Dec 12, 1984 - Sentinel Ducks and Domestic Turkeys in Minnesota ... (iv) cooler surface water temperature, allowing prolonged virus viability; (v) groundwater contamination from .... the onset of infection in ducks occurs near the end of July.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1985, 0099-2240/85/040914-06$02.00/0 Copyright © 1985, American Society for Microbiology
p.
Vol. 49, No. 4
914-919
Epizootiology of Avian Influenza: Effect of Season on Incidence in Sentinel Ducks and Domestic Turkeys in Minnesota D. A.
HALVORSON,'*
C. J. KELLEHER,1 AND D. A. SENNE2 College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108,1 and National Veterinary Services
Laboratories, Ames, Iowa 500102 Received 17 September 1984/Accepted 12 December 1984
Sentinel ducks and domestic turkey flocks were monitored for influenza infection during a 4-year period. The onset of infection among ducks was similar each year, occurring in late July or early August. Influenza in turkeys was also shown to be seasonal, but the usual onset was 6 to 8 weeks after the detection of influenza in
sentinel ducks. Possible explanations for the delayed infection in turkeys are (i) increased waterfowl activity associated with fledging and congregating in late summer and early fall; (ii) vectors transmitting virus from the waterfowl habitat to poultry farms; (iii) cooler environmental temperature, allowing prolonged virus viability; (iv) cooler surface water temperature, allowing prolonged virus viability; (v) groundwater contamination from contaminated surface water; and (vi) virus adaptation in domestic turkeys before infection is detected. We conclude that ducks are not only a natural reservoir of influenza but also have a seasonal infection that appears to be related to seasonal influenza outbreaks in domestic turkeys in Minnesota. However, only some influenza A virus isolates circulating among waterfowl at any given time appear capable of causing detectable infection in turkeys. It is speculated that the seasonal infection in migratory waterfowl may also be related to seasonal influenza infections in other species including humans.
Influenza is a seasonal disease of domestic turkeys in Minnesota (5) and is also reported to be a seasonal disease of swine, horses, and humans (4, 6) as well as a seasonal infection in mink (13). Influenza outbreaks among turkeys in Minnesota most commonly occur between July and November, with the majority of outbreaks occurring during September and October; they also occur when millions of turkeys are being raised in close proximity to large numbers of waterfowl that are marshalling for migration. There are numerous reports of influenza A viruses being detected in wild waterfowl (1, 5, 7, 9, 15, 18, 20), and as a result there is considerable speculation about their role in the epidemiology and epizootiology of influenza. However, the role of the waterfowl reservoir in outbreaks of influenza in turkeys (or other hosts) is not fully understood at present. Therefore, an influenza surveillance project was initiated to investigate the interspecies transmission of influenza A virus between wild waterfowl and domestic turkeys and to determine the role of the season in the epizootiology of avian influenza.
1,000 m of turkey farms which had both range and confinement-reared turkeys. Turkeys. Fifty turkeys representing all flocks on the farm were randomly selected every other week from each of the nearby turkey farms for monitoring for the presence of avian influenza virus. A representative number of blood samples, cloacal swabs, and tracheal swabs were collected from each flock. Sentinel turkeys. Seven- to nine-week-old confinementreared turkeys were placed in adapted sentinel duck pens at two locations during the third and fourth years of the study. The ducks and turkeys were allowed to comingle, share feed sources, and drink pond water. The sentinel turkeys were swabbed weekly and bled biweekly. Serum samples. The double immunodiffusion test was performed on all turkey serum samples (2). All positive samples were tested for hemagglutinin and neuraminidase activity at the National Veterinary Services Laboratories, Ames, Iowa, as previously described (3, 19). Tracheal and cloacal swabs. UM ducks were swabbed cloacally, and the swabs were placed in individual transport tubes containing media with antibiotics as previously described (5). Sentinel turkeys and turkeys from commercial flocks were swabbed cloacally and tracheally. Samples from sentinel turkeys were placed in individual tubes, and samples from commercial flocks were pooled five per tube. Samples were kept on ice in the field and at 4°C in the laboratory before egg inoculation. Nine-day-old embryonated chicken eggs were inoculated via the allantoic route with 0.2 ml of the medium (two eggs per sample) and incubated at 35 to 37°C. The eggs were candled daily for 3 days and chilled, and a hemagglutination test was performed on allantoic fluid. Hemagglutination-positive samples were tested for hemagglutinin and neuraminidase activity at the National Veterinary Services Laboratories (3, 19). Steps to avoid contamination and transmission. Precautions were taken to avoid spreading avian influenza virus from one
MATERIALS AND METHODS Sentinel ducks. One-day-old mallard ducks (Anas platyrhynchos) were raised in isolation facilities at the University of Minnesota (UM) for 6 to 7 weeks and were determined to be negative for avian influenza virus by cloacal swabbing. Pens were constructed on selected marshes that allowed contact of 10 to 20 captive UM ducks with released UM ducks and wild ducks, as previously described (5). Ducks were placed in the pens between mid-May and mid-July each year for 4 years. Cloacal swab samples were collected from individual sentinel ducks each week. Selection of sites. Market turkey production in Minnesota is concentrated in the west-central part of the state. We selected ponds within this region that were within 100 to
*
Corresponding author. 914
VOL. 49, 1985
SEASONAL EFFECT ON INCIDENCE OF AVIAN INFLUENZA
each year and coincides with a previously described increase in the number of wild ducks observed at the sentinel duck locations (5). Influenza infection of sentinel turkeys. Figure 2 shows the weekly infection rates in sentinel ducks and sentinel turkeys in 1982, the third year of the study. At one site, H4N6 and H3N6 subtypes were detected in 2 of 10 sentinel turkeys 1 week after being detected in sentinel ducks, but H4N2, H12N5, and H1ON5, found in sentinel ducks, were not detected in sentinel turkeys. At the second site, H4N8 and H6N8 were detected in sentinel ducks but not in comingled sentinel turkeys. In 1983, influenza A virus was detected in ducks at one site, but no influenza virus was detected in sentinel turkeys at either site. Influenza infection of turkey flocks. Figure 3 shows the number of turkey flocks in which influenza was detected each week for each of the 4 years of the study. Again, as with the onset of infection in sentinel ducks (Fig. 1), there is an obvious similarity in the pattern each year, with the onset of infection in domestic turkeys usually occurring near the end of September. Thus, each year there was a delay of about 8 weeks between the initial detection of virus in sentinel ducks and the initial detection in turkey flocks. A 4-year summary of the isolation rate in UM ducks and the number of turkey flocks with influenza is presented in Fig. 4. Influenza subtypes detected. Table 1 presents the number of influenza isolates from UM ducks and the number of turkey flocks in which influenza was detected. A total of 26 subtypes were identified, and 5 were incompletely characterized. The number of isolates from ducks of a given subtype does not correlate with the number of outbreaks in turkeys or the number of turkey flocks infected. Frequencies of antigenic subtypes. Some influenza virus subtypes were detected often, and other possible H and N combinations (i.e., H2N2) were not detected at all during the course of this study. The most common hemagglutinin subtypes detected in sentinel ducks were H3 (25%), H4 (25%), and H6 (21%), and the most common neuraminidase subtypes detected in sentinel ducks were N8 (34%), N6 (26%), and N2 (19%). The most common hemagglutinin
1980
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-2
11-16
11-30
FIG. 1. Weekly summary of influenza A virus isolations from sentinel ducks in Minnesota. The dotted line indicates the sampling period.
location to another and to avoid contaminating one group of samples with another (5). RESULTS Influenza infection of sentinel ducks. The weekly infection rates in UM ducks are shown for each of the 4 years in Fig. 1. The similarity between the onset of infection (late July to mid-August) for each of the 4 years is striking. Interestingly, the onset of infection in ducks occurs near the end of July 90
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FIG. 2. Weekly summary of influenza A virus isolations from sentinel ducks and sentinel turkeys
in 1982.
HALVORSON, KELLEHER, AND SENNE
916
subtypes causing influenza outbreaks in turkeys were H6 (28%), H4 (22%), and H5 (17%), and the most common neuraminidase subtypes causing influenza outbreaks in turkeys were N2 (41%), Ni (24%), and N8 (24%). Wild ducks in Minnesota. Each year the Minnesota Department of Natural Resources conducts an aerial census of wild ducks nesting in Minnesota (11). The results of the DNR census are presented in Table 2 along with the influenza isolation rate in sentinel ducks, the number of turkey flocks infected with influenza, and the number of influenza subtypes (the number of outbreaks) in turkey flocks. There appears to be a correlation among the number of wild ducks that nest in Minnesota, the prevalence of influenza infection among sentinel ducks, the number of turkey flocks infected, and the number of antigenic subtypes detected. DISCUSSION The yearly placement of young, isolation-reared mallard ducks onto selected marshes frequented by nesting and migratory waterfowl and the subsequent detection of influenza A virus among the sentinel ducks presumably reflect the natural ecology of influenza A virus among wild waterfowl at discrete locations. However, it should be noted that there are hundreds of thousands of ducks and thousands of marshes in Minnesota, and that we have monitored no more than 100 ducks and six locations annually during this study. As a result, the subtypes we detected in our sentinel ducks cannot be expected to reflect the total number of subtypes present among the wild waterfowl population in Minnesota at any given time or to completely reflect the subtypes detected in turkey flocks. Furthermore, the same subtypes of virus were often detected in ducks and turkeys within a particular year, and the frequency of detection of the H and N subtypes was found to be similar but not identical for each species; however, we could rarely detect the same subtype in both ducks and turkeys in close proximity. The interspecies transmission of influenza between 20 18
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wild waterfowl and commercial turkeys has not been conclusively demonstrated; however, cross-infection between sentinel ducks and sentinel turkeys with certain isolates has been shown to occur. Proof of the link between infection in ducks and turkeys would require not only detection of the same subtype in ducks before its detection in turkeys but also genetic analysis to show them to be identical. The results demonstrate that influenza A virus is detected in sentinel ducks according to the season and that the onset of infection occurs at nearly the same time each year-late July or early August. The reason for this may be related to the wild duck life cycle and the emergence of newly hatched, susceptible wild ducks from hiding, and the intermingling of various broods. The high rate of influenza virus isolation in late September and early October coincides with an influx of migrating waterfowl into Minnesota and the marshaling of large numbers of ducks for migration. Similarly, Sinnecker et al. (17) reported that an "influenza season" began in August among sentinel ducks in two different geographical areas in Europe and that a high rate of reinfection occurred among those birds in October. During a 3-year survey of wild ducks in Canada, Hinshaw and Webster (7) reported a high frequency of influenza A virus isolates from juvenile ducks (18 to 60%) compared with mature ducks (4 to 27%) during the late summer. These researchers suggested that influenza A viruses are maintained in the wild waterfowl population by passage to susceptible birds, particularly young ducks as they congregate for fall migration. In our work, the detection of influenza virus in domestic turkeys was also seasonal, but there was a 6- to 8-week lag period from the onset of infection in sentinel ducks and turkey flocks. Despite the lag period, influenza in turkeys appears to be a seasonal, not an endemic, infection, because current turkey management practices are such that there is frequent depopulation of farms due to marketing. Thus the disease cycle is interrupted, thereby preventing influenza from becoming endemic. In addition, farms are restocked with immunologically virgin turkey poults, resulting in a high-density population (i.e., 100,000 per flock) of highly susceptible turkeys. Furthermore, the results from virological and serological monitoring of hundreds of turkey flocks on a year-round basis have provided no evidence that influenza is endemic among turkeys in Minnesota (D. A.
Halvorson, unpublished data). Possible explanations for the 6- to 8-week delay between
10
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APPL. ENVIRON. MICROBIOL.
2
10 20 18 16 14 12010W 8 6W 4 2
It 1983 5-18 6-1 6-15 6-29 7-13 7-27 8-10 8-24 9-7 9-21 10-5 10-1911-2 11-16 11-30 12-14 12-28
FIG. 3. Weekly summary of the number of turkey flocks detected with influenza in Minnesota.
the onset of infection in sentinel ducks and detection in
turkeys that occurred for each of 4 years are as follows. (i) Waterfowl activity. It may be that outbreaks in turkeys (Fig. 3) are more related to duck movement during late summer-early fall than to duck infection rates (Fig. 1). An
increase in waterfowl movement associated with the fledgling of local ducks and the influx of migrating birds could increase the opportunity for direct duck-to-turkey transmission. Even though the incidence of infection is high in waterfowl from mid-July to early August, many of the wild ducks are flightless during this period, and opportunity for direct interspecies transmission is greatly reduced. The data
in Table 2 suggest that the more wild ducks there are, the more avian influenza virus there will be in both sentinel
ducks and turkeys flocks. (ii) Other vectors or vehicles. Conceivably the delay between infection of the sentinel ducks and commercial turkeys could be related to the time required for one or more vectors or vehicles to transport influenza virus from the duck habitat to the poultry farm. For example, barn swallows, gulls, raccoons, or duck hunters could do this. Some
VOL. 49, 1985
SEASONAL EFFECT ON INCIDENCE OF AVIAN INFLUENZA at
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FIG. 4. Summary over 4 years of the weekly influenza virus isolations from sentinel ducks and the weekly number of turkey flocks detected with influenza.
species of mammals that are found in waterfowl habitat and poultry farms (i.e., skunks, ground squirrels, and raccoons) have been experimentally infected with avian influenza A viruses (C. Bailey, M.S. thesis, University of Minnesota, St. Paul, 1983). In addition, there is evidence that avian influenza viruses can infect mink by contact (12), and seasonal influenza infection in mink from Augtust to November has also been reported (13).
(iii) Environntenti1 temperature, Influenza virus survives much longer at 4°C than at 22°C (21). In midsummer wild ducks excrete large amounts of virus, and some of it may be deposited directly in the turkeys' environment, but high environmental temperatures may inactivate the virus almost immediately as it is excreted. As environmental temperatures decline in autumnn, the virus excreted by ducks could survive for 1 month or more. Consequently, the amount of
on
TABLE 1. Isolation of influenza A virus from sentinel ducks and detection of influenza in domestic turkeys in Minnesota (1980 to 1983) Virus type
HlNl H1N2 H3N2 H3N6 H3N8 H4N2 H4N6 H4N8 HSN1 H5N2 H5N9 H5N? H6N1 H6N2 H6N5 H6N6 H6N8 H6N? H7N3 H7N8
1980 UM ducks Turkey flocks
1
H11N9 H12N4 H12N5 H?N2 H?N6
1983 Turkey flocks
6 21
14
20
17
2 8
1
1 1
29 3 7 1 17
7 1 22 15
31
27 2 8 2
1
11
1 2
1 23
12
18
19
8 1
27 6 3 1 1 7 1 4
1
11
10 1 4
1 1
H?N9
1
Total
73 9
Subtypes
UM ducks
8 9
H8N4 H9N2 H1ON5 H1ON7 H11N8
No. of isolates and no. of flocks in: 1981 1982 UM ducks UM ducks Turkey flocks Turkey flocks 2 12
47 5
140
15
50 3
83 7
59 9
18 2
2 1
APPL. ENVIRON. MICROBIOL.
HALVORSON, KELLEHER, AND SENNE
918
TABLE 2. Relationship between the number of wild ducks and influenza in Minnesota sentinel ducks and turkey flocks No. of wild ducks nesting in Minnesotaa
Yr
1980 1981 1982 1983 a
b
756,000 671,000 799,000 462,000
% Sentinel ducks positive
for
virusb 12.6 10.3 14.5 4.6
No. of turkey flocks
No. of subtypes in
infected
turkey flocks
47 50 59 2
5 3 9 1
Minnesota Department of Natural Resources aerial census data.
July to November.
viable virus in the environment could increase until the minimum infectious dose was reached. (iv) Water temperature. Like environmental temperature, the ambient surface water temperature also declines in the fall (5). A single duck may excrete as much as 1010 50% egg infective doses of influenza virus in a 24-h period (21). Therefore, as influenza virus is continually excreted into the water by locally produced ducks and as migrating ducks amplify the amount excreted, then lake, marsh, and pond water could become increasingly infectious for poultry (and other animals) as water temperatures decline. Hypothetically, 100 ducks, each excreting 1010 50% egg infective doses of influenza virus per day for a few weeks, could contaminate a small pond with several hundred 50% egg infective doses of influenza virus per ml of pond water in cold conditions. Virus-contaminated pond water could then serve as a source of infection for domestic turkeys by one or more of the previously mentioned vehicles or vectors or perhaps by aerosol. (v) Groundwater contamination. Under certain hydrogeological conditions, virus contamination of surface-water ponds could result in contamination of nearby groundwater supplies which serve as a source of drinking water for turkeys. The contamination of groundwater and well-derived drinking water by human enteric viruses and animal wastes has been documented (10, 14, 16). The lag period demonstrated during this study may reflect the time required for virus to be transported from surface water through groundwater to turkeys via well-derived drinking water. (vi) Virus adaptation. Even though the route(s) of transmission between waterfowl and turkeys has not been clearly identified, serial passage of a particular influenza isolate among subclinically infected turkeys may be required before infections are detected. Results obtained in this study from sentinel ducks and turkeys suggest that only certain influenza isolates that are circulating among wild waterfowl are able to infect turkeys (Fig. 2). In 1982, nine subtypes were detected in sentinel ducks, but only two of those subtypes were detected in comingled sentinel turkeys. Furthermore, only 2 of 10 sentinel turkeys had a detectable infection. Similar findings have been obtained under laboratory conditions when turkeys that were experimentally exposed to a variety of influenza isolates obtained from waterfowl failed to become infected (C. J. Kelleher and D. A. Halvorson, unpublished data). Such findings suggest that domestic turkeys in Minnesota may actually be exposed to a vast array of influenza isolates, but only a very small percentage of these are capable of infecting turkeys. In addition, virus adaptation through repeated passage among turkeys within a flock may be required before a clinical infection is detected.
As a result, undetected subclinically infected flocks could account for the 6- to 8-week delay. Although influenza in turkeys usually began between mid-September and mid-October, 3 of 18 outbreaks in the 4-year period began between early May and mid-June. Interestingly, the onset of these early outbreaks in turkeys coincided with the period of detection of influenza virus in sentinel ducks in 1981. Northward -waterfowl migration through Minnesota began in March, and we believe that a low prevalence of influenza A virus existed in wild waterfowl before our monitoring in May. However, it was difficult to determine the prevalence of infection in northbound ducks or to demonstrate a seasonal effect during March, April, and May because of the staggered nature of the northward migration and the unfavorable conditions that exist for surveillance. It is worth noting the efforts of Hope-Simpson (8), who used World Health Organization records for a 28-year period to show that year after year outbreaks of human influenza are globally ubiquitous, occurring from October through March in the northern hemisphere and from April through September in the southern hemisphere, and that epidemic loci and antigenic variations move smoothly to and fro with the time of least solar radiation across the surface of the earth almost every year. He proposed that lack of solar radiation may affect the virus or the human host in some way that allows an epidemic to progress. Our findings of a high influenza infection rate in avian species around the period of maximal solar radiation would suggest that solar radiation is not directly affecting influenza A virus survival among ducks. Seasonal onsets of influenza infections, similar to those in the sentinel ducks in the present study, have also been demonstrated in domestic swine and mink. Hinshaw et al. (6) found that swine in the north-central United States became infected with influenza A virus in October and that the highest prevalence of infection occurred from October through December. The serological monitoring of a commercial mink farm in Japan revealed evidence of infection among mink from August to November (13). It would not be surprising to find similar seasonal influenza infections in other wild and domestic species. We conclude that wild ducks are a natural reservoir of influenza A virus, but only certain duck-derived isolates are capable of infecting domestic turkeys. In addition, sentinel ducks and domestic turkeys were found to have similar seasonal influenza infections that appear to be related. Because of the seasonal infection of wild waterfowl with influenza viruses infectious for other species and their wideranging migrations (a seasonal phenomenon related to variations in solar radiation), it is speculated that wild waterfowl may play an important role in the seasonal influenza infections of other animals, including humans. ACKNOWLEDGMENTS We gratefully acknowledge the assistance of Satish Nivas and staff, Peter Poss and staff, Nancy Schaeppi, Elizabeth Ellert, and Jane Sprangers. This work was supported in part by grants from the U.S. Science and Education Administration and the Minnesota Turkey Growers Association. LITERATURE CITED 1. Bahl, A. K., B. S. Pomeroy, S. Mangundimedjo, and B. C. Easterday. 1977. Isolation of type A influenza and Newcastle disease viruses from migratory waterfowl in the Mississippi
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SEASONAL EFFECT ON INCIDENCE OF AVIAN INFLUENZA
flyway. J. Am. Vet. Med. Assoc. 171:949-951. 2. Beard, C. W. 1970. Avian influenza antibody detection by immunodiffusion. Avian Dis. 14:337-341. 3. Carbary, E. A., C. W. Beard, R. Cooper, R. P. Hanson, and B. S. Pomeroy. 1974. Hemagglutination and hemagglutination inhibition tests with Newcastle disease virus microtiter technique. Proceedings of the 17th Annual American Association of Veterinary Laboratory Diagnosticians. American Association of Veterinary Laboratory Diagnosticians, Madison, Wis. 4. Easterday, B. C. 1975. Animal influenza, p. 449-482. In E. D. Kilbourne (ed.), The influenza viruses and influenza. Academic Press, Inc., New York. 5. Halvorson, D. A., D. Karunakaran, D. A. Senne, C. J. Kelleher, C. Bailey, A. Abraham, V. S. Hinshaw, and J. A. Newman. 1983. Epizootiology of avian influenza-simultaneous monitoring of sentinel ducks and turkeys in Minnesota. Avian Dis. 27:77-85. 6. Hinshaw, V. S., W. J. Bean, Jr., R. G. Webster, and B. C. Easterday. 1978. The prevalence of influenza viruses in swine and the antigenic and genetic relatedness of influenza viruses from man and swine. Virology 84:51-62. 7. Hinshaw, V. S., and R. G. Webster. 1980. The perpetuation of orthomyxoviruses and paramyxoviruses in Canadian waterfowl. Can. J. Microbiol. 26:622-629. 8. Hope-Simpson, R. E. 1981. The role of season in the epidemiology of influenza. J. Hyg. 86:35-46. 9. Laver, W. G., and R. G. Webster. 1979. Ecology of influenza viruses in lower mamnmals and birds. Br. Med. Bull. 35:23-33. 10. Marzouk, Y., S. M. Goyal, and C. P. Gerba. 1979. Prevalence of enteroviruses in groundwater in Israel. Groundwater 17:487-491. 11. Minnesota Department of Natural Resources. 1983. Status of wildlife populations, p. 43. Minnesota Department of Natural Resources, St. Paul, Minn.
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12. Okazaki, K., R. Yanagawa, and H. Kida. 1983. Contact infection of mink with 5 subtypes of avian influenza virus. Arch. Virol. 77:265-269. 13. Okazaki, K., R. Yanagawa, H. Kida, and H. Noda. 1983. Human influenza virus infection in mink: serological evidence of infection in summer and autumn. Vet. Microbiol. 8:251-257. 14. Pye, V. I., and R. Patrick. 1983. Groundwater contamination in the United States. Science 221:713-721. 15. Rosenberger, J. K., and W. C. Krauss. 1974. Isolation of Newcastle disease and type A influenza viruses from migratory waterfowl in Atlantic flyway. Avian Dis. 18:610-613. 16. Scheuerman, P. R., G. Bitten, A. R. Overmary, M. Asce, and G. E. Gifford. 1979. Transport of viruses through organic soils and sediments. J. Environ. Eng. Div. 4:629-640. 17. Sinnecker, H., R. Sinnecker, E. Zilske, and D. Koehler. 1982. Detection of influenza A viruses and influenza epidemics in wild pelagic birds by sentinels and population studies. Zentralbl. Bakteriol. Mikrobiol. Hyg. 1 Abt. Orig. A 253:297-304. 18. Slemons, R. D., D. C. Johnson, J. S. Osborn, and F. Hayes. 1974. Type A influenza viruses isolated from wild free-flying ducks in California. Avian Dis. 18:119-124. 19. Van Deusen, R. A., V. S. Hinshaw, D. A. Senne, and D. Pellacani. 1983. Micro neuraminidase inhibition assay for classification of influenza A virus neuraminidases. Avian Dis. 27:745-750. 20. Webster, R. G., M. Morita, C. Pridgen, and B. Tumova. 1976. Ortho- and paramyxoviruses from migrating feral ducks: characterization of a new group of influenza viruses. J. Gen. Virol. 32:217-225. 21. Webster, R. G., M. Yakhno, V. S. Hinshaw, W. J. Bean, and K. G. Murti. 1978. Intestinal influenza: replication and characterization of influenza viruses in ducks. Virology 84:268-278.
