Potential of low pathogenicity avian influenza viruses of wild bird origin to establish experimental infections in turkeys and chickens.

The potential of low pathogenicity (LP) avian influenza virus (AIV) isolates of wild bird origin to establish infection in commercial turkeys and broiler chickens was studied. Isolates, representing subtypes H5N1, H7N3, H6N2, and H3N6, were recovered in 2005 and 2006 from waterfowl and shorebirds in the Delmarva Peninsula region of the east coast of the United States. The LP AIV isolates were not pathogenic for 2-wk-old meat-type turkeys and broiler chickens. No mortality, clinical signs, or gross lesions were observed following intratracheal and conjunctival sac routes of exposures with 10(6.0) EID50 (embryo infectious dose) per bird. Isolates resulting in an established infection based on virus isolation were: A/mallard/Maryland/1159/ 2006 (H5N1) in the upper respiratory tract of turkeys; A/mallard/Delaware/418/2005 (H7N3) in the upper respiratory and intestinal tracts of turkeys and chickens; and A/shorebird-environment/Delaware/251/2005 (H3N6) in the upper respiratory and intestinal tracts of chickens. Infections were also confirmed by production of AIV-specific serum antibodies detected by hemagglutination inhibition.

Evidence of avian metapneumovirus subtype C infection of wild birds in Georgia, South Carolina, Arkansas and Ohio, USA.

Metapneumoviruses (MPVs) were first reported in avian species (aMPVs) in the late 1970s and in humans in 2001. Although aMPVs have been reported in Europe and Asia for over 20 years, the virus first appeared in the United States in 1996, leaving many to question the origin of the virus and why it proved to be a different subtype from those found elsewhere. To examine the potential role of migratory waterfowl and other wild birds in aMPV spread, our study focused on determining whether populations of wild birds have evidence of aMPV infection. Serum samples from multiple species were initially screened using a blocking enzyme-linked immunosorbent assay. Antibodies to aMPVs were identified in five of the 15 species tested: American coots, American crows, Canada geese, cattle egrets, and rock pigeons. The presence of aMPV-specific antibodies was confirmed with virus neutralization and western blot assays. Oral swabs were collected from wild bird species with the highest percentage of aMPV-seropositive serum samples: the American coots and Canada geese. From these swabs, 17 aMPV-positive samples were identified, 11 from coots and six from geese. Sequence analysis of the matrix, attachment gene and short hydrophobic genes revealed that these viruses belong to subtype C aMPV. The detection of aMPV antibodies and the presence of virus in wild birds in Georgia, South Carolina, Arkansas and Ohio demonstrates that wild birds can serve as a reservoir of subtype C aMPV, and may provide a potential mechanism to spread aMPVs to poultry in other regions of the United States and possibly to other countries in Central and South America.

Evolution of H5 subtype avian influenza A viruses in North America.

The phylogenetic relationships of the hemagglutinin (HA) and non-structural (NS) genes from avian influenza (AI) H5 subtype viruses of North American origin are presented. Analysis of the HA genes of several previously uncharacterized isolates from waterfowl and turkeys provided clear evidence of significant sequence variation and existence of multiple virus clades or sub-lineages, maintained in migratory waterfowl. Phylogenetic analysis of NS gene sequences further demonstrated multiple sub-lineages and also demonstrated re-assortment of two NS alleles in wild duck populations. Based on currently available HA1 gene sequences, at least four clades exist with waterfowl isolates included in three of the four groups. The most genetically unstable of these sub-lineages is composed of recent poultry isolates from the outbreak of AI in Central Mexico. This group of viruses, which replicated unabated in chickens for at least 16 months, exhibited an increased rate of mutation in both the HA and NS gene. Comparison of the HA1 sequence data for all available North American H5 subtype viruses demonstrated minimal variation both in and around the amino acids predicted to be involved in the HA receptor binding site. The sequences also revealed that migratory waterfowl, live poultry market chicken, and turkey isolates uniformly lack a glycosylation site at amino acid 236 in the HA protein which is present in commercial chicken isolates.

Evaluation of an enzyme-linked immunosorbent assay licensed by the USDA for use in cattle for diagnosis of ovine paratuberculosis.

A commercially available Mycobacterium phlei-absorbed enzyme-linked immunosorbent assay (ELISA) approved to detect antibodies to Mycobacterium paratuberculosis in cattle was evaluated for its applicability in sheep. The potential for interference with ELISA results from cross-reacting antibodies to Corynebacterium pseudotuberculosis was also investigated. Serum samples were randomly selected from a collection of samples obtained in 1986-1991 from 6 infected and 5 noninfected sheep flocks varying in breed, age, and geographic origin. Tests were performed on sera from 27 paratuberculous sheep, confirmed by histopathology, bacteriologic culture, and/or acid-fast staining of ileal mucosal smears, and on sera from 246 noninfected sheep. The optical density of each sample was expressed as a percentage of the optical density of a known positive sheep serum sample tested on the same plate. These values were log-transformed to achieve normality of distribution, and sensitivity and specificity estimates were calculated based on 2 and 3 standard deviations above the mean of the percent positive value (PPV) of the noninfected sheep. A cutoff value of PPV > or = 55.74 resulted in an estimated sensitivity of 0.48 and a specificity of 0.95. Sera from 10 noninfected sheep with PPV above the cutoff level of 55.74% were absorbed with heat-treated C. pseudotuberculosis organisms in addition to M. phlei antigens. Sera from 14 ELISA-positive paratuberculous sheep and 23 ELISA-negative noninfected sheep were similarly treated, and results were compared. Absorption with C. pseudotuberculosis resulted in a significant decrease in PPV in all 3 groups of sheep sera, but a greater decrease was observed in the noninfected sheep with PPV above the cutoff level when compared with noninfected sheep with PPV below that level. Results of this study suggest that ELISA may be of value in screening sheep flocks for paratuberculosis, but further experimentation is needed to optimize the sensitivity and specificity of the assay. Exposure to C. pseudotuberculosis may confound results obtained by M. phlei-absorbed ELISA for paratuberculosis.

Replication of a waterfowl-origin influenza virus in the kidney and intestine of chickens.

Intravenous inoculation of chickens with a waterfowl-origin type A influenza virus resulted in high titers of virus in kidney tissues and viral nucleoprotein in renal tubular epithelial cells and in intestinal mucosal epithelial cells. Virus titers in kidneys of four of eight clinically normal chickens sampled on days 3 and 5 postinoculation (PI), one dead chicken on day 3 PI, and one dead chicken on day 7 PI exceeded 10(6) mean embryo infectious dose per gram of tissue. Using immunofluorescent and immunoperoxidase staining, viral nucleoprotein was identified in the cytoplasm and nucleus of tubular epithelial cells in kidneys and in nucleus of mucosal epithelial cells lining villi in the lower small intestine. Based on the low intravenous pathogenicity index for this virus (0.3) along with the high virus titers in kidney tissues and localization of viral antigen in kidney important site for replication of avian influenza (AI) virus of low pathogenicity. Recovery of type A influenza viruses from cloacal swabs could result from viral replication in kidneys as well as in the lower intestine and/or the bursa of Fabricius.

Renal pathology in specific-pathogen-free chickens inoculated with a waterfowl-origin type A influenza virus.

Five-week-old specific-pathogen-free chickens inoculated intravenously with a waterfowl-origin type A influenza virus (A/mallard/Ohio/184/86) had swollen and mottled kidneys on days 3, 5, and 7 postinoculation (PI) and multiple raised nodules on days 5, 10, and 20 PI. Histologically, the kidneys had multifocal heterophilic tubulointerstitial nephritis with epithelial necrosis on day 3 PI, lymphoplasmacytic tubulointerstitial nephritis on day 5 PI, and fibrosing interstitial nephritis with cortical lobular collapse, atrophic tubules, glomerular aggregates, and interstitial lymphoid follicles and aggregates on days 7, 10, and 20 PI. Heterophilic intratubular medullary-cone nephritis was present in dead or moribund chickens on days 3 and 5 PI. Furthermore, the presence of mild multifocal heterophilic tubulointerstitial nephritis on day 20 PI suggests that a waterfowl-origin strain of type A influenza virus of low pathogenicity has the potential to produce acute and chronic active nephritis in the chicken and that the kidney is a potential site for influenza viral persistence. The acute, subacute, and chronic histopathologic renal lesions of this influenza virus in chickens are similar to lesions reported for some nephropathogenic infectious bronchitis viruses and avian nephritis picornavirus.

Evaluation of the kidney as a potential site of avian influenza virus persistence in chickens.

One-day-old chickens were inoculated intravenously with one of three low-pathogenicity avian-origin influenza isolates. On day 5 postinoculation (PI), the frequency of influenza virus isolation from cloacal swabs following challenge with each isolate ranged from 83% to 100% for clinically normal euthanatized chickens. Influenza virus was also frequently isolated from kidneys of these chickens (47%) and from chickens that died (100%). Kidneys positive for virus isolation had lesions of nephrosis and/or acute nephritis, and influenza viral nucleoprotein was demonstrated in nuclei and cytoplasm of necrotic renal tubule epithelium. On sampling days 28 and 45/60 PI, influenza virus was neither isolated from nor immunohistochemically demonstrated in kidneys (0/125); however, the kidneys (47%) did have chronic histologic lesions that suggested previous influenza virus infection of the kidneys. Influenza virus was isolated from cloacal swabs of two of 44 chickens on day 28 PI, but all cloacal swabs were negative for virus recovery on sampling day 45/60 PI (0/81). These results indicate that replication of influenza virus in renal tubule epithelial cells did not result in persistence of type A influenza virus in this immunologically privileged site.

Nephrotropic properties demonstrated by A/chicken/Alabama/75 (H4N8) following intravenous challenge of chickens.

Tissue tropism properties of A/chicken/Alabama/75 (H4N8) were examined after intravenous inoculation of 5-week-old specific-pathogen-free chickens. From 14 clinically normal chickens euthanatized on days 1-20 postinoculation, the frequencies of virus recovery were highest for cloacal swabs (86%), bursal swabs (64%), and kidney tissues (64%) and lowest for tracheal swabs (14%), thymus tissues (14%), bone-marrow swabs (7%), and brain tissues (0%). Evidence that the high frequency of virus recovery from kidney tissues was associated with virus replication in the kidney tissues was provided by high virus titers, ranging up to 10(9.5) mean embryo infectious dose per gram of kidney tissue, and by identification of intranuclear and intracytoplasmic type A influenza nucleoprotein in kidney cells using immunohistochemistry. Virus-recovery and virus titer results from three chickens that died on days 4 and 5 postinoculation paralleled the results from the clinically normal chickens. These findings indicate that A/chicken/Alabama/75 has nephrotropic properties similar to nephrotropic properties previously reported for waterfowl-origin type A influenza viruses and provide evidence that kidney lesions could be manifestations of systemic influenza infections in commercial laying chickens.

Biological properties of waterfowl-origin type A influenza viruses in chickens.

The replicative abilities and tissue tropism properties of 13 non-pathogenic or low-pathogenic waterfowl-origin type A influenza isolates recovered in 1986 were examined in chickens. Following intravenous challenge, reisolation of challenge virus was attempted from swabs of the luminal surfaces of the cloaca, jejunum, ileum, bursa, trachea, and air sacs and from swabs of bone marrow and liver tissues. Virus-isolation attempts were also accomplished on brain, thymus, spleen, pancreas, gonad, kidney, blood, and lung tissues. The overall frequency of influenza virus recovery for each experiment ranged from 3.1% to 49.3%. For all experiments combined, 58.3% of the kidney tissues and 62.9% of the cloacal swab samples collected on days 1 to 10 postinoculation were positive for challenge virus recovery. Virus titers up to 10(8.7) mean embryo infective dose per gram of kidney tissue were demonstrated in clinically normal chickens. Distinct biological variations and nephrotropism appear to exist among the corporate properties of virus populations making up each of the 13 waterfowl-origin type A influenza isolates.

Virus replication in the digestive tract of ducks exposed by aerosol to type-A influenza.

Enteric infection and cloacal shedding of influenza virus was demonstrated in ducks exposed experimentally to an aerosol of an avirulent type-A influenza virus. The fluorescent-antibldy technique was used to identify sites of virus replication in the epithelial cells of the digestive tract and the bursa.

Tissue tropism and replicative properties of waterfowl-origin influenza viruses in chickens.

Waterfowl-origin influenza (WFOI) viruses were evaluated for their tissue tropism and replicative properties in chickens. The 14 WFOI isolates used in this study represented 13 different hemagglutinin-neuraminidase combinations recovered during 1987 and 1988 and included isolates possessing the H5 and H7 hemagglutinin subtypes and one isolate possessing the H5N2 combination. Following intravenous challenge, the frequencies of virus recovery within individual experiments were generally higher for the lower digestive tract and kidney samples. Virus titers ranged up to 10(8.5) mean embryo infective doses per gram of kidney tissue in clinically normal chickens. Differences in frequencies of virus recovery and virus titers in tissues indicated that some of these uniformly nonpathogenic and low-pathogenicity WFOI virus isolates replicated more extensively in chickens than did others. This enhanced ability to replicate in chickens should be further evaluated as a potential factor associated with the threat WFOI viruses present to poultry.

Detection of proteolytic bacteria in the upper respiratory tract flora of poultry.

Proteolytic bacteria were readily demonstrated among the upper respiratory tract flora of poultry in two chicken flocks and two turkey flocks. Between 20% and 50% of birds in each flock had highly proteolytic bacteria in the upper respiratory tract flora, and the amount of the bacterial flora determined to be highly proteolytic in any given bird ranged from none to a majority. Highly proteolytic bacteria recovered included five species of staphylococci, as well as two gram-negative bacterial species, Flavobacterium sp. and Vibrio alginolyticus. All species of staphylococci isolated have previously been recovered from animal sources, whereas Flavobacterium sp. and V. alginolyticus are usually reported to be associated with soil and surface-water origins. The protocols used to screen for proteolytic bacteria among the flora and to assess protease activity required minimal supplies and equipment.

Comparative pathology of intravenously inoculated wild duck- and turkey-origin type A influenza viruses in chickens.

Five-week-old specific-pathogen-free chickens were inoculated intravenously with one of 16 low-pathogenicity type A influenza virus isolates; 14 were of wild duck origin, and two were of turkey origin. Tubulointerstitial nephritis was the most frequent specific histopathologic change. The frequency and severity of kidney lesions were independent of the virus hemagglutinin-neuraminidase subtype or titer of the challenge virus. Influenza nucleoprotein was most frequently demonstrated in the kidney and was consistently localized to necrotic proximal and/or distal renal tubule epithelium. Common nonspecific histopathologic changes were lymphoid hyperplasia of the spleen and cecal tonsils, as well as lymphocyte depletion in the cloacal bursa. Uncommon histopathologic changes, in decreasing order of frequency, were interstitial pneumonia, lymphoid follicular hyperplasia in the myocardium, and lymphocytic tracheitis. Histopathologic changes were rare or absent in the jejunum, duodenum, pancreas, and brain. The low-pathogenicity avian-origin type A influenza virus isolates were epitheliotropic in chickens, primarily nephrotropic. Such findings were dissimilar from findings with highly pathogenic avian-origin type A influenza virus isolates both in severity and in tissue distribution of histopathologic changes and influenza viral antigen.

Assessing pathogenicity potential of waterfowl-origin type A influenza viruses in chickens.

Intravenous pathogenicity index (IVPI) tests on 29 wild duck-origin type A influenza viruses, two turkey-origin type A influenza viruses, and one chicken-origin type A influenza virus resulted in indices ranging from 0.0 to 0.49. Most of the wild duck-origin viruses and the two turkey-origin viruses had indices of 0.0, indicating they are not pathogenic. Six of the duck-origin viruses had indices ranging from 0.25 to 0.49, and the IVPI for A/chicken/Alabama/75 (H4N8) was 0.49, indicating they had low pathogenic potential. An IVPI of 1.25 up to the maximum score of 3.0 is necessary for a type A influenza virus to be classified as highly pathogenic. Gross lesions observed in chickens dying following intravenous viral challenge included kidney swelling with more prominent lobular patterns, but visceral urate deposits were not present. The usefulness of the IVPI test in evaluating the pathogenicity potential of nonpathogenic and low-pathogenic strains of avian influenza virus may be limited.

Pathological studies of A/chicken/Alabama/7395/75 (H4N8) influenza virus in specific-pathogen-free laying hens.

Specific-pathogen-free laying hens were inoculated with avian influenza virus (AIV) A/chicken/Alabama/7395/75 (H4N8) either intratracheally (IT) or intravenously (IV). IT inoculation produced a localized infection of the upper and lower respiratory tracts with lesions of tracheitis, bronchitis, airsacculitis, and pneumonia around the secondary bronchi. IV inoculation produced a systemic infection with major lesions of nephritis, interstitial pneumonia, salpingitis, and splenic and hepatic necrosis. In IV-inoculated hens, AIV nucleo-protein was demonstrated within renal tubule epithelium, in luminal surface and glandular oviduct epithelium, and in mononuclear cells within pulmonary blood capillaries. However, no virus was recovered from internal contents of eggs laid between days 1.5 and 5 postinfection. These data indicate that A/chicken/Alabama/7395/75 has tissue tropism and pathogenicity for the respiratory and urogenital systems of reproductively active laying hens. Site and severity of lesion development are determined by the localized or systemic nature of AIV infection.

Type A influenza virus surveillance in free-flying, nonmigratory ducks residing on the eastern shore of Maryland.

Virus surveillance in free-flying, nonmigratory ducks living on the eastern shore of Maryland indicated that influenza A viruses were introduced into the area or that the prevalence of endemic infections increased between July 15 and August 27, 1998. Cloacal swabs collected between May 28 and July 15, 1998, were negative for influenza A virus recovery (0/233), whereas 13.9% (29/209) of swabs collected between August 27 and September 2, 1998, were positive for influenza A virus recovery. Five hemagglutinin subtypes (H2, H3, H6, H9, and H12), six neuraminidase subtypes (N1, N2, N4, N5, N6, and N8), and nine HA-NA combinations were identified among 29 influenza A isolates. Interestingly, 18 of the 29 isolates initially appeared to contain two or more HA and/or NA subtypes. The free-flying, nonmigratory ducks served as excellent sentinels for the early detection of type A influenza viruses in the southern half of the Atlantic Migratory Waterfowl Flyway during the earliest phase of the yearly southern migration.

Type A influenza viruses in waterfowl in Ohio and implications for domestic turkeys.

Because ducks are considered an important reservoir for type A influenza virus, and type A influenza viruses had not been recovered from ducks in Ohio, a 3-year virus surveillance study was conducted in Ohio waterfowl and waterfowl passing through Ohio to determine if domestic turkeys were at risk of exposure to avian influenza (AI) viruses from the waterfowl reservoir. The prevalence of AI infections in ducks during the fall migration averaged about 5.9%. The 55 waterfowl-origin type A influenza viruses recovered from ducks during fall 1986, 1987, and 1988 represented 23 different hemagglutinin-neuraminidase sub-type combinations of type A influenza viruses. Virus recovery frequencies ranged from 3.6% to 7.8% between years, from 2.0% to 8.2% between study sites, from 0.0% to 16.7% for sampling days, and from 0.0% to 14.3% among species of ducks sampled.

Acute renal failure as the cause of death in chickens following intravenous inoculation with avian influenza virus A/chicken/Alabama/7395/75 (H4N8).

One-day-old and 5-week-old commercial leghorn, specific-pathogen-free leghorn, and broiler chickens were inoculated intravenously with either avian influenza virus isolate A/chicken/Alabama/7395/75 (H4N8) (Ck/AL) or sterile diluent. Ck/AL infection resulted in a 44% mortality rate, reduced weight gains, and necrosis of proximal renal tubules and/or tubulointerstitial nephritis. The renal tubule necrosis was more severe and widespread in chickens that died than in chickens that were euthanatized. Hyperuricemia, hypercalcemia, and hyperphosphatemia were present in 5-week-old chickens at day 5 postinfection. Influenza virus isolate Ck/AL was nephropathogenic, and death was associated with acute severe renal damage and failure. Some data suggested that the pathogenicity of Ck/AL may be more severe in leghorns than broilers.

Assessment of the ability of ratite-origin influenza viruses to infect and produce disease in rheas and chickens.

Pathobiologic characteristics were determined for three mildly pathogenic (MP) ratite-origin avian influenza viruses (AIVs). Ratite-origin AIVs produced respiratory disease in rheas, and virus was reisolated from oropharyngeal and cloacal swabs on days 2-6 postinoculation. Inoculation of two ratite-origin AIVs in the upper respiratory tract of chickens resulted in viral infections, but the mean chicken infectious dose (CID50) for A/emu/Texas/39924/93 (H5N2) (Emu/Texas) virus was 500-fold lower than the CID50 for the A/rhea/North Carolina/39482/93 (H7N1) virus. In ovo and in vivo passage of the MP parent Emu/Texas isolate resulted in emergence of a highly pathogenic (HP) variant that had high plaquing efficiency in chicken embryo fibroblast cultures and was highly lethal in chicken pathotyping tests. This variant virus produced gross lesions in chickens similar to those reported for other HP AIVs. These findings demonstrated that ratite-origin AIVs can produce significant clinical disease in rheas and have a realistic potential for interspecies transmission to domestic poultry. Furthermore, HP variants can emerge from MP H5 ratite-origin AIVs if introduced and allowed to circulate in chicken populations.

Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains.

Wild waterfowl that were captured between 1915 and 1919 and preserved in 70% ethyl alcohol were tested for influenza A virus RNA. Most of the HA1 domain of the hemagglutinin (HA) gene segment was sequenced from one bird, captured in 1917, that was infected with a virus of the same HA subtype as the 1918 human pandemic virus. The 1917 HA sequence is closely related to modern avian HA sequences, suggesting little drift in avian sequences in 80 years and that the 1918 pandemic virus probably did not acquire its hemagglutinin directly from a bird. A 151-bp fragment of the nucleoprotein gene segment was sequenced from two pre-1918 birds and compared to avian and mammalian influenza strains. The 1917 avian NP sequences are also closely related to modern avian sequences and distinct from the mammalian clade in which the 1918 NP sequence is found.