clinical signs of disease every 24 h over a period of 10 days, and each bird was scored as described above. Tissue samples from the brain of all dead or euthanized birds were collected for virus isolation and titration. 2.3. RT-PCR and virus titration Every 2 g of tissue samples were homogenized in 4 ml of transport medium by the use of a porcelain mortar and pestle. RNA extraction and RT-PCR were performed as described (Munster et al., 2005). RNA from swabs and tissue suspensions was extracted by using QIAGEN RNeasy Mini Kit as recommended by the producer (QIAGEN GmbH, D40724 Hiden). Real-time RT-PCR assays were performed on a BioRad iQ5 Sequence Detection System machine by using QIAGEN One Step RT-PCR Kit Cat, no. 210212 (QIAGEN GmbH, D-40724 Hiden). The test used a hybridization probe (6-FAM-TCAGGCCCCCTCAAAGCCGA-TAMRA) and specified primers (forward: 50 -ARAT-GAGTCTTCTRACCGAGGTCG-30 and reverse: 50 -TGCAAAGACATCYTCAAGYYTCTG-30 ) to detect the matrix gene segment AIV as previously described (Runstadler et al., 2007). For each run, the samples were prepared and processed in parallel with several negative and positive control samples. Virus was quantified by inoculation of serial 10-fold dilutions of supernatant from the homogenized tissue samples and swabs in quadruplicate of MDCK cell cultures, as described by Rimmelzwaan et al. (1998). To determine possible modification of the inoculated virus strain in the HA gene during the infections in the reference infection experiment, 11 isolates were obtained throughout the study from the brain and the intestine. Samples were obtained from both dead and killed chickens and ducks until day 7. In addition, virus was isolated from contact ducks and chickens day 2–5 post contact.
2.4. Sequencing Sequencing was done according to previous reports (Slomka et al., 2007). The PCR primers KHA-1 and KHA-3 were used both as initial RT-PCR primers and as sequencing primers. The resulting sequences were approximately 350 bp long and including HA0 cleavage site. The sequences were assembled and aligned by the Invitrogen software Vector NTI version 10.3. 2.5. Serological test Pre- and post-infection serums were tested for H5 antibodies by using standard procedures (Thayer and Beard, 2008). The HI tests were performed by using a 0.5% suspension of chicken erythrocytes in phosphate-buffered saline. HI was assessed using 25 ml each of a series of serum dilutions 1:2, and 25 ml of HA antigen, standardized at 8 hemagglutination units (HAU) by hemagglutination titration. Serum was incubated for 30 min at 56 8C in a water bath to eliminate the nonspecific inhibitors, and the first dilution on the test plate was 1:8. All HI titers >8 were considered positive. None of the birds used in the infection studies were antibody positive to H5 prior to infections. 3. Results 3.1. Experimental infections Except for the strain M.dk/vn/06, all the strains used in the study caused clinical signs, mortality and gross lesions typical of HPAI following intranasal inoculation in both mallard ducks and Muscovy ducks. Although M.dk/vn/06 did not cause any mortality in mallards and only mild clinical signs, but it affected Muscovy ducks at a similar level with the other strains used. The clinical signs observed throughout the study varied from mild to severe (Table 1). Differences in mortality between mallard ducks and Muscovy ducks were observed following inoculation with Dk/vn/04 and M.dk/vn/06 and were also expressed in the level of mortality observed in the contact birds. Following infection with Dk/vn/04, no mortality was observed in mallards in contact with inoculated mallards while 66% of the Muscovy ducks in contact with inoculated Muscovy ducks died. M.dk/vn/06 showed low virulence to mallards and their contacts but showed full virulence for Muscovy ducks and their contacts, with 40% of the contact Muscovy ducks being fatal. It was also observed that the strains Dk/vn/04 and Dk/vn/07, both belong to genotype Z but to different antigenic clades differed in their virulent properties in mallards. Dk/vn/07 was in contrast to Dk/vn/ 04 in that the former is more capable of causing high mortality than the latter in contact mallards. In general observations, the mean death time (MDT) in Muscovy ducks is shorter than of those in mallards, regardless of virus strains. Mortality of the contact chickens was not observed after the second introduction of chickens to inoculated mallards; whereas the third group of chickens in contact with inoculated Muscovy ducks died (no further introduction of the fourth group) (Tables 2 and 3).
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Table 2 Reference infection study on susceptibilities of mallards to A/duck/Vietnam/40D/04. Group of birds
MDT (in days) Inoculate mallard
Inoculate group Contact group.1 (day 1 p.i.)
b
Contact mallard
Contact chicken
No dead
Inoculate mallard
Clinical score
Contact mallard
Contact chicken
4.5 (4–5)
Contact chicken
Contact mallard
Contact chicken
0/2
2/2
Low
High
3–6
NDa
No dead
2 (2)
0/2
2/2
Low
High
3–6
ND
No dead
No dead
0/2
0/2
Low
Low
NSb
NS
No dead
No dead
0/2
0/2
None
None
NS
NS
8/15
Inoculate mallard
Shedding time
Contact mallard
5.4 (5–6)
Contact group.2 (day 5 p.i.) Contact group.3 (day 10 p.i.) Contact group.4 (day 15 p.i.) a
No. dead/tatal
Medium
Inoculate mallard 7–13
ND: not done. NS: not shed.
As shown in Tables 2 and 3, none of the inoculated surviving mallards and Muscovy ducks shed virus for more than 13 and 14 days, respectively. The investigation into the pathogenicity of virus isolated late in the main infection study did not reveal any changes, compared to the initial inoculate.
found that the virus titers obtained from vital tissues of Muscovy ducks were higher than those of mallards at day 5 and 6. In mallards alone, it was observed that the titers were higher in intestines than in tracheas (day 5 and 6), whereas, titers obtained from the trachea of Muscovy ducks were always higher than those in the intestines.
3.2. Virus in tissues
3.3. Sequencing
The virus titers obtained from different tissues of all groups of birds that died at day 5 post infection (or contact) or killed day 5 due to severe clinical signs are shown in Fig. 1. The most obvious difference between the groups of birds was in relation to the titer obtained from the brain tissue, which is significantly higher in the inoculated Muscovy ducks than in any other group. Furthermore, the contact Muscovy ducks also developed high titers in the brain. The titers obtained from the inoculated mallards were more than 50% lower in Muscovy ducks, and no positive titers were recorded from the brain of the contact mallards (Fig. 1). A more detailed comparison of the titers obtained from different tissues of the infected mallards and Muscovy ducks is shown in Fig. 2. As no mallards died before day 5 post infection, no data were obtained during the first sampling points for this type of ducks. However, the data that were obtained from dead Muscovy ducks demonstrated high titers of the virus in lung and trachea, especially during courses of per-acute/ acute of infections. The brain and muscles also showed high titers. General speaking, when comparing data, it was
The partial sequencing of the HA gene of 11 isolates of Dk/vn/04 obtained from brain and intestinal samples showed no differences – all sequences were identical. 3.4. Serology In the survival cases, both the infected mallards together with their contacts and the infected Muscovy ducks together with their contacts developed a positive antibody titer (HI titer (log 2) >6–7) at day 7. No seropositive birds were observed before day 7. However, the surviving mallards and chickens in contact groups 3 and 4 did not develop a positive titer, but only the two of them in contact group 3 were positive to virus in tissues. 4. Discussions Few reports are available on HPAI and Muscovy ducks. However, Capua and Mutinelli (2001) reported that during the 413 outbreaks of HPAI (H7N1) occurring in Italy 1999– 2000, Muscovy ducks and geese were the only species of waterfowl being affected in spite of the fact that several of
Table 3 Reference infection study on susceptibilities of Muscovy to A/duck/Vietnam/40D/04. Group of birds
MDT (in days) Inoculate Muscovy
Inoculate group Contact ggroup.1 (day 1 p.i.) Contact ggroup.2 (day 5 p.i.) Contact ggroup.3 (day 10 p.i.) a
ND: not done.
No. dead/tatal
Contact Muscovy
Contact chicken
4
Clinical score
Contact Muscovy
Contact chicken
3–4
1/2
4
4
2–3
3
4.6 (4–6)
Inoculate Muscovy
Shedding time
Contact Muscovy
Contact chicken
2/2
High
1/2
2/2
2/2
2/2
8/10
Inoculate Muscovy
Contact Muscovy
Contact chicken
High
NDa
ND
High
High
ND
ND
High
High
ND
ND
High
Inoculate Muscovy 9–14
[(Fig._1)TD$IG]
D.Q. Phuong et al. / Veterinary Microbiology 148 (2011) 168–174
172 8.0
Virus titer (log10 TCID50/ml)
7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 Inoculated mallard group (3 birds)
Inoculated Muscovy group (4 birds)
contact mallard group (5 birds)*
contact Muscovy group (4 birds)**
Contact chicken group (3 birds)***
Groups of birds Brain
Lung
Spleen
Intestine
* Include contact duck group 1, 2 and 3 **Include contact Muscovy duck group 1 and 2 *** Include contact chicken group 1 and 2 Fig. 1. Virus titer in different tissues of the different groups of birds in the main study at day 5 post inoculation with A/duck/Vietnam/40D/04 (included dead and killed birds). * Include contact duck groups 1, 2 and 3. **Include contact Muscovy duck groups 1 and 2. *** Include contact chicken group 1 and 2.
the flocks also contained mallard ducks. Few histopathological lesions were observed in the dead Muscovy ducks, and if they were present, they were mild ones. The previous challenge of 6 weeks old Muscovy ducks with a strain of H5N1 resulted in 100% mortality within 1 week,
[(Fig._2)TD$IG]
Virus titer (log TCID50/ml)
a
but no further characterization of the infection was done (Steensels et al., 2007). In this study, however, the mortality of the inoculated Muscovy ducks was found to be at least 80% regardless of the virus strain employed. In contrast, the mortality of the
Virus titer in different tissues of dead mallard ducks 7 6 5 4 3 2 1 0
No mallard ducks died before day 5 p.i
Day 2
Day 3
Day 4
Day 5
Day 6
(0 birds)
(0 birds)
(0 birds)
(5 birds)
(3 birds)
Time after infection (day) Brain
Spleen
Intestine
Trachea
Muscular
Virus titer in different tissues of dead Muscovy ducks
b Virus titer (log TCID50/ml)
Lung
7 6 5 4 3 2 1 0 Day 2
Day 3
Day 4
Day 5
Day 6
(1 bird)
(1 bird)
(4 birds)
(4birds)
(2birds)
Time after infection (day) Brain
Lung
Spleen
Intestine
Trachea
Muscular
Fig. 2. Virus titer in different tissues of dead mallard and Muscovy ducks at period of time post inoculation with A/duck/Vietnam/40D/04 (included inoculated and contact birds).
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inoculated mallards ranged from nil to 100%, which suggests that Muscovy ducks are more susceptible to HPAI H5N1 virus infection with regard to the development of disease and mortality. A shorter MDT of the Muscovy ducks further substantiated this. The virulence of a particular virus following the infection of either Muscovy ducks or Mallards was, to some degree, reflected in the mortality that was observed among the contacts birds. In the main infection study, Dk/vn/04 was found to be moderately virulent for infected mallards but of low virulence for the contact mallards. The same isolate was found highly virulent for infected as well as contact Muscovy ducks. The mortality of chickens following contact to infected mallards and Muscovy ducks was 100% for the first 2 groups of contact chickens (introduced on day 1 and day 5). After that, no chickens in the third contact group died when brought into contact with the infected mallards at day 10 post infection. Only three contact groups were introduced to the infected Muscovy ducks and they all died. As it was demonstrated that the isolate shed from the mallards at day 10 was as virulent as the initial strain. The results also indicated that the level of excretion from the inoculated birds and the susceptibility of the contacts were the main reasons for the disease/ mortality pattern that was observed during the experiment rather than a reduced virulence of the initial strain following the challenge. Both the mallards and the Muscovy ducks shed virus for up to from 13 to 14 days, which is in accordance with the results from a previous study (Hulse-Post et al., 2005). Thus, the last introduced contacts (at days 10 and 15) were probably exposed to lower concentrations of the virus, as suggested by Hulsepost et al. (2005), and only the highly susceptible contacts (Muscovy ducks and chickens) contracted fatal infections. Our results show that higher titers develop in vital organs of Muscovy ducks in comparison with mallards, particularly in the brain. It is clear that the ability to produce severe diseases and mortality is associated with high virus replication titers in the host, especially in vital tissues such as brain and heart tissues (Lo¨ndt et al., 2008; Swayne, 2007; Pantin-Jackwood and Swayne, 2007). Muscovy ducks are the only domestic ducks that are not derived from Mallard stock. They are a species of South American origin and may genetically differ from mallards regarding susceptibility to infections. This has previously been documented for diseases such as parvovirus infection and duck virus hepatitis (Hoekstra et al., 1973; Hwang, 1974). The virus strains used in the study differ in their virulence properties. All the investigated strains demonstrated high virulence for Muscovy ducks, whereas a difference was observed between the strains for mallards. Even the strains belonging to the same genotype (Z) differed as e.g. Dk/vn/04 was moderately virulent for mallards whereas Dk/vn/05 and Dk/vn/07 were highly virulent. This suggests that only minor genetic differences may be responsible for the differences in virulence observed in the present study. Different strains of genotype Z have previously been shown to differ in virulence for ducks. Kim et al. (2008) also found that an isolate of clade 2.3.4 was of extreme virulence to Pekin ducks whereas a clade 1 virus was of lower virulence, which is in accordance
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with our results concerning mallards. However, our study also demonstrates a difference in virulence between Dk/ vn/04 and Dk/vn/05 strains in clade 1 for mallards, which underlines that highly related strains may differ in virulence properties. It was also observed that a virus originally isolated from Muscovy ducks (genotype G) was of low virulence to mallards. Genotype G, which differs from genotype Z viruses in the source of the PB2 gene, has only been isolated since 2004 but no reports concerning virulence for this genotype seem to be available (Smith et al., 2006). In the present study, the strain Dk/vn/04 was stable in the HA gene segment being investigated following passage of the virus in four groups of contacts of the main infection study. Previous studies have shown that modification of the virus may happen quickly. Hulse-Post et al. (2005) demonstrated that HA amino acid differences may develop following repeated infections in ducks. Thus, Dk/vn/04 was shown to develop four amino acid differences in HA following 13 days of infection in one mallard. Modification was not observed with other virus strain infections in the same study, suggesting that modification is a quite unpredictable event. In conclusion, the present study shows that Muscovy ducks have a susceptibility similar to chickens in terms of disease development and mortality to different genotypes of H5N1. Consequently, there is no indication that this species acts as a ‘‘silent reservoir’’ for H5N1 in endemic areas. Once again, it was also demonstrated that mallards differ significantly concerning their susceptibility to different H5N1 viruses, underlining the risk that this species may pose a risk as a reservoir for some H5N1 viruses. The mechanisms behind the major differences observed concerning virulence properties of closely related viruses for mallards need further investigation. Acknowledgements This research was supported by the Danish Foreign Ministry (DANIDA) through the grant: ‘‘Epidemiology of avian influenza in Viet Nam – the role of ducks and survival of virus in water’’; and the MARD of Viet Nam. We would like to express our thanks to the staff members of the Virology Department from the NIVR (Hanoi – Viet Nam) for their technical assistance. References Anonymous, 2007. Vietnam Department of Animal Heath, Epidemiology Division. In: Seminar of Evaluating the Strategy of AI Prevention and Controlling, and Petition for Solution, Hanoi, 27 July. Capua, I., Mutinelli, F., 2001. Mortality in muscovy ducks (Cairina moschata) and domestic geese (Anser anser var. domestica) associated with natural infection with a highly pathogenic avian influenza virus of H7N1 subtype. Avian Pathology 30, 1979–1983. Delquigny, T., Edan, M., Nguyen, D.H., Pham, T.K., Gautier, P., 2004. Impact of Avian Influenza Epidemic and Description of the Avian Production in Vietnam. Food and Agriculture Organization, Rome, Italy, p. 119. Hoekstra, J., Smit, T., van Brakel, C., 1973. Observations on the host range and control of goose virus hepatitis. Avian Pathology 2, 169–178. Hulse-Post, D.J., Sturm-Ramirez, K.M., Humberd, J., Seiler, P., Govorkova, E.A., Krauss, S., Scholtissek, C., Puthavathana, P., Buranathai, C., Nguyen, T.D., Long, H.T., Naipospos, T.S.P., Chen, H., Ellis, T.M., Guan, Y., Peiris, J.S.M., Webster, R.G., 2005. Role of domestic ducks in the propagation and biological evolution of highly pathogenic H5N1
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