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.

Using RRT-PCR analysis and virus isolation to determine the prevalence of avian influenza virus infections in ducks at Minto Flats State Game Refuge, Alaska, during August 2005.

This study describes surveillance for avian influenza viruses (AIV) in the Minto Flats State Game Refuge, high-density waterfowl breeding grounds in Alaska. Five hundred paired cloacal samples from dabbling ducks (Northern Pintail, Mallard, Green Wing Teal, and Widgeon) were placed into ethanol and viral transport medium (VTM). Additional ethanol-preserved samples were taken. Of the ethanol-preserved samples, 25.6% were AIV RNA-positive by real-time RT-PCR. The hemagglutinin (HA) and neuraminidase (NA) subtypes were determined for 38 of the first-passage isolates, and four first-passage isolates could not be definitively subtyped. Five influenza A virus HA-NA combinations were identified: H3N6, H3N8, H4N6, H8N4, and H12N5. Differences in the prevalence of AIV infections by sex and by age classes of Northern Pintail and Mallard ducks were detected, but the significance of these differences is undefined. In the 500 paired samples, molecular screening detected positive birds at a higher rate than viral isolation (chi(2) = 8.35, p = 0.0035, df = 1); however, 20 AIV isolates were recovered from PCR-negative ducks. Further research is warranted to compare the two screening protocols' potential for estimating true prevalence in wild birds. Our success during 2005 indicates Minto Flats will be a valuable study site for a longitudinal research project designed to gain further insight into the natural history, evolution, and ecology of AIV in wild birds.

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.

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.

Synergistic effects of concurrent challenge with bovine respiratory syncytial virus and 3-methylindole in calves.

OBJECTIVE: To evaluate the potential synergy between bovine respiratory syncytial virus (BRSV) and 3-methylindole (3MI) in inducing respiratory disease in cattle. ANIMALS: 20 mixed-breed beef calves. PROCEDURE: A 2 X 2 factorial design was used, with random assignment to the following 4 treatment groups: unchallenged control, BRSV challenge exposure (5 X 10(4) TCID50 by aerosolization and 5.5 X 10(5) TCID50 by intratracheal inoculation), 3MI challenge exposure (0.1 g/kg of body weight, PO), and combined BRSV-3MI challenge exposure. Clinical examinations were performed daily. Serum 3MI concentrations, WBC counts, PCV, total plasma protein, and fibrinogen concentrations were determined throughout the experiment. Surviving cattle were euthanatized 7 days after challenge exposure. Pulmonary lesions were evaluated at postmortem examination. RESULTS: Clinical respiratory disease was more acute and severe in cattle in the BRSV-3MI challenge-exposure group than in cattle in the other groups. All 5 cattle in this group and 3 of 5 cattle treated with 3MI alone died or were euthanatized prior to termination of the experiment. Mean lung displacement volume was greatest in the BRSV-3MI challenge-exposure group. Gross and histologic examination revealed that pulmonary lesions were also most severe for cattle in this group. CONCLUSIONS AND CLINICAL RELEVANCE: Feedlot cattle are commonly infected with BRSV, and 3MI is produced by microflora in the rumen of all cattle. Our results suggest that there is a synergy between BRSV and 3MI. Thus, controlling combined exposure may be important in preventing respiratory disease in feedlot cattle.

Effect of moderate exercise on the severity of clinical signs associated with influenza virus infection in horses.

The purpose of this experiment was to determine if exercising horses, infected with influenza virus, exacerbates the severity of clinical disease. Eight horses were trained on a treadmill for 42 days and then challenged with aerosolised influenza A/equine/Kentucky/91 (H3N8). Following challenge, 4 horses (exercise group) continued training for 28 days, while the other 4 horses (nonexercise group) were confined to their stalls. All horses developed clinical signs within 36 h of challenge (fever, coughing, and mucopurulent nasal discharge) and clinical scores were greater in the exercise group. Horses developed fever from Days 1-11 post challenge (PC) and were tachypnoeic and tachycardic from Days 1-14 PC. All horses lost weight within 4 days PC, but the exercise group lost an average of 20 kg more than the nonexercise group. All horses developed pneumonia, and ultrasonography revealed pulmonary consolidation and oedema by Day 7 PC that was resolving by Day 14 PC. Endoscopy and transtracheal aspirates showed airway inflammation for up to 21 days PC. While the exercise group exhibited more severe signs of clinical disease, resolution occurred for both groups on approximately Day 14 PC, and no adverse effects were noted at the end of the study. However, the potential long term effects of exercising horses acutely infected with influenza virus are unknown. Until further research is conducted in this area, it appears prudent not to exercise affected horses.

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.

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.

Evaluation of an agar gel immunodiffusion test kit for detection of antibodies to Mycobacterium paratuberculosis in sheep.

OBJECTIVE: To determine whether a commercially available agar gel immunodiffusion test approved for detecting antibodies to Mycobacterium paratuberculosis in cattle could be used for sheep. DESIGN: Experimental trial. SAMPLE POPULATION: Serum samples from 27 sheep confirmed to have paratuberculosis by means of acid-fast staining of smears of ileal mucosa, histologic examination of tissues, or bacteriologic culture; 7 sheep with clinical signs of paratuberculosis; and 55 sheep from 5 uninfected flocks. PROCEDURE: Serum samples were tested concurrently with the commercially available test and with a previously validated agar gel immunodiffusion test. Multiple samples collected from 13 infected sheep over a period of 6 years were also tested so that each test's ability to detect onset of seropositivity could be compared. RESULTS: For both tests, results for samples from all 55 uninfected sheep were negative, results for samples from 32 of the 34 sheep with paratuberculosis were positive, and results for the remaining 2 sheep with paratuberculosis were negative. Results of both tests were in agreement for 50 of 54 samples obtained from 13 infected sheep over time. The 4 samples for which results of the 2 tests disagreed were the fourth, eighth, and ninth of 10 samples from 1 sheep and the first of 6 samples from a second sheep. For all 4 samples, the commercially available assay yielded a weak-positive result, but the previously described test yielded a negative result. CLINICAL IMPLICATIONS: The commercially available agar gel immunodiffusion test approved for use in cattle may be useful in the differential diagnosis of paratuberculosis in sheep.

Effect of vitamin D on testicular CaBP28K expression and serum testosterone in chickens.

Vitamin D is known to reverse infertility in male and female rats. This study was an investigation of the effects of vitamin D deficiency on calbindin-D28K (CaBP28K) and testosterone levels in male chickens. Chickens were raised from 1 day of age to 8 wk of age on a normal or a vitamin D-deficient diet. A radioreceptor assay showed that serum vitamin D levels were significantly higher in chickens fed a normal diet than in those fed a vitamin D-deficient diet. The morphology of the seminiferous tubules was not different between the vitamin D-replete and vitamin D-deficient chickens. Immunohistochemical studies revealed that CaBP28K was present in spermatogonia and spermatocytes of the seminiferous tubules. A few interstitial Leydig cells were positive for CaBP28K. RIA was used to quantify the amount of CaBP28K in the testes, which was threefold higher in chickens raised on a normal diet than in chickens raised on a vitamin D-deficient diet. Testosterone concentration in serum, determined by RIA, was not different between the two groups. Neither serum calcium nor phosphorus levels were different between the two groups. This investigation represents the first demonstration of the effect of vitamin D deficiency on CaBP28K expression in chicken testes. The results indicate that the decrease in testicular CaBP28K concentration was attributable to vitamin D deficiency despite normal serum testosterone and calcium levels in 8-wk-old chickens.

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.

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.

Comparative pathology of a chicken-origin and two duck-origin influenza virus isolates in chickens: the effect of route of inoculation.

Forty-nine 5-week-old chickens were inoculated by the intravenous (i.v.), intratracheal (IT), or intranasal (IN) routes with either a chicken-origin or one of two duck-origin type A influenza virus isolates. Twelve control chickens were inoculated with sterile chorioallantoic fluid. For all viruses, i.v. inoculation produced predominate lesions of renal tubule necrosis (nephrosis) and nephritis, and influenza virus nucleoprotein was localized in nuclei and cytoplasm of necrotic renal tubule epithelium. Chickens inoculated by the IT route, and to a lesser extent the IN route, had mild to severe tracheitis, bronchitis, and ventromedial pneumonia associated with secondary bronchi but lacked renal tubule necrosis and nephritis. These data indicate low-virulence avian-origin influenza viruses were nephrotropic during simulated systemic infection (i.v. inoculation) and pneumotropic during simulated local infection (IT and IN inoculation). Gross and histologic kidney lesions produced by i.v. inoculation of the chicken-origin influenza virus were similar to changes reported in outbreaks of low-virulence influenza virus in laying chickens.

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.

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.

Immunocytochemical localization of type A influenza virus nucleoprotein in chicken kidney, using freeze substitution technique for tissue fixation.

Kidney tissues were removed from euthanatized mature White Leghorn chickens 4 days after IV inoculation with type A influenza virus. The kidney tissues were then fixed at -70 C, using a freeze substitution technique. Type A influenza virus nucleoprotein was readily detected in the nuclei and cytoplasm of the proximal and distal tubular epithelial cells by immunocytochemistry, and the sharpness of the immunomarker in the cells indicated minimal antigen migration during fixation and tissue section preparation. This tissue fixation technique also resulted in good preservation of cellular morphology. The freeze substitution technique of tissue fixation is an excellent alternative to cryostat-cut acetone-fixed tissue sections or conventional chemical fixation of paraffin-embedded tissues for in situ immunocytochemical localization of type A influenza virus nucleoprotein antigen.

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.