Duck Isolates Research Articles

Characterisation and comparison of avian Pasteurella multocida strains by conventional and ERIC-PCR assays

Sixty-one avian strains of Pasteurella multocida were characterised and compared by biochemical tests, capsular PCR typing and ERIC-PCR. The strains were recovered from various avian species (goose, duck, Muscovy duck, turkey, chicken and pheasant) and represented different geographic locations in Hungary. Forty-two strains (69%) were identified as P. multocida subsp. multocida and 19 strains (31%) as P. multocida subsp. septica. The strains were grouped into 7 different biovars (1, 2, 3, 4, 5, 6 and 7). The most prevalent biovars were 1 (25%), 3 (21%) and 6 (21%). Most of the duck isolates (90%) belonged to biovar 1 or 6. The most frequent capsular type was A (93.5%). Type F represented only a small number (6.5%) of the strains. Other capsular types were not identified. From the 61 isolates 24 different fingerprint patterns were generated by ERIC-PCR assay. Based on cluster analysis the strains could be grouped into four larger and four mini-clusters that showed considerable correlation with the geographical origin and the host species. The results indicate that ERIC-PCR may be a suitable technique for studying the host adaptation of P. multocida and the epidemiology of fowl cholera.

Chlamydial infections in duck farms associated with human cases of psittacosis in France

Five severe cases of psittacosis in individuals associated with duck farms were notified in France between January and March 2006. Diagnostic examination included serology and/or molecular detection by PCR from respiratory samples. As a consequence, we investigated all duck flocks ( n = 11) that were housed in the three farms where human infections occurred. While serology by complement fixation test was negative for all samples, cloacal and/or tracheal chlamydial excretion was detected by PCR in all three units. Notably, one duck flock was tested strongly positive in 2 of the 3 affected farms, and Chlamydophila ( C.) psittaci strains were isolated from cloacal and/or tracheal swab samples from both farms. Human samples and duck isolates exhibited the same PCR-RFLP restriction pattern, which appeared to be an intermediate between genotypes A and B. Analysis of ompA gene sequences and comparison to those of the type strains showed that the isolates could not be strictly assigned to any of the generally accepted genotypes of C. psittaci. Further analysis by MLVA of the PCR-positive human samples revealed two distinct patterns, which were related to previously isolated C. psittaci duck strains.

Molecular epidemiological investigation of Newcastle disease virus from domestic ducks in Korea

To expand the epidemiological understanding of Newcastle disease virus (NDV) found in domestic ducks in Korea, 14 NDV isolates from apparently healthy domestic ducks were biologically and genetically characterized. Thirteen and 1 isolates of NDV were categorized into lentogenic and velogenic viruses, respectively, based on in vivo pathogenicity tests. Twelve lentogenic viruses showed HA activity to horse RBCs, while 1 lentogenic virus and the velogenic virus were negative. Lentogenic viruses ( n = 13) had sequence motifs of 112ERQERL 117 ( n = 1) or 112GRQGRL 117 ( n = 12) at the F0 cleavage site, while the velogenic virus ( n = 1) had a sequence motif of 112RRQKRF 117 at the same site. Phylogenetic analysis revealed that at least three distinct genotypes may exist in domestic ducks in Korea; one class I genotype (genotype 2), and two class II (genotypes I and VII) genotypes. The class I virus was most closely related to strains of genotype 2 which were isolated in birds from the USA, Germany and Denmark. Twelve lentogenic class II viruses were grouped together in genotype I, and were then divided into at least three clusters, namely Aomori-like, Ulster2C-like, and V4-like. The velogenic class II virus was assigned to genotype VII which represents viruses responsible for recent epidemics in many Asian countries including Korea. The epidemiological importance of domestic duck isolates of NDV in Korea is discussed.

Development of Goose- and Duck-Specific DNA Markers To Determine Sources of Escherichia coli in Waterways

The contamination of waterways with fecal material is a persistent threat to public health. Identification of the sources of fecal contamination is a vital component for abatement strategies and for determination of total maximum daily loads. While phenotypic and genotypic techniques have been used to determine potential sources of fecal bacteria in surface waters, most methods require construction of large known-source libraries, and they often fail to adequately differentiate among environmental isolates originating from different animal sources. In this study, we used pooled genomic tester and driver DNAs in suppression subtractive hybridizations to enrich for host source-specific DNA markers for Escherichia coli originating from locally isolated geese. Seven markers were identified. When used as probes in colony hybridization studies, the combined marker DNAs identified 76% of the goose isolates tested and cross-hybridized, on average, with 5% of the human E. coli strains and with less than 10% of the strains obtained from other animal hosts. In addition, the combined probes identified 73% of the duck isolates examined, suggesting that they may be useful for determining the contribution of waterfowl to fecal contamination. However, the hybridization probes reacted mainly with E. coli isolates obtained from geese in the upper midwestern United States, indicating that there is regional specificity of the markers identified. Coupled with high-throughput, automated macro- and microarray screening, these markers may provide a quantitative, cost-effective, and accurate library-independent method for determining the sources of genetically diverse E. coli strains for use in source-tracking studies. However, future efforts to generate DNA markers specific for E. coli must include isolates obtained from geographically diverse animal hosts.

Colonizing Capability of Campylobacter jejuni Genotypes from Low-Prevalence Avian Species in Broiler Chickens

Genetic variations in Campylobacter jejuni or host factors result in low prevalence rates among nonchicken poultry species. The objective of this study was to determine the colonizing potential, in broiler chickens, of C. jejuni that was recovered from low-prevalence avian species. Twenty-day-old Campylobacter-negative broiler chicks were inoculated by oral gavage with genetically different primary isolates of C. jejuni recovered from squab, duck, or chicken. Serial sampling and microbiologic testing of ceca were used to determine the level of colonization and the prevalence of positive chickens. All isolates were recovered from chickens by 10 days postinoculation. The C. jejuni strains recovered from challenged birds were genetically identical to the inoculated strains. By 10 days postinoculation, treatment groups inoculated with duck or control chicken isolates were 100% positive. The level of colonization by the squab isolate on day 2 postinoculation was significantly less than the duck or chicken isolates and had not colonized all birds by day 10 postinoculation.

Diversity of flaA Genotypes among Campylobacter jejuni Isolated from Six Niche-Market Poultry Species at Farm and Processing

PCR–restriction fragment length polymorphism of the flagellin (flaA) gene in Campylobacter jejuni was used to determine the relationships of isolates collected at the farm and throughout processing for six niche-market poultry species. This study focused on two specialty chicken products, poussin and free range, and four other specialty products, squab, duck, guinea fowl, and quail. Cloacal and carcass samples were collected from three flocks from each of the six niche species. Three processing plants in California participated in a 2-year investigation. A total of 773 isolates from farm, posttransport, and the processing plants were genotyped, yielding a total of 72 distinct flaA profiles for the six commodities. Genetic diversity of C. jejuni at the farm was greatest for ducks with up to 12 distinct flaA types in two flocks and least for squab 1 flaA type between two farms. For two of the guinea fowl flocks, one free-range flock, two squab flocks, and all three poussin flocks, the flaA types recovered at the prepackage station matched those from the farm. Cross-contamination of poultry carcasses was supported by the observation of flaA types during processing that were not present at the farm level. New C. jejuni strains were detected after transport in ducks, guinea fowl, and free-range chickens. Postpicker, postevisceration, and prewash sampling points in the processing plant yield novel isolates. Duck and free-range chickens were the only species for which strains recovered within the processing plant were also found on the final product. Isolates recovered from squab had 56 to 93% similarity based on the flaA types defined by PCR–restriction fragment length polymorphism profiles. The 26 duck isolates had genetic similarities that ranged from 20 to 90%. Guinea fowl and free-range chickens each had 40 to 65% similarity between isolates. Poussin isolates were 33 to 55% similar to each other, and quail isolates were 46 to 100% similar. Our results continue to emphasize the need to clean processing equipment and posttransport crates in order to decrease cross contamination between flocks. This study also determined that several strains of C. jejuni had unique flaA types that could only be recovered in their host species.

Phylogenetic Analysis of the Sigma 2 Protein Gene of Turkey Reoviruses

The open reading frame of the S3 segment encoding the sigma2 protein of four turkey reovirus field isolates was analyzed for sequence heterogeneity. The turkey reoviruses we present here have a 97% amino acid identity to turkey NC 98. The S3 nucleotide and amino acid sequence similarity was < or =61% and 78%-80%, respectively, when compared to the chicken reovirus isolates. Comparison of amino acid sequences from chickens and turkeys with that of a duck isolate revealed a 53% and 55% similarity, respectively. Phylogenetic analyses, based on both nucleotide and amino acid sequence, resulted in three major groups among the avian reoviruses; these groups were clearly separated by species. The results of this study provide further evidence, based on the deduced sigma2 sequence, that turkey reoviruses form a distinct, separate group relative to chicken and duck isolates. In addition, as a result of the limited sequence identity with their avian counterparts, turkey reoviruses could potentially be considered a separate virus species within subgroup 2 of the Orthoreovirus genus.

Restriction enzyme analysis of Indian isolates of egg drop syndrome 1976 virus recovered from chicken, duck and quail.

Egg drop syndrome 1976 (EDS-76) is caused by a haemagglutinating adenovirus belonging to group III of the genus Aviadenovirus in the family Adenoviridae. All isolates are serologically identical, but have been divided into three groups based on restriction endonuclease (RE) analysis. In this study the viral DNA of various Indian EDS-76 viral isolates (CEDS-A, CEDS-B, EDS-M, EDS-ML, EDS-1/AD/86, EDS-KC and QEDS) obtained from different avian species and different geographical regions were digested with restriction endonucleases viz., EcoRI, BamHI, HindIII and PstI. The results showed that one Indian isolate obtained from duck (DEDS-KC) was different from all other chicken and quail counterparts. All other isolates were identical to the reference viral strain BC-14, which belong to group I of EDS-76 viruses. The duck isolate EDS-KC could not be placed in any of the three groups reported earlier.

Isolation and molecular characterization of Toxoplasma gondii from chickens and ducks from Egypt

The prevalence of Toxoplasma gondii in free range chickens is a good indicator of the prevalence of T. gondii oocysts in the environment because chickens feed from the ground. In the present study, prevalence of T. gondii in 121 free range chickens ( Gallus domesticus) and 19 ducks ( Anas sp.) from a rural area surrounding Giza, Egypt was assessed. Blood, heart, and brain from each animal were examined for T. gondii infection. Antibodies to T. gondii, assayed with the modified agglutination test (MAT), were found in 49 (40.4%) chickens in titers of 1:5 in 11, 1:10 in four, 1:20 in four, 1:40 in eight, 1:80 in 10, and 1:160 or more in 12 chickens. Antibodies were found in three ducks each with a titer of 1:80. Hearts and brains of seropositive (MAT≥1:5) chickens and ducks were bioassayed in mice. Additionally, hearts and brains of seronegative (MAT<1:5) animals were bioassayed in T. gondii-free cats. T. gondii was isolated from 19 of 49 seropositive chickens (one with a titer of 1:5, two with a titer of 1:20, one with a titer of 1:40, five with a titer of 1:80, three with a titer of 1:160, and seven with a titer of ≥1:360). One cat fed tissues pooled from 15 seronegative chickens shed T. gondii oocysts, while two cats fed tissues of 34 seronegative chickens did not shed oocysts. T. gondii was isolated from one of the seropositive ducks by bioassay in mice. The two cats fed tissues from 16 seronegative ducks did not shed oocysts. Genotyping of 20 chicken isolates of T. gondii using the SAG 2 locus indicated that 17 isolates were type III and three were type II. The duck isolate of T. gondii was type III. The mice inoculated with tissue stages of all 21 isolates of T. gondii from chickens and ducks remained asymptomatic, indicating that phenotypically they were not type I because type I strains are lethal for mice. Infections with mixed genotypes were not found.

Epidemiological studies of Brachyspira pilosicoli in two Australian piggeries

The epidemiology of infection with the intestinal spirochaete Brachyspira pilosicoli within pig herds is incompletely understood. To investigate this further, cross-sectional and cohort studies were undertaken on two piggeries. Faeces were subjected to selective culture, and DNA was extracted from growth on the primary media and amplified by polymerase chain reaction (PCR). On one farm, samples from other animal species and the environment were also examined. Isolates were subjected to multilocus enzyme electrophoresis (MLEE) and pulsed field gel electrophoresis (PFGE). The prevalence on farm A (>2000 sows) was 2.4% (95% confidence interval (CI): 0.3, 4.4%). Infection was largely confined to grower/finisher pigs. The six isolates of B. pilosicoli recovered belonged to a single MLEE electrophoretic type (ET) and a single PFGE type. On piggery B, an 80-sow unit located on a research farm, the prevalence amongst growers and finishers was 12.2% (95% CI: 4.7, 19.6%). There was also evidence that weaners were being infected. Ten isolates obtained were genetically heterogeneous, being divided into six ETs and seven PFGE types. One of four isolates in one ET had an identical PFGE type to those on piggery A, and may have been introduced to piggery B in stock from piggery A. On farm B, B. pilosicoli was also detected by PCR in chickens, effluent pond water and wild ducks on the pond. An isolate from the pond belonged to the same ET as one from a pig, whereas the duck isolates were distinct. This study demonstrates the complex epidemiology of B. pilosicoli infections in piggeries.

Isolation of avian pneumovirus from mallard ducks that is genetically similar to viruses isolated from neighboring commercial turkeys

Our earlier studies demonstrating avian pneumovirus (APV) RNA in wild geese, sparrows, swallows, starlings and mallard ducks suggested that wild birds might be involved in the circulation of APV in the United States. To determine whether turkey virus can be transmitted to the free flying birds, we placed APV-negative mallard ducks next to a turkey farm experiencing a severe APV outbreak and in an area with a large population of waterfowls. The sentinel ducks did not develop clinical APV disease but infectious APV (APV/MN-12) was recovered from choanal swabs after 2 weeks, and anti-APV antibodies detected after 4 weeks. Four APV isolates recovered from the neighboring turkeys that were experiencing an APV outbreak at the same time shared 95–99% nucleotide identity and 97–99% predicted amino acid identity with the duck isolate. In addition experimental infection of turkey poults with APV/MN-12 resulted in detection of viral RNA in nasal turbinates and APV-specific IgG in serum. These results indicate that the APV isolates from turkeys and ducks shared a common source, and the viruses from different avian species can cross-infect.

Use of repetitive DNA sequences and the PCR To differentiate Escherichia coli isolates from human and animal sources.

The rep-PCR DNA fingerprint technique, which uses repetitive intergenic DNA sequences, was investigated as a way to differentiate between human and animal sources of fecal pollution. BOX and REP primers were used to generate DNA fingerprints from Escherichia coli strains isolated from human and animal sources (geese, ducks, cows, pigs, chickens, and sheep). Our initial studies revealed that the DNA fingerprints obtained with the BOX primer were more effective for grouping E. coli strains than the DNA fingerprints obtained with REP primers. The BOX primer DNA fingerprints of 154 E. coli isolates were analyzed by using the Jaccard band-matching algorithm. Jackknife analysis of the resulting similarity coefficients revealed that 100% of the chicken and cow isolates and between 78 and 90% of the human, goose, duck, pig, and sheep isolates were assigned to the correct source groups. A dendrogram constructed by using Jaccard similarity coefficients almost completely separated the human isolates from the nonhuman isolates. Multivariate analysis of variance, a form of discriminant analysis, successfully differentiated the isolates and placed them in the appropriate source groups. Taken together, our results indicate that rep-PCR performed with the BOX A1R primer may be a useful and effective tool for rapidly determining sources of fecal pollution.

Nucleotide Sequence and Vaccinia Expression of the Nucleoprotein of a Highly Virulent, Neurotropic Strain of Newcastle Disease Virus

The nucleoprotein (NP) of Newcastle disease virus (NDV) was selected to study the relative importance of an internal structural protein in the avian immune response. The NP gene of the virulent, neurotropic NDV Texas GB (TGB) strain was cloned and sequenced. Nucleotide sequence data for the NP gene allowed comparison of the deduced amino acid sequences for the NP genes of NDV-TGB and the avirulent duck isolate NDV-D26. These comparisons demonstrated an 89% nucleotide sequence homology and a 97% homology between the deduced amino acid sequences. The NDV-TGB NP expressed in recombinant vaccinia virus (rVAC) was electrophoretically and immunologically identical to the wild-type NDV-TGB. Although inoculation of chickens with the recombinant vaccinia virus expressing the NDV NP gene elicited anti-NDV antibodies in higher titers than in birds inoculated with live LaSota NDV, this strong anti-NDV response did not protect against lethal challenge with NDV-TGB.

An epidemiological study of influenza viruses among Chinese farm families with household ducks and pigs.

To examine the possibility of interspecies transmission and genetic reassortment of influenza viruses on farms in Southern China, we surveyed 20 farm families living outside the city of Nanchang who raised pigs and ducks in their homes. Weekly interviews of family members and virus isolation studies of throat swabs and faecal samples, collected from September 1992 to September 1993, established the seasonal pattern of respiratory tract infections in these families and identified 11 influenza viruses (6 in humans and 5 in ducks). Most of the human isolates were type A of H3N2 subtype. Serologic studies of farm pigs indicated infection by the same human viruses circulating in family members, but there was no evidence that either swine or avian viruses had been transmitted to pigs. Eight of 156 human serum samples inhibited the neuraminidase activity of two of the duck isolates, raising the possibility of interspecies transmission of these avian viruses. Genotype analysis of duck and human isolates provided no evidence for reassortment. Our finding support the concept that intermingling of humans, pigs and ducks on Chinese farms is favourable to the generation of new, potentially hazardous strains of influenza virus.

Comparison of avian Chlamydia psittaci isolates by restriction endonuclease analysis and serovar-specific monoclonal antibodies

Avian Chlamydia psittaci isolates were examined by restriction endonuclease analysis and serovar-specific monoclonal antibodies and compared with ovine abortion and polyarthritis isolates. The avian isolates were divided into four serovars (turkey, psittacine, pigeon, and duck) based on their reactivity to the monoclonal antibodies. The DNA digest patterns were similar across the four avian serovars; most bands were identical when the isolates were tested with PstI, BamHI, and EcoRI restriction endonuclease enzymes. The turkey group restriction endonuclease analysis patterns were distinguished from those of the other avian strains by three to four band differences with all enzymes. The duck and pigeon isolates showed only minor DNA pattern differences when compared with the psittacine isolates. Four psittacine isolates from various locations in Texas had an extra band with the EcoRI restriction enzyme, suggesting that they were from a common source; however, they were indistinguishable from the other psittacine isolates when examined with the monoclonal antibodies. The avian isolates were distinctly different from either abortion or polyarthritis isolates by both restriction endonuclease analysis and monoclonal antibody analysis. The data demonstrate that the avian isolates form a distinct group or separate biovar with at least four serovars.

Antigenic stability of H3 influenza viruses in the domestic duck population of southern China

An antigenic analysis was carried out on 145 duck influenza virus isolates of the H3 haemagglutinin subtype obtained over five years continuous surveillance from the region of southern China, a hypothetical influenza epicentre. This was done using a panel of twelve monoclonal antibodies raised to an early human strain of the H3 subtype. We demonstrate the existence of an extensive range of antigenic profiles, broadly similar but not identical to the human H3 strain, which persisted over the five year period. This variability was as great during discrete twelve month periods as over the whole five years. Hierarchic progression (observed with human strains) was not evident and no correlation of antigenic drift, in either positive or negative direction, was observed with the domestic duck isolates over time. Changing dominant antigenic profiles were, however, observed in faecal isolates with time within a single farm. The much broader range of profiles detected in pond water samples from the same farm suggested the existence of a heterogeneous antigenic reservoir. Local switching of dominant profiles may occur due to changes of cohorts as birds are taken to market. In vitro and in vivo passage experiments revealed a high degree of heterogeneity in antigenic profiles in progeny of uncloned isolates, whereas the profiles of cloned isolates were largely conserved. These results suggested that particular antigenic profiles in primary isolates may result from mixtures of subpopulations of the wild type virus in natural duck infections. Switching between reactivity profiles of different progeny is likely to be largely a result of regrouping of these subpopulations with lesser effects due to mutation. Hypervariability in some of the cloned isolates was observed with a few monoclonal antibodies recognising a region of HA reported to be hypervariable in swine influenza virus. Reactivity with one particular antibody was correlated with passage in chicken eggs. The ability of this enormously varied pool of duck influenza H3 strains to cross the species barrier to man and give rise to viruses with hierarchic capabilities was considered.

Differentiation of egg drop syndrome virus isolates by restriction endonuclease analysis of virus dna

Thirteen isolates of egg drop syndrome (EDS) virus were compared by restriction endonuclease analysis of the virus DNA. One virus, an Australian chicken isolate, was distinguished from the others using the endonucleases EcoRl, BamUl, Kpnl, Hindlll, Pstl and Pvull, all of which recognise six base pair DNA sequences. Polyacrylamide gel restriction fragment patterns generated by Haelll, Hhall and TaqI, which recognise four base pairs, allowed further differentiation of the virus isolates. Nine chicken viruses isolated in the United Kingdom and Belgium in the period 1976 to 1987 were identical and could be distinguished from three United Kingdom duck isolates. The Australian isolate, in addition to possessing a DNA deletion (0.4 kbp) at one end of the genome (32.6 kbp) also differed from the European isolates at base sequence level. Genome maps for EcoRl, BamHl, Kpnl and Pstl are reported for the 127 isolate of EDS virus.

Antigenic and genetic conservation of H3 influenza virus in wild ducks

The hemagglutinins of H3 influenza viruses isolated from migratory ducks on the Pacific flyway in Japan during the period 1977 to 1985 were analyzed antigenically and genetically. Antigenic analysis using monoclonal antibodies to the hemagglutinins of A/Aichi/2/68 (H3N2) and A/duck/Hokkaido/8/80 (H3N8) viruses showed that antigenic drift occurred extensively in human strains, whereas the hemagglutinins of duck viruses were highly conserved. It was also found that the hemagglutinins of duck viruses were antigenically closely related to that of human 1968 H3 prototype strains. Nucleotide sequence analysis of seven duck H3 hemagglutinin genes showed a limited number of changes among the six duck isolates and between these duck isolates and Aichi/68. The deduced amino acid sequence revealed amino acid changes randomly distributed throughout the molecule and not confined to antigenic sites. These findings indicate that the duck virus hemagglutinin genes are conserved in nature and that viruses of different lineages cocirculate.

Isolation of ortho- and paramyxoviruses from domestic poultry in Hong Kong between November 1977 and October 1978 and comparison with isolations made in the preceding two years

Regular surveillance of domestic poultry from Hong Kong and southern China for influenza A viruses was conducted in Hong Kong from November 1977 to October 1978. Tracheal or cloacal swabs taken from 2844 ducks, geese and fowls yielded 141 influenza viruses of which 135 were derived from ducks. The isolation rate from ducks was twice as high from the cloaca (12.1 per cent) as from the trachea (5.8 per cent). The overall frequency of isolation was 10.3 per cent and 2.3 per cent for poultry originating from southern China and Hong Kong, respectively. Fourteen of the influenza antigenic combinations recorded in this period of surveillance had been obtained in the previous two years while five were new--H2N2, H2Nav6, Hav5N2, Hav6Nav1 and Hav7Nav4. The number of combinations recorded over three years was 39, the most frequent being Hav4Nav1 which comprised one fifth of the total. All the combinations found in geese and fowl isolates, apart from Hav7Nav6 found in a fowl, have been recorded in duck isolates. Faecal/oral transmission of virus from infected pond water was thought to be an important factor in the high incidence of influenza in ducks. The existence of a large number of combinations of haemagglutinin and neuraminidase antigens and the isolation of two viruses from the cloaca suggest that recombination is occurring in these birds. Reasons for the limited isolation of some antigenic combinations are considered. The paramyxoviruses, Newcastle disease virus and the Hong Kong avian paramyxovirus, were also isolated. Over the three year period the isolation rate of influenza and paramyxoviruses from ducks appeared to be cyclical and seasonal. Influenza was found mainly in the summer and paramyxoviruses in the winter.

The ecology of influenza viruses in ducks and analysis of influenza viruses with monoclonal antibodies

It has been suggested that influenza A viruses from lower animals may be involved in the origin of human pandemic strains. To test this hypothesis, we have been analyzing the influenza A viruses present in lower mammals and birds. Over the past few years, it has become increasingly evident that a major reservoir of influenza viruses exists in both domestic and feral avian species, particularly ducks. For example, studies on migratory ducks in Canada in 1978 showed that 50% of these healthy birds were infected with influenza viruses. These viruses included 16 different combinations of hemagglutinin and neuraminidase subtypes, including those related to human strains as hemagglutinins, Hav7, H2, and Hsw1 and neuraminidases, N1 and N2. The duck isolates which were antigenically related to the Hsw1N1 viruses from pigs and man were evaluated by in vivo pathogenicity tests in ferrets, ducks, pigs, and chickens; the results indicated that these avian Hsw1N1 viruses replicated in both avian and mammalian species, underlining their potential for interspecies transmission. Since the reassortment of genetic information between human and animal influenza A viruses has been suggested as the mechanism for the origin of new human strains, we also examined the migratory ducks for evidence of mixed infections, i.e. infection with two antigenically distinct viruses to determine if this phenomenon occurs in nature. Antisera to the original isolate was used to select for antigenically different viruses in the original cloacal sample. Mixtures of two and, on rare occasions, three distinct subtypes (e.g., Hav7N2, Hav6N2, and Hav7Neq2) were isolated from one bird. Analysis of the RNAs of these viruses by polyacrylamide gel electrophoresis suggested that genetic reassortment between viruses in the mixedly infected birds was occurring. These findings agree with laboratory studies in which recombinant viruses were isolated from mixedly infected ducks. These data indicate that mixed infections do occur in the natural setting, thus establishing the situation where genetic reassortment between viruses could occur. The avian species, particularly ducks, continue to represent an impressive reservoir of influenza A viruses and may well contribute to the appearance of related influenza viruses in other species, including humans.