Comparative susceptibility of chickens and turkeys to avian influenza A H7N2 virus infection and protective efficacy of a commercial avian influenza H7N2 virus vaccine

During the spring of 2002, a low pathogenic avian influenza (LPAI) A (H7N2) virus caused a major outbreak among commercial poultry in Virginia and adjacent states. The virus primarily affected turkey flocks, causing respiratory distress and decreased egg production. Experimentally, turkeys were more susceptible than chickens to H7N2 virus infection, with 50% bird infectious dose titers equal to 10(0.8) and 10(2.8-3.2), respectively. Comparison of virus shedding from the cloaca and oropharynx demonstrated that recent H7N2 virus isolates were readily isolated from the upper respiratory tract but rarely from the gastrointestinal tract. The outbreak of H7N2 virus raised concerns regarding the availability of vaccines that could be used for the prevention and control of this virus in poultry. We sought to determine if an existing commercial avian influenza (AI) vaccine prepared from a 1997 seed stock virus could provide protection against a 2002 LPAI H7N2 virus isolated from a turkey (A/turkey/Virginia/158512/02 [TV/02]) in Virginia that was from the same lineage as the vaccine virus. The inactivated AI vaccine, prepared from A/chicken/ Pennsylvania/21342/97 (CP/97) virus, significantly reduced viral shedding from vaccinated turkeys in comparison with sham controls but did not prevent infection. The protective effect of vaccination correlated with the level of virus-specific antibody because a second dose of vaccine increased antiviral serum immunoglobulin G and hemagglutination inhibition (HI) reactivity titers in two different turkey age groups. Serum from CP/97-vaccinated turkeys reacted equally well to CP/97 and TV/02 antigens by HI and enzyme-linked immunosorbent assay. These results demonstrate the potential benefit of using an antigenically related 1997 H7N2 virus as a vaccine candidate for protection in poultry against a H7N2 virus isolate from 2002.     

The pathogenicity of an avian influenza virus isolated in Victoria

An influenza virus (H7N7) isolated from an outbreak of disease in chickens in Victoria, was examined for its ability to cause disease in inoculated chickens, turkeys and ducks. The virus was highly pathogenic in chickens and turkeys but produced no clinical disease in ducks. Transmission of infection occurred from inoculated chickens to those in direct contact but other chickens separated by a distance of 3m directly downwind developed neither clinical disease nor antibody to the virus.     

Comparative pathobiology of low and high pathogenicity H7N3 Chilean avian influenza viruses in chickens

Chickens were intranasally inoculated with Chilean H7N3 avian influenza (AI) viruses of low pathogenicity (LP) (H7N3/LP), high pathogenicity (HP) (H7N3/HP), and a laboratory derivative (02-AI-15-#9) (H7N3/14D) from the LPAI virus to determine pathobiologic effects. All chickens inoculated with H7N3/HP AI virus became infected and abruptly died 2 or 3 days postinoculation, but a few showed moderate depression before death. The H7N3/HP AI virus produced focal hemorrhages of the comb, petechial hemorrhage at the esophageal-proventricular junction and proventricular mucosa, edema and congestion of the lung, petechiation of the spleen, and generalized decrease in body fat. Histologically, severe necrosis, hemorrhage, and inflammation were primarily identified in lungs and the lymphoid tissues. All tissues sampled from the H7N3/HP AI group were positive for the AI viral antigen, predominantly in endothelium of blood vessels throughout most tissues and less frequently in histiocytes and cellular debris of lymphoid tissues. Even less consistently, cardiac myocytes, hepatocytes, Kupffer cells, glandular epithelial cells, microglial cells, and neurons became infected. These studies suggest the Chilean H7N3/LP AI virus was poorly infectious for chickens and may have been recently introduced from a nongalliform host. By contrast, the H7N3/HP AI virus was highly infectious and lethal for chickens. The H7N3/HP AI virus had a strong tropism for the cardiovascular system, principally vascular endothelium, which is similar to the viral tropism demonstrated previously with other H5 and H7 HPAI viruses. Interestingly, the H7N3/LP AI virus on intravenous inoculation replicated in cardiac myocytes, a feature of HPAI and not LPAI viruses, which further supports the theory that the H7N3/LP AI virus was in transition from LP to HP.     

Experimental pathogenesis for chickens, turkeys, and pigeons of exotic Newcastle disease virus from an outbreak in California during 2002-2003

Exotic Newcastle disease virus (NDV) isolated from chickens during the 2002-2003 California outbreak (CA exotic Newcastle disease [END] virus) was inoculated into 4-week-old specific-pathogen-free (SPF) White Leghorn chickens, 3-week-old SPF Beltsville White turkeys, 6-week-old commercial Broad Breasted White turkeys, and 10- to 20-week-old racing pigeons, and the clinicopathologic features of disease were compared. Birds were monitored clinically and euthanized sequentially with collection of tissues. Tissues were examined by histopathology, by immunohistochemistry to detect viral nucleoprotein, and by in situ hybridization to detect viral mRNA. Clinically, infected chickens and SPF turkeys showed severe depression, and all died or were euthanized because of severe clinical signs by day 5 postinoculation. In these birds, histologic lesions were widespread and virus was detected in multiple organs. All infected commercial turkeys showed mild depression, and incoordination was observed in some birds. Histologic lesions were mild, and viral distribution was limited. In pigeons, only 1 bird showed overt clinical disease, and histologic lesions and viral distribution were present in limited organs. Consequently, susceptibility to highly virulent NDV was shown to vary among chickens, SPF turkeys, commercial turkeys, and pigeons. Additionally, we have evidence of CA END virus subclinical infections that suggest pigeons could be subclinical carriers of other virulent NDV.     

Measurement of systemic and local respiratory cell-mediated immunity after influenza infection in chickens

The detection of ChIFN production after ex vivo antigenic-stimulation of T-lymphocytes has been evaluated for the first time, as a tool to assess cell-mediated immunity (CMI) after avian influenza (AI) infection in 10-day-old SPF chickens. Preliminarily, recall antigens have been produced either by concentrating and inactivating the whole virus or by dissociating the viral proteins. Biologically and structurally intact forms of the viral proteins were isolated by non-ionic detergents while heating, chemical agents and ionic detergent used for virus inactivation altered the antigenic viral components. The n-octyl-B-D-gluco-pyranoside treatment at low temperature was very efficient to produce AI antigenic proteins used for evaluation of ChIFN production after ex vivo antigenic-stimulation of splenic and peripheral lymphocytes. In addition, protocols to isolate lymphocytes from the respiratory tract - the trachea and the lung - have been adapted for local CMI evaluation after similar ex vivo recall assay. Specific AI CMI in the spleen, the blood and the lung was detected for 5 weeks after low pathogenic AI (LPAI) infection in chickens, while further development is needed for tracheal CMI measurement.     

Highly pathogenic or low pathogenic avian influenza virus subtype H7N1 infection in chicken lungs: small differences in general acute responses

ABSTRACT: Avian influenza virus can be divided into two groups, highly pathogenic avian influenza virus (HPAI) and low pathogenic avian influenza virus (LPAI) based on their difference in virulence. To investigate if the difference in clinical outcome between LPAI and HPAI in chickens is due to immunological host responses in the lung within the first 24 hours post infection (hpi), chickens were infected with LPAI or HPAI of subtype H7N1. Virus was found in the caudal and cranial part of the lung. With LPAI, virus was localised around the intrapulmonary bronchus and secondary bronchi. In sharp contrast, HPAI was detected throughout the whole lung. However, based on viral RNA levels, no quantitative difference was observed between LPAI and HPAI infected birds. In infected areas of the lungs, an influx of CD8α+ cells as well as KUL01+ macrophages and dendritic cells (DC) occurred as fast as 8 hpi in both infected groups. No major difference between LPAI and HPAI infected birds in the induction of cytokines and interferons at mRNA level in lung tissue was found.In conclusion, the differences in lethality for chickens infected with LPAI or HPAI could be ascribed to difference in location of the virus. However similar amounts of viral RNA, similar cytokine mRNA levels, and similar influxes of CD8α+ and KUL01+ macrophages and DC were found between HPAI and LPAI in the lungs. A cytokine storm at mRNA level as described for mammals was not observed in the lungs of HPAI infected birds within 24 hpi.     

Investigation of H7N2 avian influenza outbreaks in two broiler breeder flocks in Pennsylvania, 2001-02

An avian influenza (AI) outbreak occurred in meat-type chickens in central Pennsylvania from December 2001 to January 2002. Two broiler breeder flocks were initially infected almost simultaneously in early December. Avian influenza virus (AIV), H7N2 subtype, was isolated from the two premises in our laboratory. The H7N2 isolates were characterized as a low pathogenic strain at the National Veterinary Services Laboratories based on molecular sequencing of the virus hemagglutinin cleavage site and virus challenge studies in specific-pathogen-free leghorn chickens. However, clinical observations and pathologic findings indicated that this H7N2 virus appeared to be significantly pathogenic in meat-type chickens under field conditions. Follow-up investigation indicated that this H7N2 virus spread rapidly within each flock. Within 7 days of the recognized start of the outbreak, over 90% seroconversion was observed in the birds by the hemagglutination inhibition test. A diagnosis of AI was made within 24 hr of bird submission during this outbreak using a combination of virus detection by a same-day dot-enzyme-linked immunosorbent assay and virus isolation in embryonating chicken eggs. Follow-up investigation revealed that heavy virus shedding (90%-100% of birds shedding AIV) occurred between 4 and 7 days after disease onset, and a few birds (15%) continued to shed virus at 13 days post-disease onset, as detected by virus isolation on tracheal and cloacal swabs. AIV was not detected in or on eggs laid by the breeders during the testing phase of the outbreak. The two flocks were depopulated at 14 days after disease onset, and AIV was not detected on the two premises 23 days after depopulation.     

Using mean infectious dose of high- and low-pathogenicity avian influenza viruses originating from wild duck and poultry as one measure of infectivity and adaptation to poultry

The mean infectious doses of selected avian influenza virus (AIV) isolates, determined in domestic poultry under experimental conditions, were shown to be both host-dependent and virus strain-dependent and could be considered one measure of the infectivity and adaptation to a specific host. As such, the mean infectious dose could serve as a quantitative predictor for which strains of AIV, given the right conditions, would be more likely transmitted to and maintained in a given species or subsequently cause an AI outbreak in the given species. The intranasal (IN) mean bird infectious doses (BID50) were determined for 11 high-pathogenicity AIV (HPAIV) isolates of turkey and chicken origin for white leghorn (WL) chickens, and for low-pathogenicity AIV (LPAIV) isolates of chicken (n = 1) and wild mallards (n = 2) for turkeys, and WL and white Plymouth rock (WPR) chickens, domestic ducks and geese, and Japanese quail. The BID50 for HPAIV isolates for WL chickens ranged from 10(1.2) to 10(4.7) mean embryo infectious dose (EID50) (median = 10(2.9)). For chicken-origin HPAIV isolates, the BID50 in WL chickens ranged from 10(1.2) to 10(3.0) EID50 (median = 10(2.6)), whereas for HPAIV isolates of turkey origin, the BID50 in WL chickens was higher, ranging from 10(2.8) to 10(4.7) EID50 (median = 10(3.9)). The BID50 of 10(4.7) was for a turkey-origin HPAIV virus that was not transmitted to chickens on the same farm, suggesting that, under the specific conditions present on that farm, there was insufficient infectivity, adaptation, or exposure to that virus population for sustained chicken transmission. Although the upper BID50 limit for predicting infectivity and sustainable transmissibility for a specific species is unknown, a BID50 < 10(4.7) was suggestive of such transmissibility. For the LPAIVs, there was a trend for domestic ducks and geese and Japanese quail to have the greatest susceptible and for WL chickens to be the most resistant, but turkeys were susceptible to two LPAIV tested when used at moderate challenge doses. This suggests domestic ducks and geese, turkeys, and Japanese quail could serve as bridging species for LPAIVs from wild waterfowl to chickens and other gallinaceous poultry. These data do provide support for the commonly held and intuitive belief that mixing of poultry species during rearing and in outdoor production systems is a major risk factor for interspecies transmission of AIVs and for the emergence of new AIV strains capable of causing AI outbreaks because these situations present a more diverse host population to circumvent the natural host dependency or host range of circulating viruses.     

Low pathogenicity avian influenza viruses infect chicken layers by different routes of inoculation

In order to develop better control measures against avian influenza, it is necessary to understand how the virus transmits in poultry. In a previous study in which the infectivity and transmissibility of the pandemic H1N1 influenza virus was examined in different poultry species, we found that no or minimal infection occurred in chicken and turkeys intranasally (IN) inoculated with the virus. However, we demonstrated that the virus can infect laying turkey hens by the intracloacal (IC) and intraoviduct (IO) routes, possibly explaining the drops in egg production observed in turkey breeder farms affected by the virus. Such novel routes of exposure have not been previously examined in chickens and could also explain outbreaks of low pathogenicity avian influenza (LPAI) that cause a decrease in egg production in chicken layers and breeders. In the present study, 46-wk-old specific-pathogen-free chicken layers were infected by the IN, IC, or IO routes with one of two LPAI viruses: a poultry origin virus, A/chicken/CA/1255/02 (H6N2), and a live bird market isolate, A/chicken/NJ/12220/97 (H9N2). Only hens IN inoculated with the H6N2 virus presented mild clinical signs consisting of depression and anorexia. However, a decrease in number of eggs laid was observed in all virus-inoculated groups when compared to control hens. Evidence of infection was found in all chickens inoculated with the H6N2 virus by any of the three routes and the virus transmitted to contact hens. On the other hand, only one or two hens from each of the groups inoculated with the H9N2 virus shed detectable levels of virus, or seroconverted and did not transmit the virus to contacts, regardless of the route of inoculation. In conclusion, LPAI viruses can also infect chickens through other routes besides the IN route, which is considered the natural route of exposure. However, as seen with the H9N2 virus, the infectivity of the virus did not increase when given by these alternate routes.     

Experimental study to determine if low-pathogenicity and high-pathogenicity avian influenza viruses can be present in chicken breast and thigh meat following intranasal virus inoculation

Two low-pathogenicity (LP) and two high-pathogenicity (HP) avian influenza (AI) viruses were inoculated into chickens by the intranasal route to determine the presence of the AI virus in breast and thigh meat as well as any potential role that meat could fill as a transmission vehicle. The LPAI viruses caused localized virus infections in respiratory and gastrointestinal (GI) tracts. Virus was not detected in blood, bone marrow, or breast and thigh meat, and feeding breast and thigh meat from virus-infected birds did not transmit the virus. In contrast to the two LPAI viruses, A/chicken/Pennsylvania/1370/1983 (H5N2) HPAI virus caused respiratory and GI tract infections with systemic spread, and virus was detected in blood, bone marrow, and breast and thigh meat. Feeding breast or thigh meat from HPAI (H5N2) virus-infected chickens to other chickens did not transmit the infection. However, A/lchicken/Korea/ES/2003 (H5N1) HPAI virus produced high titers of virus in the breast meat, and feeding breast meat from these infected chickens to other chickens resulted in Al virus infection and death. Usage of either recombinant fowlpox vaccine with H5 AI gene insert or inactivated Al whole-virus vaccines prevented HPAI virus in breast meat. These data indicate that the potential for LPAI virus appearing in meat of infected chickens is negligible, while the potential for having HPAI virus in meat from infected chickens is high, but proper usage of vaccines can prevent HPAI virus from being present in meat.     

Marek's disease in turkeys. I. A seven-year survey of commercial flocks and experimental infection using two field isolates

Marek's disease virus (MDV) causes immunosuppression and tumors in chickens, but the turkey is an unusual host for the virus, and tumors caused by MDV in turkeys are unique. We describe the prevalence of turkey tumors in Israel between 1993 and 2000, their molecular diagnosis by polymerase chain reaction (PCR), and the natural distribution of herpesvirus of turkeys (HVT). Most clinical cases with tumors in commercial turkeys were diagnosed as MDV. The reproduction of Marek's disease (MD) in turkeys by two turkey MDV strains, Ar and La, was analyzed, and it was shown that these strains can induce tumors in experimental trials. The severity of experimental disease differed from those features of the original outbreak, since a less severe disease was recorded.     

Re-evaluation of the pathogenicity of A/chicken/Alabama/75 (H4N8) influenza virus.

Avian influenza (AI) virus A/chicken/Alabama/7395/75 (H4N8), a putatively non-pathogenic virus associated with a self-limiting outbreak of severe disease in commercial layers, was selectively passed in chickens or in cell cultures and then in chickens to determine whether virus with increased pathogenicity would emerge. When 20 derivatives of the parental virus were each inoculated intranasally and intratracheally in leghorn hens, mortality rates ranged from zero (0/24) to 25% (6/24); mortality was 4% (1/24) for hens inoculated with the parental virus. Many virus reisolates (51/144) from hens that died exhibited high pathogenicity, killing at least six of eight intravenously inoculated 4-week-old chickens. Most derivatives examined produced plaques in trypsin-free cell cultures more efficiently than the parental virus, but the highest plaquing efficiencies observed (10%) were lower than would be expected (100%) for highly pathogenic subtype H5 or H7 AI viruses. These results confirm that the Alabama H4N8 virus can acquire increased pathogenicity upon passage in chickens and suggest that it may have acted alone in producing the severe disease observed in laying chickens in Alabama.     

A comparison of three serological tests for the diagnosis of B. ovis infection in rams

The complement fixation test (CFT), the enzyme labelled immunosorbent assay (ELISA) and the gel diffusion precipitin test (GD) were compared, for the diagnosis of Brucella ovis infection in rams. The sensitivities of the tests in 109 rams which were shedding B. ovis in their semen were: CFT 96.3%; ELISA 97.2%; GD 91.7%. The specificities of the tests in 141 rams from non-infected flocks were: CFI 99.3%; ELISA 98.6%; GD 100%. Predictive values of the three tests were measured in 285 rams from infected flocks. Thirty-eight percent of these rams were shedding B. ovis in their semen. Predictive values of positive tests were: CFT 75.5%; ELISA 66.7%; GD 72.5%. Predictive values of negative tests were: CFI 97.1%; ELISA 97.6%; GD 93.8%.     

Improvements to the hemagglutination inhibition test for serological assessment of recombinant fowlpox-H5-avian-influenza vaccination in chickens and its use along with an agar gel immunodiffusion test for differentiating infected from noninfected vaccinated animals

In general, avian influenza (AI) vaccines protect chickens from morbidity and mortality and reduce, but do not completely prevent, replication of wild AI viruses in the respiratory and intestinal tracts of vaccinated chickens. Therefore, surveillance programs based on serological testing must be developed to differentiate vaccinated flocks infected with wild strains of AI virus from noninfected vaccinated flocks in order to evaluate the success of vaccination in a control program and allow continuation of national and international commerce of poultry and poultry products. In this study, chickens were immunized with a commercial recombinant fowlpox virus vaccine containing an H5 hemagglutinin gene from A/turkey/Ireland/83 (H5N8) avian influenza (AI) virus (rFP-H5) and evaluated for correlation of immunological response by hemagglutination inhibition (HI) or agar gel immunodiffusion (AGID) tests and determination of protection following challenge with a high pathogenicity AI (HPAI) virus. In two different trials, chickens immunized with the rFP-H5 vaccine did not develop AGID antibodies because the vaccine lacks AI nucleoprotein and matrix genes, but 0%-100% had HI antibodies, depending on the AI virus strain used in the HI test, the HI antigen inactivation procedure, and whether the birds had been preimmunized against fowlpox virus. The most consistent and highest HI titers were observed when using A/turkey/Ireland/83 (H5N8) HPAI virus strain as the beta-propiolactone (BPL)-inactivated HI test antigen, which matched the hemagglutinin gene insert in the rFP-H5 vaccine. In addition, higher HI titers were observed if ether or a combination of ether and BPL-inactivated virus was used in place of the BPL-inactivated virus. The rFP-H5 vaccinated chickens survived HPAI challenge and antibodies were detected by both AGID and HI tests. In conclusion, we demonstrated that the rFP-H5 vaccine allowed easy serological differentiation of infected from noninfected birds in vaccinated populations of chickens when using standard AGID and HI tests.     

Development of enzyme-linked immunosorbent assay with nucleoprotein as antigen for detection of antibodies to avian influenza virus

During the avian influenza outbreak of 2003-04 in Southeast Asia, two avian influenza viruses (AIV), one of H5N1 subtype and the other H9N2 subtype, were isolated and identified from local farms. The nudeoprotein (NP) gene of the H5N1 AI isolate was cloned, and the segment encoding amino acid 47-384, which covers its major antigenic domains, was subcloned and expressed in E. coli. Subsequently, the NP (47-384) expression product was purified and used as the diagnostic antigen to develop a NP-based type-specific indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to AI from chicken sera. The ELISA is shown to be specific for AIV and does not cross-react with chicken sera that has antibodies to other avian viruses. The NP(47-384)-ELISA was compared with a hemagglutination inhibition test and a commercial AIV ELISA kit in evaluating 150 sera samples from experimentally AIV-infected or vaccinated specific-pathogen-free (SPF) chickens. Our NP(47-384)-ELISA was more sensitive than the two tests and showed an 82% agreement ratio with the HI test and an 80.67% agreement ratio with the commercial kit. The NP(47-384)-ELISA and the commercial AIV ELISA were used to evaluate 448 field sera samples from diseased chickens or vaccinated chickens during the 2003-04 AI outbreak in China. The two ELISA tests had a 95% agreement ratio. We conclude that the NP(47-384)-ELISA developed in our laboratory was specific and sensitive and it has great application potential in China's long-term prevention and control of AI.     

The pathogenicity of three Australian fowl plague viruses for chickens,turkeys and ducks

The pathogenicity of three Australian fowl plague viruses, FPV-1, FPV-2, FPV-3, isolated during a natural outbreak of the disease varied for chickens, turkeys and ducks. FPV-1 and FPV-2 were pathogenic for chickens and turkeys, but not for ducks. However, these viruses were not highly pathogenic as they failed to cause illness or death in all birds that became infected. FPV-3 was non-pathogenic for the three species tested.The viruses spread from infected to in-contact birds, and more readily to ducks than to chickens or turkeys. All chickens and turkeys infected with the fowl plague viruses developed specific serum haemagglutination-inhibiting antibody which persisted for up to 85 days after infection. The titre of this antibody wan ed in six of 16 ducks over an 85-day period and two ducks failed to produce detectable specific HaI antibody despite being infected with the virus.