Comparison of capsid gene sequences of turkey astrovirus-2 from poult-enteritis-syndrome-affected and apparently healthy turkeys.

This study was conducted to determine genetic variations in the capsid gene of turkey astrovirus-2 (TAstV-2) detected in apparently healthy and poult enteritis syndrome (PES)-affected turkeys. Capsid genes of astroviruses obtained from 30 PES-affected and 45 apparently healthy turkey flocks had sequence homologies of 73.4-100% and 72.4-100% at the nucleotide levels, respectively. The analysis of deduced amino acid sequences revealed one amino acid deletion at position 552 in 28 (93.3%) of 30 PES-affected cases. However, there were two deletions (at positions 551 and 552) in 31 (68.9%) of 45 TAstV-2 from apparently healthy flocks. The TAstV-2 (6.7%) from two PES-affected cases had two amino acid insertions each between positions 552 and 553, while TAstV-2 from 14 (31.1%) of 45 healthy flocks had two insertions at the same position. Phylogenetic analysis based on nucleotide sequences revealed that the astroviruses in this study were closely related to most of the previously published TAstV-2 isolates. The sequence homology of TAstV-2 in this study ranged from 70.4% to 99.4% at the nucleotide level with those of previously published TAstV-2 isolates. The variations at the amino acid level in the capsid gene suggest the possibility of the existence of different serotypes of turkey astrovirus. The close relationship of turkey astroviruses from apparently healthy flocks to those from PES-affected cases in capsid gene phylogeny necessitates further studies to compare complete capsid gene sequences from both types of flocks from different geographic areas for better understanding of TAstV circulating in turkeys.

Detection and molecular characterization of enteric viruses from poult enteritis syndrome in turkeys.

This study was conducted to detect and characterize enteric viruses [rotavirus, turkey astrovirus-2 (TAstV-2), reovirus, and turkey coronavirus] from cases of poult enteritis syndrome (PES) in Minnesota turkeys. Of the intestinal contents collected from 43 PES cases, 25 were positive for rotavirus and 13 for small round viruses by electron microscopy (EM). Of the enteric virus-positive cases by EM (n=27), 16 cases had rotavirus or small round viruses alone and the remaining 11 cases had both viruses. None of the cases were positive for reovirus or coronavirus by EM. However, with reverse transcription-PCR (RT-PCR), 40 cases (93%) were positive for rotavirus, 36 (84%) for TAstV-2, and 17 (40%) for reovirus. None of the cases were positive for turkey coronavirus by RT-PCR. The viruses from all cases were detected either alone or in combination of 2 or 3 by RT-PCR. Thus, 8 (19%) cases were positive for a single virus, whereas a combination of viruses was detected in the remaining 35 (81%) cases. The rota-TAstV-2 combination was the most predominant (n=18 cases). Fifteen cases were positive for all 3 viruses. The rotaviruses had sequence homology of 89.8 to 100% with previously published sequences of turkey rotaviruses at the nucleotide level. The TAstV-2 had sequence homology of 84.6 to 98.7% with previously published TAstV-2, whereas reoviruses had sequence homology of 91.6 to 99.3% with previously published sequences of turkey reoviruses. Phylogenetic analysis revealed that rota- and reoviruses clustered in a single group, whereas TAstV-2 clustered in 2 different groups. In conclusion, a larger number of PES cases was positive for rotavirus, TAstV-2, and reovirus by RT-PCR than with EM. The presence of more than one virus and changes at the genetic level in a virus may affect the severity of PES in turkey flocks.

Experimental reproduction of poult enteritis syndrome: clinical findings, growth response, and microbiology.

Poult enteritis syndrome (PES) is an infectious disease of turkey poults characterized by diarrhea, dullness, and depression. Five experiments were conducted to reproduce the disease in turkey poults using intestinal contents of PES-affected birds. In all experiments, poults at 14 d of age were divided into 4 groups and were orally given 2 mL of unfiltered supernatant, filtered supernatant, sediment dissolved in PBS, or PBS alone. Inocula in experiments 1, 3, and 5 consisted of intestinal contents from PES-affected birds of less than 2 wk of age, whereas those in experiments 2 and 4 consisted of intestinal contents from PES-affected birds of 4 to 6 wk of age. Poults in all groups were observed daily for clinical signs. The BW and microbiological criteria in experiments 1, 3, and 5 were evaluated at 5, 10, and 15 d postinoculation, whereas in experiments 2 and 4, these observations were made at 10 and 20 d postinoculation. Rotavirus, astrovirus, and Salmonella were present in all 5 inocula. Diarrhea and depression were the major signs in poults given PES material. Significant retardation of growth was observed in poults given any of the 3 PES materials, but this effect was more pronounced in poults given the sediment inoculum. Rotavirus, astrovirus, and Salmonella were detected in poults given PES material. In some cases, enterovirus was also detected. No major difference was noticed in experimental reproduction of PES when intestinal contents from different age birds were used as the inoculum.

A review of the 1996-98 nonpathogenic H7N2 avian influenza outbreak in Pennsylvania.

The nonpathogenic avian influenza (AI) outbreak in Pennsylvania began in December 1996 when there was a trace back from a New York live bird market to a dealer's flock. A total of 18 commercial layer flocks, two commercial layer pullet flocks, and a commercial meat turkey flock were diagnosed with nonpathogenic AI (H7N2) viral infection with an economic loss estimated at between 3 and 4 million dollars. Clinical histories of flocks infected with the disease included respiratory disease, elevated morbidity and mortality throughout the house, egg production drops, depression, and lethargy. A unique gross lesion in the commercial layers was a severe, transmural oviduct edema with white to gray flocculent purulent material in the lumen. Layer flocks on two separate premises were quarantined but permitted to remain in the facilities until cessation of virus shed was determined through virus isolation. Several months later, clinical AI appeared again in these flocks. It is not known whether the recurrence of disease in these cases is due to persistence of the organism in the birds or the environment. In addition to serologic testing and virologic testing by chicken embryo inoculation, an antigen capture enzyme immunoassay was evaluated as a diagnostic tool for AI. Research projects related to disinfection, burial pits, and geographical system technology were developed because of questions raised concerning transmission, diagnosis, and control of nonpathogenic AI (H7N2).

Epidemiology, production losses, and control measures associated with an outbreak of avian influenza subtype H7N2 in Pennsylvania (1996-98).

An outbreak of H7N2 low-pathogenicity (LP) avian influenza (AI) occurred in a two-county area in Pennsylvania from December of 1996 through April of 1998. The outbreak resulted in infection of 2,623,116 commercial birds on 25 premises encompassing 47 flocks. Twenty-one (one premise with infection twice) of the twenty-five infected premises housed egg-laying chickens and one premise each had turkeys, layer pullets, quail, and a mixed backyard dealer flock. Despite dose proximity of infected flocks to commercial broiler flocks, no infected broilers were identified. Experimentally, when market age broilers were placed on an influenza-infected premise they seroconverted and developed oviduct lesions. The outbreak was believed to have originated from two separate introductions into commercial layer flocks from premises and by individuals dealing in sales of live fowl in the metropolitan New York and New Jersey live-bird markets. Source flocks for these markets are primarily in the northeast and mid-Atlantic areas, including Pennsylvania. Mixed fowl sold include ducks, geese, guinea hens, quail, chukar partridges, and a variety of chickens grown on perhaps hundreds of small farms. Infections with the H7N2 AI virus were associated with variable morbidity and temporary decreases in egg production ranging from 1.6% to 29.1% in commercial egg-laying chickens. Egg production losses averaged 4.0 weeks duration. Mortality ranged from 1.5 to 18.3 times normal (mean of 4.3 times normal). Duration of mortality ranged from 2 to 13 weeks (average of 3.9 weeks) in flocks not depopulated. Lesions observed were primarily oviducts filled with a mucous and white gelatinous exudates and atypical egg yolk peritonitis. Quarantine of premises and complete depopulation were the early measures employed in control of this outbreak. Epidemiological studies suggested that depopulation furthered the spread of influenza to nearby flocks. Thereafter, later control measures included quarantine, strict biosecurity, and controlled marketing of products.

Nephropathogenic infectious bronchitis in Pennsylvania chickens 1997-2000.

Nephropathogenic infectious bronchitis (NIB) was diagnosed in 28 infectious bronchitis virus (IBV)-vaccinated commercial chicken flocks in Pennsylvania from December 1997 to July 2000. Early dinical signs were increased flock mortality and urinary water loss (polyuria and pollakiuria) leading to wet litter. Daily mortality ranged from 0.01% in layers to 2.45% in broilers, with total broiler mortality as high as 23%. Severe renal swelling and accumulation of urates in the tubules were commonly seen. Visceral gout and urolithiasis were less frequently observed. Histopathologic changes included characteristic tubular epithelial degeneration and sloughing with lymphoplasmacytic interstitial nephritis. Minimal respiratory disease signs were noted in broilers. Egg production and shell quality declined in layers. Confirmatory diagnosis of NIB was made by IBV antigen-specific immunohistochemical staining of the renal tubular epithelium and virus isolation. Sequencing of the S1 subunit gene of 21 IBV isolates showed the NIB outbreak to be associated with two unique genotypes, PA/Wolgemuth/98 and PA/171/99. The cases from which the genotypes were isolated were clinically indistinguishable. The NIB viruses were unrelated to previously recognized endemic strains in Pennsylvania and were also dissimilar to each other. Genotype PA/Wolgemuth/98 was isolated almost exclusively during the first 14 mo of the outbreak, whereas PA/171/99 was recovered during the final 18 mo. The reason for the apparent replacement of PA/Wolgemuth/98 by PA/171/99 is not known.

Protection of chickens after live and inactivated virus vaccination against challenge with nephropathogenic infectious bronchitis virus PA/Wolgemuth/98.

Protection provided by live and inactivated virus vaccination against challenge with the virulent nephropathogenic infectious bronchitis virus (NIBV) strain PA/Wolgemuth/98 was assessed. Vaccinations with combinations of live attenuated strains Massachusetts (Mass) + Connecticut (Conn) or Mass + Arkansas (Ark) were given by eyedrop to 2-wk-old specific-pathogen-free leghorn chickens. After live infectious bronchitis virus (IBV) vaccination, some chickens at 6 wk of age received an injection of either an oil emulsion vaccine containing inactivated IBV strains Mass + Ark or an autogenous vaccine prepared from NIBV PA/Wolgemuth/98. Challenge with PA/Wolgemuth/98 was given via eyedrop at 10 wk of age. Serum IBV enzyme-linked immunosorbent assay antibody geometric mean titers (GMTs) after vaccination with the combinations of live attenuated strains were low, ranging from 184 to 1,354, prior to NIBV challenge at 10 wk of age. Both inactivated vaccines induced an anamnestic response of similar magnitudes with serum GMTs of 6,232-12,241. Assessment of protection following NIBV challenge was based on several criteria virus reisolation from trachea and kidney and renal microscopic pathology and IBV-specific antigen immunohistochemistry (IHC). Live attenuated virus vaccination alone with combinations of strains Mass + Conn or Mass + Ark did not protect the respiratory tract and kidney of chickens after PA/Wolgemuth/98 challenge. Chickens given a live combination vaccination of Mass + Conn and boosted with an inactivated Mass + Ark vaccine were also susceptible to NIBV challenge on the basis of virus isolation from trachea and kidney butshowed protection on the basis of renal microscopic pathology and IHC. Live IBV-primed chickens vaccinated with an autogenous inactivated PA/Wolgemuth/98 vaccine had the highest protection against homologous virulent NIBV challenge on the basis of virus isolation.

Eustrongylidiasis in eastern great blue herons (Ardea herodias).

The nematode Eustrongylides ignotus was found in peritoneal lesions of several great blue herons (Ardea herodias) submitted for necropsy from a wildlife rehabilitation center in northern Delaware. Prior to death, signs of disease included ataxia, emaciation, weakness, and anemia. Blood collection was not uniformly performed, but in cases where it was performed, affected birds demonstrated abnormal clinical hematology. Postmortem findings included numerous lesions associated with verminous peritonitis. Significant histologic granulomatous response to the presence of these organisms was noted, particularly in the proventricular specimens. Other organs involved included intestine, spleen, pancreas, and liver.

Evaluation of disinfectants with the addition of antifreezing compounds against nonpathogenic H7N2 avian influenza virus.

In the winter of 1997 and 1998, in the midst of the H7N2 avian influenza outbreak in Pennsylvania, producers added antifreeze or windshield washer fluid to disinfectant solutions in wash stations to prevent freezing. The purpose of this study was to determine if the addition of these products to the disinfectant solutions would have deleterious effects. Four disinfectants (two phenols, one quarternary ammonium, and one combination product: quarternary ammonium and formaldehyde) and one sodium hypochlorite detergent product currently used in the poultry industry were studied. Each product was diluted according to the manufacturer's recommendation in sterile distilled water and compared with dilutions of the disinfectants with the addition of antifreeze products (ethylene glycol or propylene glycol) or windshield washer fluid for their effectiveness in killing nonpathogenic H7N2 avian influenza virus. All products diluted according to the manufacturer's recommendation killed the nonpathogenic H7N2 avian influenza virus in this test system. The phenol products and the quaternary ammonium product were still efficacious with the addition of the antifreeze containing ethylene glycol. Both the combination product and the sodium hypochlorite detergent had decreased efficacy when the ethylene glycol product was added. When the propylene glycol product was added, the efficacy of all disinfectants remained unaffected, whereas the efficacy of the sodium hypochlorite detergent decreased. With the addition of the windshield washer fluid (methyl alcohol), all products remained efficacious except for the combination product.

Economic analysis of an outbreak of avian influenza, 1997-1998.

OBJECTIVE: To determine economic losses associated with an outbreak of avian influenza in flocks in Pennsylvania during 1997 and 1998. SAMPLE POPULATION: 5 premises containing avian influenza-infected layer, pullet, and turkey flocks. PROCEDURE: Losses incurred before depopulation, those incurred at the time of depopulation, and those that were attributable to depopulation (unrealized loss of income) were evaluated. Results were extrapolated to provide values for all infected flocks. RESULTS: Extrapolating the costs determined on the basis of age and number of birds from the 5 sample flocks to all other flocks infected with nonpathogenic avian influenza H7N2 yielded an estimated total cost to the Pennsylvania poultry industry of $3.5 million. CLINICAL IMPLICATIONS: The H7N2 virus is not highly pathogenic. If the pathogenicity of the virus does not change, then the poultry industry and state and federal governments will not have severe economic losses for the 1997-1998 outbreak similar to those for the 1983-1984 avian influenza outbreak in Pennsylvania. To decrease the potential for financial losses that could result from future outbreaks of avian influenza, it is essential that the commercial industry and livebird market system be separated via increased use of biosecurity measures.

Characteristics of H7N2 (nonpathogenic) avian influenza virus infections in commercial layers, in Pennsylvania, 1997-98.

Between January 1997 and March 1998, 11 cases of H7N2 avian influenza (nonpathogenic) were diagnosed at the Laboratory of Avian Medicine and Pathology, Kenneth Square, PA. These cases involved either commercial leghorn laying hens or leghorn pullets raised in Pennsylvania. Grossly and histologically, the most striking lesion associated with disease was salpingitis, usually with edema and occasionally with oviduct necrosis. Fluid, fibrinous, and egg yolk material in the peritoneum (egg yolk peritonitis) as well as pulmonary congestion and pulmonary edema were also frequently seen. Oviduct lesions have rarely been described in association with avian influenza infections in previous outbreaks. Mortality in affected houses was mild to moderate (less than 4% total mortality during the outbreak), with concurrent mild to moderate egg production declines (2%-4% at the time of disease onset).

Comparison of an antigen-capture enzyme immunoassay with virus isolation for avian influenza from field samples.

The standard tests used to detect avian influenza (AI) viral infection include virus isolation from tissues of the infected birds and the detection of AI antibody in blood or egg yolk. A new application of an existing human test to rapidly detect the presence of any influenza A virus is now possible. A commercially available antigen-capture enzyme immunoassay (AC-EIA), developed for the detection of influenza A in humans was tested for relative sensitivity and specificity and for speed of use in diagnosing nonpathogenic H7N2 AI in naturally infected poultry. During the recent nonpathogenic H7N2 AI epornitic, the AC-EIA was used for rapid diagnosis and quarantine decisions. Between February and August 1997, 1524 samples from 295 commercial layer, pullet, and broiler flocks were submitted to the Laboratory of Avian Medicine and Pathology, New Bolton Center, for AI virus isolation and testing by AC-EIA. The relative specificity of the AC-EIA was 100% and the relative sensitivity was 79%. We believe that the AC-EIA will be a useful adjunct to standard AI diagnostic tests.