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.

Field observations with Salmonella enteritidis bacterins.

A study involving 11 commercial layer flocks was conducted to determine the efficacy of Salmonella enteritidis bacterins (autogenous or federally licensed). The criterion for evaluation of vaccine efficacy was the presence or absence of S. enteritidis in the environment, the organs of the bird (including ovary and oviduct), and eggs. Environmental, rodent, and organ specimens from dead birds as well as eggs were cultured throughout the life of the flock. All layers were obtained from pullet sources that were negative for S. enteritidis, as determined by organ and environmental cultures. Despite the use of S. enteritidis vaccination, 63.6% of the houses had S. enteritidis-positive environmental cultures and 100% of the flocks had S. enteritidis organ-culture-positive birds. The range of positive cultures for S. enteritidis in the environment in vaccinated flocks was between 0 and 45.5%. Birds in vaccinated flocks were organ-culture positive for S. enteritidis between 10% and 40% of the time. The unvaccinated portion of flocks in the same house and the unvaccinated flock in a complex had similar results compared with the vaccinated portion of the flocks.

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.

Invasion of chicken reproductive tissues and forming eggs is not unique to Salmonella enteritidis.

Experiments were conducted in which Salmonella enteritidis Phage Type 8, Phage Type 2, and RDNC (reaction does not conform) or three isolates of Salmonella typhimurium of diverse origin were fed to adult laying hens to determine if S. enteritidis has a selective advantage over S. typhimurium, which is now rarely isolated from chicken eggs, in its capacity to invade reproductive tissues. The results revealed that S. enteritidis and S. typhimurium may be equal in their potential to colonize the tissues of the reproductive tract and eggs that are forming in the oviduct prior to oviposition. S. enteritidis, but not S. typhimurium, was isolated from egg contents after oviposition. The degree to which intestinal, hepatic, splenic, or reproductive tissues were colonized by either serotype was not seen to affect the rate of colonization of eggs forming in the oviduct or the contamination of eggs after oviposition. Virulence factors related to the difference in the association of S. enteritidis and S. typhimurium with egg-borne salmonellosis remain to be defined.

Evaluation of disinfectants against Salmonella enteritidis.

Five classes of disinfectants (phenol, quaternary ammonium, chlorine, glutaraldehyde, and a combination of quaternary ammonium and formaldehyde) were diluted in "field" water (well, stream, or pond water) and compared with dilutions of the disinfectants in laboratory-grade water for their efficacy against the AOAC (Association of Official Agricultural Chemists) test organism Salmonella cholerasuis (ATCC 10708), S. enteritidis isolated from the spleen of an infected laying hen, and an egg-invasive S. enteritidis isolate. In all cases when S. cholerasuis was used, there was a significant association between the use of well, pond, and stream water and the growth of the bacterium. If we exclude glutaraldehyde, there was also a significant association between the use of "field" water and the growth of both isolates of S. enteritidis. There was no significant association when glutaraldehyde was used. There was a significant association between the use of lab water and the growth of S. enteritidis. The results suggested that the inability to remove S. enteritidis from layer houses may in part be associated with the source of water. Variables in pH, hardness, conductivity, nitrate content, or bacterial contamination of the water did not appear to affect the ability of the disinfectant to kill S. enteritidis. If "field" water is used for disinfection against S. enteritidis, the use of quaternary ammonium, the combination (quaternary ammonium/formaldehyde), or phenol should be considered.

Salmonella enteritidis colonization of the reproductive tract and forming and freshly laid eggs of chickens.

Salmonella enteritidis colonizes the tissues of the chicken ovary and oviduct, presumably contaminating eggs and thereby contributing to human outbreaks of salmonellosis. In this study, commercial adult laying hens were given an oral inoculation of 10(8) S. enteritidis organisms. Tissues from various organs, the intestines, and the reproductive tract, including freshly laid eggs, were collected daily for up to 40 days postinoculation (p.i.). Within 2 days p.i. S. enteritidis was detected by culture in pools of the spleen, liver, heart, and gallbladder tissues, in intestinal tissues of all infected birds, and in various sections of the ovary and oviduct. Detection of organisms by immunohistochemical staining was rare for most tissues in spite of their culture-positive status, suggesting a low level of tissue colonization. However, S. enteritidis could be detected by immunohistochemical staining in oviduct tissues associated with four forming eggs, indicating the possibility of a heavier colonization in the egg during its development. In two subsequent experiments, forming eggs taken from the oviduct with their associated tissue, were found to be culture positive for S. enteritidis at a rate of 27.1 and 31.4%, while freshly laid eggs in these experiments were culture positive at the rate of 0 and 0.6%. These observations suggest that while forming eggs are significantly colonized in the reproductive tract, factors within the eggs may control the pathogen before the eggs are laid. The data show that prior to egg deposition, forming eggs are subject to descending infections from colonized ovarian tissue, ascending infections from colonized vaginal and cloacal tissues, and lateral infections from colonized upper oviduct tissues. The data are consistent with an ascending infection of freshly laid eggs from the cloaca, as the incidence of positive eggs in experiments 1 and 3 coincided with heavily contaminated cloacal tissues (50.7 and 80%, respectively), while no positive eggs were detected in experiment 2 when cloacal colonization was low (8.3%). The data do not support the possibility of egg invasion by bacterial translocation from the peritoneal cavity.

Comparison of environmental monitoring protocols for the detection of Salmonella in poultry houses.

Environmental monitoring has been used as a screening method to detect Salmonella enteritidis infection in laying hens. Several transport protocols (buffered peptone water, skim milk, asparagine, double distilled water, and no media), to be used for the detection of Salmonella in environmental samples from poultry houses, were compared for their ability to preserve the integrity of specimens. The isolation rates of Salmonella using the various transport protocols, including double-strength skim milk and no media (dry), were similar. Use of dry swabs is more convenient than a media transport system and should be adopted as an alternative method.

Duck viral enteritis in domestic muscovy ducks in Pennsylvania.

Duck viral enteritis (DVE) outbreaks occurred at two different locations in Pennsylvania in 1991 and 1992. In the first outbreak, four ducks died out of a group of 30 domestic ducks; in the second outbreak, 65 ducks died out of a group of 114 domestic ducks, and 15 domestic geese died as well. A variety of species of ducks were present on both premises, but only muscovy ducks (Cairina moschata) died from the disease. On necropsy, gross lesions included hepatomegaly with petechial hemorrhages, petechial hemorrhages in the abdominal fat, petechial hemorrhages on the epicardial surface of the heart, and multifocal to coalescing areas of fibrinonecrotic material over the mucosal surface of the trachea, esophagus, intestine, and cloaca. Histologically, the liver had random multifocal areas of necrosis and eosinophilic intranuclear inclusion bodies in hepatocytes. DVE virus was isolated and identified using muscovy duck embryo fibroblast inoculation and virus neutralization.

Monoclonal antibody-based detection system for Salmonella enteritidis.

Two monoclonal antibodies that react with Salmonella enteritidis in chicken tissue, eggs, and environmental samples were used to develop a rapid enzyme-linked immunosorbent assay (ELISA) screen and agglutination assay for the specific detection of S. enteritidis. S. enteritidis was detected in 100% of egg samples and 99.8% of various field and research samples by both ELISA and traditional microbiological isolation and identification techniques. Results of titer experiments indicate that as few as 10(4) organisms can be detected by ELISA.

Differences among restriction endonuclease DNA fingerprints of Pennsylvania field isolates, vaccine strains, and challenge strains of infectious laryngotracheitis virus.

Restriction endonuclease fingerprints of infectious laryngotracheitis virus (ILTV) DNA from 13 Pennsylvania field isolates, embryo-propagated and tissue-culture-propagated vaccine strains, and three reference strains were compared. These comparisons were made to evaluate the possible contribution of mutation of ILTV vaccine strains to recent outbreaks of infectious laryngotracheitis (ILT) in Pennsylvania. Six different restriction enzymes were used to generate the fingerprints. Differences in DNA banding patterns were revealed between the currently used ILTV vaccine strains and six of the 13 field isolates. Even greater DNA banding pattern differences were found between the older ILTV reference strains and the vaccine strains. The ILTV DNA fingerprints generated in the present study suggest that at least five different strains of ILTV have contributed to the outbreaks of ILT that have occurred since 1987 in Pennsylvania.

Characterization of avian retroviruses carrying activated transforming human c-src genes and of steps involved in expression of activated src-PKases in vitro.

We have isolated four activated transforming human c-src mutants derived spontaneously from viruses carrying the normal human c-src (SRC) genes in the form of Rous sarcoma virus. These mutants induced transformed cell morphology distinguishable from each other in vitro as well as tumors in chicks, whereas normal SRC-carrying viruses did not. Analyses of the transforming SRC proteins together with the normal SRC protein showed that levels of the carboxy-terminal Tyr-phosphorylation were negatively correlated with both transforming ability and protein kinase (PKase) activity as determined by in vitro autophosphorylation. It was observed that two cell lysis methods, that is, NP-40 and RIPA, yielded two different phosphorylated forms of transforming SRC proteins: one possessed low levels of phosphorylation at the autophosphorylation site and the other possessed high levels of phosphorylation at this site. Using the two types of transforming SRC preparations, we have studied in vitro SRC-PKase reactions in relation to in vivo and in vitro autophosphorylation and in vitro phosphorylation of an exogenous substrate. A possible functional relationship between autophosphorylation and SRC-PKase expression is discussed.

Laryngotracheitis in chickens: the length of the preinfectious and infectious periods.

Transmission studies to measure the length of the infectious period and the interval between virus inoculation and infectiousness were conducted using the standard National Veterinary Services Laboratory laryngotracheitis (LT) challenge virus (Log 10(6.7) EID50 per ml). Previously unexposed sentinel chickens were placed in contact with chickens inoculated intratracheally with LT virus. Transmission of virus to the sentinel birds was assessed by studying clinical signs and results of virus isolation and challenge. Chickens began to shed infective quantities of virus 2-4 days postinoculation and continued until day 6.

Laryngotracheitis in chickens: infection studies and the efficacy of a tissue-culture vaccine in chicks less than four weeks old.

Challenge studies using the standard National Veterinary Services Laboratory laryngotracheitis (LT) challenge virus (Log 10(6.7) EID50 per ml) were conducted to assess the presence of maternal protection in chicks of various ages (1, 7, 14, 21, and 28 days). Chicks from vaccinated and unvaccinated breeders were challenged by intratracheal inoculation. Chicks of all these ages irrespective of origin were susceptible to infection. Similarly derived chicks were vaccinated by eyedrop with a commercially available tissue-culture vaccine. Chicks vaccinated at 21 and 28 days were adequately protected from challenge (77-94% protection), whereas less than 60% of chicks vaccinated at 1, 7, or 14 days were protected.

A standardized enzyme-linked immunosorbent assay for infectious bronchitis virus: comparison with hemagglutination-inhibition and virus-neutralization assays for measuring protective antibody levels in chickens.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure antibodies to infectious bronchitis virus (IBV) in chickens. The results are reported in IBV standard ELISA values calculated by comparing antibody levels in test sera with antibody levels in a series of standard reference sera. The IBV standard ELISA values were good indicators of responses to vaccination and the immune status of experimentally challenged birds. Although the assay was not serotype-specific, the sensitivity makes it ideally suited for determining the immune status of poultry flocks. The assay results compared favorably with other laboratory results, including virus-neutralization titers, hemagglutination-inhibition levels in sera, virus isolation from vaccinated/challenged birds, and the tracheal ring test results.