重组禽流感病毒灭活疫苗(H5N1亚型,Re-1株)免疫鸡抗体消长规律及免疫程序的初步研究

研究新型重组H5N1亚型禽流感灭活疫苗对种鸡和肉鸡的免疫原性,并对雏鸡母源抗体和免疫后的抗体进行动态观察,根据试验结果推荐该疫苗对鸡的免疫程序。用HI方法检测种鸡、肉鸡的母源抗体和免疫抗体,根据母源抗体的衰减和免疫抗体的消长规律确定首免和再免日龄。结果表明种鸡的母源抗体约能维持10 d多;0.3 mL/羽首免后10 d HI抗体就可达到6.40 log2,3-5周达到高峰期,至免疫17周后(19周龄)HI抗体水平仍然维持在4.88 log2;19周龄时0.5 mL/羽进行二免,有效抗体能维持约20周;280日龄0.5 mL/羽三免后抗体水平均一,下降缓慢,至种鸡淘汰时(三免后29周)抗体水平仍能维持在5.32 log2。肉鸡母源抗体约能维持7d,10日龄时0.3 mL/羽免疫,有效抗体能维持到上市。新型重组H5N1亚型禽流感灭活疫苗对鸡的免疫原性确实。     

Isolation of H13N2 influenza A virus from turkeys and surface water.

This is the first report of the isolation of H13N2 avian influenza virus (AIV) subtype from domestic turkeys. This subtype was also isolated from nearby surface water. The observation of large numbers of gulls in close association with turkeys on range before the virus isolations suggests that this virus subtype was transmitted from gulls to range turkeys. Turkey flocks infected by this virus subtype did not show any clinical signs of the disease, although seroconversion did occur. The H13N2 isolates were found to be non-pathogenic in chickens.     

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.     

Failure of a recombinant fowl poxvirus vaccine containing an avian influenza hemagglutinin gene to provide consistent protection against influenza in chickens preimmunized with a fowl pox vaccine

Vaccines against mildly pathogenic avian influenza (AI) have been used in turkeys within the United States as part of a comprehensive control strategy. Recently, AI vaccines have been used in control programs against highly pathogenic (HP) AI of chickens in Pakistan and Mexico. A recombinant fowl pox-AI hemagglutinin subtype (H) 5 gene insert vaccine has been shown to protect specific-pathogen-free chickens from HP H5 AI virus (AIV) challenge and has been licensed by the USDA for emergency use. The ability of the recombinant fowl pox vaccine to protect chickens preimmunized against fowl pox is unknown. In the current study, broiler breeders (BB) and white leghorn (WL) pullets vaccinated with a control fowl poxvirus vaccine (FP-C) and/or a recombinant fowl poxvirus vaccine containing an H5 hemagglutinin gene insert (FP-HA) were challenged with a HP H5N2 AIV isolated from chickens in Mexico. When used alone, the FP-HA vaccine protected BB and WL chickens from lethal challenge, but when given as a secondary vaccine after a primary FP-C immunization, protection against a HP AIV challenge was inconsistent. Both vaccines protected against virulent fowl pox challenge. This lack of consistent protection against HPAI may limit use to chickens without previous fowl pox vaccinations. In addition, prior exposure to field fowl poxvirus could be expected to limit protection induced by this vaccine.     

Pathogenesis of rotavirus infection in various age groups of chickens and turkeys: clinical signs and virology

Age-related susceptibility patterns of turkeys, broilers, and specific pathogen-free (SPF) White Leghorn chickens to experimentally induced infection with turkey or chicken rotavirus isolates were compared. The following determinants were evaluated: clinical signs, onset and duration of virus production, viral titers, involvement of intestinal villi in the replication of the virus, and the development of antibodies against the virus. Older turkeys and chickens were more susceptible than were their younger counterparts, turkeys were more susceptible than were broiler and White Leghorn chickens (regardless of age), and broiler chickens were slightly more susceptible than were age-matched White Leghorn chickens. Turkeys developed diarrhea, accompanied by high viral titers within 1 day after inoculation with virus. Viral antigen was found in the epithelial cells of the intestinal villi throughout the intestinal tract and some cells of the cecal tonsils. Antibodies could be detected as early as 4 to 5 days after inoculation. These findings were more pronounced in turkeys inoculated at 112 days of age than in birds inoculated at a younger age. Age-related susceptibility patterns were similar in White Leghorn and broiler chickens. Infection was subclinical in birds less than 56 days old, whereas older birds developed soft feces. Egg production in the White Leghorn chickens decreased after being inoculated with virus at 350 days of age.     

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.     

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.     

两株鹅源H6N2亚型禽流感广东分离株的全序列分析及致病性研究

为了解禽流感病毒(AIV)的变异情况,本研究对我国禽流感监测期间分离鉴定的两株鹅源AIVA/Goose/Guangdong/362/2009(H6N2)(GD/362/09)与A/Goose/Guangdong/244/2010(H6N2)(GD/244/10)进行全基因序列的测定和分析,并进行其对SPF鸡和BALB/c小鼠的致病性试验。序列分析显示:HA裂解位点的序列为339PQIETR↓GLFG348,表明两株病毒均为低致病力AIV。HA和NA的核苷酸同源性分别为84.5%和98.9%,另外,序列分析结果显示,GD/244/10的PA、M基因分别与高致病性AIV(HPAIV)A/aquatic bird/Korea/w74/2005(H5N2)和A/duck/Hong Kong/140/1998(H5N1)的同源性最高,表明其内部基因来源复杂,可能与H5 HPAIV发生重组或有共同的来源。病毒对动物的致病性试验结果显示:两株病毒均不能在鸡体内有效复制,在小鼠的肺脏能够有效复制,但在小鼠的鼻甲内只能检测到GD/244/10。     

Evaluation of the infectivity, length of infection, and immune response of a low-pathogenicity H7N2 avian influenza virus in specific-pathogen-free chickens

The H7N2 subtype of avian influenza virus (AIV) field isolate (H7N2/chicken/PA/3779-2/97), which caused the 1997-98 AIV outbreak in Pennsylvania, was evaluated for its infectivity, length of infection, and immune response in specific-pathogen-free (SPF) chickens. The composite findings of three clinical trials with various concentrations of virus indicated that this H7N2 subtype contained minimal pathogenicity for chickens. The concentration of the virus in the inoculum proved critical in the establishment of a productive infection in a chicken. Seven-day-old SPF chickens were not infected when inoculated with 10(0.7-2.0) mean embryo lethal dose (ELD50) of the H7N2 virus per bird. At this dose level, the immune response to this virus was not detected by the hemagglutination-inhibition (HI) test. Nonetheless, chickens at ages of 5 and 23 wk old tested were successfully infected when exposed to 10(4.7-5.7) ELD50 of H7N2 infectious doses per bird by various routes of administration and also by direct contact. Infected birds started shedding virus as early as 2 days postinoculation, and the period of virus shedding occurred mostly within 1 or 2 wk postinoculation (WPI). This H7N2 subtype of AIV induced a measurable immune response in all birds within 2 wk after virus exposure. Antibody titers were associated with AIV infectious doses and age of exposure of birds. Challenge of these infected birds with the same H7N2 virus at 5 and 10 WPI indicated the infective virus was recoverable from cloacal swabs at 3 days postchallenge and disappeared thereafter. In these challenged birds, the antibody levels as measured by the HI test spiked within 1-2 wk.     

Pathogenicity of an avian influenza virus serotype H9N2 in chickens

A low pathogenic avian influenza virus (AIV) serotype H9N2 affected many commercial flocks in the Middle East in late 1990s and early 2000s. Due to the varying pathogenicity ofAIV H9N2 reported in previous studies, this study was carried out to determine the pathogenicity of a Jordanian isolate of H9N2 in broiler and specific-pathogen-free (SPF) chickens. Mild tracheal rales were observed in the broilers but not in the SPF birds starting 3 days postinfection (DPI) and until the end of the experiment at 16 DPI. Infected chickens had gross and histologic changes limited to the respiratory system (sinuses, trachea, lungs, and air sacs) characterized by congestion and lymphoplasmacytic inflammation. However, the lesions in the broiler chickens were more severe than those in the SPF chicks. Furthermore, the virus caused significant (P = 0.004) reduction (230 g) in average body weight of the infected broiler group compared with the uninfected broiler group. Both broiler and SPF-infected groups seroconverted, and they had a geometric mean titer of 2(8.2) and 2(9.3), respectively, on the hemagglutination inhibition test at 16 DPI. Cloacal virus shedding was not detected by 9 DPI and 15 DPI in broiler and SPF-infected groups, respectively. This study demonstrated the pathogenic nature of the local Jordanian H9N2 isolate and the variation from what it has been reported in other countries of the region. Regional effort should be directed to start an eradication program of this disease because of its pathogenicity for chickens, wide distribution, and possible interference with surveillance for H5N1 serotype.     

Efficacy of vaccines in chickens against highly pathogenic Hong Kong H5N1 avian influenza.

In 1997, highly pathogenic (HP) H5N1 avian influenza virus (AIV) caused infections in poultry in Hong Kong and crossed into humans, resulting in a limited number of infections including 18 hospitalized cases and six associated deaths. The unique ability of this, AIV to infect both poultry and people raised a concern for the potential of humans to be biological as well as mechanical vectors of this AIV to poultry. The current study was undertaken to determine if existing vaccines and their technologies could be used during an outbreak to protect poultry. Commercial and experimental inactivated whole H5 AIV and baculovirus-expressed AIV H5 hemagglurinin protein vaccines provided protection from clinical signs and death in chickens after lethal challenge by human-origin HP H5N1 Hong Kong strains 156/97 and 483/97. The commercial and experimental inactivated vaccines had mean protective doses ranging from 0.25 to 0.89, which represents the milligrams of viral protein in the vaccines that provided protection from death in half of the birds. Furthermore, the vaccines reduced the ability of the challenge AIV to replicate in chickens and decreased the recovery of challenge AIV from the enteric and respiratory tracts, but the use of a vaccine will nor totally prevent AI virus replication and shedding. Existing vaccines will protect poultry from mortality and reduce virus replication from the new HP AIV strain that can infect both poultry and humans.     

Genetic relationship of H3 subtype avian influenza viruses isolated from domestic ducks and wild birds in Korea and their pathogenic potential in chickens and ducks

The H3 subtype avian influenza virus (AIV) is one of the most frequently isolated subtypes in domestic ducks, live poultry markets, and wild birds in Korea. In 2002-2009, a total of 45 H3 subtype AIVs were isolated from the feces of clinically normal domestic ducks (n=28) and wild birds (n=17). The most prevalent subtypes in domestic ducks were H3N2 (35.7%), H3N6 (35.7%), H3N8 (25.0%), and H3N1 (3.6%, novel subtype in domestic duck in Korea). In contrast, H3N8 (70.6%) is the most prevalent subtype in wild birds in Korea. In the phylogenetic analysis, HA genes of the Korean H3 AIVs were divided into 3 groups (Korean duck, wild bird 1, and wild bird 2) and all viruses of duck origin except one were clustered in a single group. However, other genes showed extensive diversity and at least 17 genotypes were circulating in domestic ducks in Korea. When the analysis expanded to viruses of wild bird origin, the genetic diversity of Korean H3 AIVs became more complicated. Extensive reassortments may have occurred in H3 subtype influenza viruses in Korea. When we inoculated chickens and ducks with six selected viruses, some of the viruses replicated efficiently without pre-adaptation and shed a significant amount of viruses through oropharyngeal and cloacal routes. This raised concerns that H3 subtype AIV could be a new subtype in chickens in Korea. Continuous surveillance is needed to prepare the advent of a novel subtype AIV in Korea.     

野生鸟类禽流感病毒感染情况的调查

为了解野生鸟类禽流感病毒(AIV)的携带感染情况,2006年～2010年,本研究在湖南省主要候鸟迁徙地收集115只野鸟组织或拭子样品、75份野鸟的新鲜粪便样品和72份血清样品。组织或拭子样品采用RT-PCR方法检测和鸡胚接种病毒分离鉴定,血清样品分别进行H5(含Re-5和Re-4)、H6、H7、H9、H10和H11抗体检测。结果表明,从斑鸠和绿头鸭组织中分别分离到H5N1亚型和H3N2亚型AIV;72份血清中有17份抗体为阳性,其中H5(Re-5)亚型5份、H5(Re-4)亚型1份、H6亚型1份、H7亚型2份和H9亚型8份,阳性率分别为6.94%、1.39%、1.39%、2.78%和11.11%。H10和H11亚型未检测到抗体阳性。     

Avian influenza A viruses in birds --an ecological, ornithological and virological view

Avian influenza A viruses (AIV) are the causative agents of the presently most important poultry disease. Ten countries in Asia and several other countries in Eastern Europe suffer high losses from the lethal effects of these viruses of the H5N1 subtype. AIV of other subtypes cause in additional countries severe losses. The threat to health and well-being of the avifauna, domestic poultry and possibly mammals including humans are worldwide of major concern. The European Union reacted with a complete import ban on untreated meat, eggs, poultry products as well as free-living and pet birds. Extensive surveillance of free-living birds and domestic poultry that is maintained in free-range and close to open waters were initiated in an attempt to gather information on the current status of infection with these viruses and to target appropriate countermeasures for the protection of domestic poultry (in-house keeping) and to safeguard food production for humans. Since the monitoring of free-living birds is labour-intensive, costly, and time-consuming, only birds should be included in the monitoring programme that harboured in the past most if not all influenza A viruses. The birds of the order Anatiformes, family Anatidae, subfamilies Anserinae and Anatinae, provided 65.9 % of all avian AIV isolates. The cosmopolitan Common Mallard (Anas platyrhynchos) is the dominant species with the highest rate of isolations among all bird species. Second in frequency is the North-American Blue-winged Teal (Spatula discors). Consequently, free-living anatiform birds of the genera Anas and Spatula should comprise the main focus for the collection of cloacal and pharyngeal swabs. With the likely exception of the most recent H5N1 viruses, signs of disease were not recorded in AIV infected anatiform birds. AIV isolations were definitely less frequently obtained from birds of the orders Phasianiformes (including domestic chickens and turkeys), Charadriiformes (plovers and lapwings), Lariformes (gulls), Columbiformes (pigeons) and Psittaciformes (psittacines) and need less attention in sampling efforts. This review presents also data on taxonomy and most suitable means for isolation and typing of haemagglutinating viruses. The different frequencies of the detection of 16 haemagglutinin (HA) subtypes and 9 subtypes of neuraminidase (NA) surface antigens are composed on the basis of extensive literature retrievals. Both antigens occure in isolates at different frequencies. Only 103 of all 144 possible HA x NA combinations were described so far. The AIV that contain the HA subtypes H3, H4, H6 are most frequently isolated whereas the AIV of the subtypes H5 and H7 were less frequently encountered. All other HAs are rather rare. AIV that possess the NA of the subtypes N2, N1, N8 and N3 are frequent and all other NAs are rarely detected.     

Isolation and characterization of a novel H9N2 influenza virus in Korean native chicken farm

An outbreak of avian influenza, caused by an H9N2 low-pathogenic avian influenza virus (AIV), occurred in a chicken farm and caused severe economic losses due to mortality and diarrhea. AIV was isolated and identified in a sample from an affected native Korean chicken. Genetic analysis of the isolate revealed a high sequence similarity to genes of novel reassortant H9N2 viruses isolated from slaughterhouses and live bird markets in Korea in 2008 and 2009. Animal challenge studies demonstrated that the replication kinetics and pathogenicity of the isolate were considerably altered due to adaptation in chickens. Vaccine protection studies indicated that commercial vaccine was not able to prevent virus shedding and clinical disease when chickens were challenged with the isolate. These results suggest that the novel H9N2 virus possesses the capacity to replicate efficiently in the respiratory system against vaccination and to cause severe disease in domestic chickens. The results also highlight the importance of appropriate updating of vaccine strains, based on continuous surveillance data, to prevent the possibility of a new H9N2 epidemic in Korea.     

A quantitative measurement of the effect of avian influenza virus on the ability of turkeys to eliminate Pasteurella multocida from the respiratory tract

The effect of avian influenza virus (AIV) infection on the ability of turkeys to eliminate Pasteurella multocida from the respiratory tract was evaluated. Four-week-old turkeys were experimentally infected with an apathogenic AIV subtype (H5N2) by the oculonasal route and subsequently superinfected with P multocida (Urbach strain) by the intranasal route three days after infection with AIV. Quantitative clearance of P multocida from the trachea and lung was determined using a pour plate technique on samples collected at intervals after infection. Samples from turkeys which had been infected with AIV were found to yield more P multocida than those from turkeys which had not been infected with AIV. The numbers of P multocida increased in infected birds to a greater extent than in birds which had not been infected with the virus. The present study suggests that AIV infection may contribute to the increased numbers and a decreased clearance of P multocida in turkeys.     

Highly pathogenic avian influenza virus subtype H5N1 in Mute swans in the Czech Republic

In order to determine the actual prevalence of avian influenza viruses (AIV) in wild birds in the Czech Republic extensive surveillance was carried out between January and April 2006. A total of 2101 samples representing 61 bird species were examined for the presence of influenza A by using PCR, sequencing and cultivation on chicken embryos. AIV subtype H5N1 was detected in 12 Mute swans (Cygnus olor). The viruses were determined as HPAI (highly pathogenic avian influenza) and the hemagglutinin sequence was closely similar to A/mallard/Italy/835/06 and A/turkey/Turkey/1194/05. Following the first H5N1 case, about 300 wild birds representing 33 species were collected from the outbreak region and tested for the presence of AIV without any positive result. This is the first report of highly pathogenic avian influenza subtype H5N1 in the Czech Republic. The potential role of swan as an effective vector of avian influenza virus is also discussed.     

Comparative serological evaluation of avian influenza vaccine in turkeys

Four- and six-week-old turkeys were vaccinated subcutaneously using avian influenza virus (AIV) A/Duck/613/MN/79 (H4N2) killed oil-emulsion vaccine. Sequential serological tests using agar gel precipitin (AGP), hemagglutination inhibition (HI), and enzyme-linked immunosorbent assay (ELISA) for measuring antibodies to AIV were performed up to 4 weeks postvaccination, when birds were challenged intranasally using A/Turkey/MN/80 (H4N2) live AIV. The ELISA was 25 to 1600 times more sensitive than the HI test and was able to detect antibody production earlier than the HI test. All turkeys with an ELISA titer of greater than or equal to 800 were protected against homologous challenge, as measured by virus recovery 3 days postchallenge. Four turkeys out of 20 serologically negative by AGP and HI tests but ELISA-positive were protected.