Renal pathology in specific-pathogen-free chickens inoculated with a waterfowl-origin type A influenza virus

Five-week-old specific-pathogen-free chickens inoculated intravenously with a waterfowl-origin type A influenza virus (A/mallard/Ohio/184/86) had swollen and mottled kidneys on days 3, 5, and 7 postinoculation (PI) and multiple raised nodules on days 5, 10, and 20 PI. Histologically, the kidneys had multifocal heterophilic tubulointerstitial nephritis with epithelial necrosis on day 3 PI, lymphoplasmacytic tubulointerstitial nephritis on day 5 PI, and fibrosing interstitial nephritis with cortical lobular collapse, atrophic tubules, glomerular aggregates, and interstitial lymphoid follicles and aggregates on days 7, 10, and 20 PI. Heterophilic intratubular medullary-cone nephritis was present in dead or moribund chickens on days 3 and 5 PI. Furthermore, the presence of mild multifocal heterophilic tubulointerstitial nephritis on day 20 PI suggests that a waterfowl-origin strain of type A influenza virus of low pathogenicity has the potential to produce acute and chronic active nephritis in the chicken and that the kidney is a potential site for influenza viral persistence. The acute, subacute, and chronic histopathologic renal lesions of this influenza virus in chickens are similar to lesions reported for some nephropathogenic infectious bronchitis viruses and avian nephritis picornavirus.     

Replication of a waterfowl-origin influenza virus in the kidney and intestine of chickens

Intravenous inoculation of chickens with a waterfowl-origin type A influenza virus resulted in high titers of virus in kidney tissues and viral nucleoprotein in renal tubular epithelial cells and in intestinal mucosal epithelial cells. Virus titers in kidneys of four of eight clinically normal chickens sampled on days 3 and 5 postinoculation (PI), one dead chicken on day 3 PI, and one dead chicken on day 7 PI exceeded 10(6) mean embryo infectious dose per gram of tissue. Using immunofluorescent and immunoperoxidase staining, viral nucleoprotein was identified in the cytoplasm and nucleus of tubular epithelial cells in kidneys and in nucleus of mucosal epithelial cells lining villi in the lower small intestine. Based on the low intravenous pathogenicity index for this virus (0.3) along with the high virus titers in kidney tissues and localization of viral antigen in kidney important site for replication of avian influenza (AI) virus of low pathogenicity. Recovery of type A influenza viruses from cloacal swabs could result from viral replication in kidneys as well as in the lower intestine and/or the bursa of Fabricius.     

Evaluation of the kidney as a potential site of avian influenza virus persistence in chickens.

One-day-old chickens were inoculated intravenously with one of three low-pathogenicity avian-origin influenza isolates. On day 5 postinoculation (PI), the frequency of influenza virus isolation from cloacal swabs following challenge with each isolate ranged from 83% to 100% for clinically normal euthanatized chickens. Influenza virus was also frequently isolated from kidneys of these chickens (47%) and from chickens that died (100%). Kidneys positive for virus isolation had lesions of nephrosis and/or acute nephritis, and influenza viral nucleoprotein was demonstrated in nuclei and cytoplasm of necrotic renal tubule epithelium. On sampling days 28 and 45/60 PI, influenza virus was neither isolated from nor immunohistochemically demonstrated in kidneys (0/125); however, the kidneys (47%) did have chronic histologic lesions that suggested previous influenza virus infection of the kidneys. Influenza virus was isolated from cloacal swabs of two of 44 chickens on day 28 PI, but all cloacal swabs were negative for virus recovery on sampling day 45/60 PI (0/81). These results indicate that replication of influenza virus in renal tubule epithelial cells did not result in persistence of type A influenza virus in this immunologically privileged site.     

Viral pathogenesis of a nephrotropic infectious bronchitis virus isolated from commercial pullets.

Infectious bronchitis was diagnosed in 3-to-4-week-old pullets from an outbreak in a commercial flock in California. The disease was characterized by head swelling, watery discharge from the eyes and nostrils, and urates in kidneys. Mortality ranged from 1.8% to 12.5% per week. The isolation of a coronavirus from a suspension of pooled kidneys from clinically ill chickens at the fifth passage in 10-day-old chicken embryos, gross and histologic renal lesions, and seroconversion by enzyme-linked immunosorbent assay in inoculated birds suggested that the virus isolated was a nephrotrophic strain of infectious bronchitis virus (IBV). The virus isolate was found to be a previously unrecognized serotype, based on virus neutralization tests performed in embryonated chicken eggs. Nephropathogenicity of the IBV isolate was confirmed by inoculation of the viral isolate into specific-pathogen-free chicks and demonstration of renal lesions. The isolation of nephrotropic strains of IBV has not been reported previously from poultry in California.     

Intracerebral pathogenicity for chickens of avian influenza viruses isolated from free-living waterfowl in Japan

The pathogenicity for chickens of 91 strains of avian influenza A virus isolated from such free-living waterfowl as whistling swan, pintail, tufted duck, mallard and black-tailed gull in Japan was tested. The majority of the virus strains infected and were pathogenic for the chickens. The virulence of these viruses seemed not to be as high as that of fowl plague virus. There were no significant differences in the intracerebral index score among the viruses belonging to the same subtype, irrespective of year of isolation or host.     

Vaccines protect chickens against H5 highly pathogenic avian influenza in the face of genetic changes in field viruses over multiple years

Inactivated whole avian influenza (AI) virus vaccines, baculovirus-derived AI haemagglutinin vaccine and recombinant fowlpoxvirus-AI haemagglutinin vaccine were tested for the ability to protect chickens against multiple highly pathogenic (HP) H5 AI viruses. The vaccine and challenge viruses, or their haemagglutinin protein components, were obtained from field AI viruses of diverse backgrounds and included strains obtained from four continents, six host species, and isolated over a 38-year-period. The vaccines protected against clinical signs and death, and reduced the number of chickens shedding virus and the titre of the virus shed following a HP H5 AI virus challenge. Immunization with these vaccines should decrease AI virus shedding from the respiratory and digestive tracts of AI virus exposed chickens and reduce bird-to-bird transmission. Although most consistent reduction in respiratory shedding was afforded when vaccine was more similar to the challenge virus, the genetic drift of avian influenza virus did not interfere with general protection as has been reported for human influenza viruses.     

Influenza virus surveillance in waterfowl in Pennsylvania after the H5N2 avian outbreak

During the latter stages of the lethal H5N2 influenza eradication program in domestic poultry in Pennsylvania in 1983-84, surveillance of waterfowl was done to determine if these birds harbored influenza viruses that might subsequently appear in poultry. From late June to November 1984, 182 hemagglutinating viruses were isolated from 2043 wild birds, primarily ducks, in the same geographical area as the earlier lethal H5N2 avian influenza outbreak. The virus isolates from waterfowl included paramyxoviruses (PMV-1, -4, and -6) and influenza viruses of 13 antigenic combinations. There was only one H5N2 isolate from a duck. Although this virus was antigenically related to the lethal H5N2 virus, genetic and antigenic analysis indicated that it could be discriminated from the virulent family of H5N2 viruses, and it did not originate from chickens. Many of the influenza viruses obtained from wild ducks were capable of replicating in chickens after experimental inoculation but did not cause disease. These studies show that many influenza A virus strains circulating in waterfowl in the vicinity of domestic poultry in Pennsylvania did not originate from domestic poultry. These influenza viruses from wild ducks were capable of infecting poultry; however, transmission of these viruses to poultry apparently was avoided by good husbandry and control measures.     

Different routes of inoculation impact infectivity and pathogenesis of H5N1 high pathogenicity avian influenza virus infection in chickens and domestic ducks

The H5N1 type A influenza viruses classified as Qinghai-like virus (clade 2.2) are a unique lineage of type A influenza viruses with the capacity to produce significant disease and mortality in gallinaceous and anseriform birds, including domestic and wild ducks. The objective of this study was to determine the susceptibility and pathogenesis of chickens and domestic ducks to A/Whooper Swan/Mongolia/224/05 (H5N1) high pathogenicity avian influenza (HPAI) virus when administered through respiratory or alimentary routes of exposure. The chickens and ducks were more susceptible to the H5N1 HPAI virus, as evidenced by low infectious and lethal viral doses, when exposed by intranasal as compared to alimentary routes of inoculation (intragastric or oral-fed infected chicken meat). In the alimentary exposure pathogenesis study, pathologic changes included hemorrhage, necrosis, and inflammation in association with virus detection. These changes were generally observed in most of the visceral organs of chickens, between 2 and 4 days postinoculation (DPI), and are similar to lesions and virus localization seen in birds in natural cases or in experimental studies using the intranasal route. Alimentary exposure to the virus caused systemic infection in the ducks, characterized by moderate lymphocytic encephalitis, necrotized hepatitis, and pancreatitis with a corresponding demonstration of virus within the lesions. In both chickens and ducks with alimentary exposure, lesions, virus, or both were first demonstrated in the upper alimentary tract on 1 DPI, suggesting that the alimentary tract was the initial site affected upon consumption of infected meat or on gavage of virus in liquid medium. However, as demonstrated in the infectivity study in chickens, alimentary infection required higher exposure doses to produce infection as compared to intranasal exposure in chickens. These data suggest that upper respiratory exposure to H5N1 HPAI virus in birds is more likely to result in virus infection and transmission than will consumption of infected meat, unless the latter contains high doses of virus, as found in cannibalized infected carcasses.     

Assessing pathogenicity potential of waterfowl-origin type A influenza viruses in chickens.

Intravenous pathogenicity index (IVPI) tests on 29 wild duck-origin type A influenza viruses, two turkey-origin type A influenza viruses, and one chicken-origin type A influenza virus resulted in indices ranging from 0.0 to 0.49. Most of the wild duck-origin viruses and the two turkey-origin viruses had indices of 0.0, indicating they are not pathogenic. Six of the duck-origin viruses had indices ranging from 0.25 to 0.49, and the IVPI for A/chicken/Alabama/75 (H4N8) was 0.49, indicating they had low pathogenic potential. An IVPI of 1.25 up to the maximum score of 3.0 is necessary for a type A influenza virus to be classified as highly pathogenic. Gross lesions observed in chickens dying following intravenous viral challenge included kidney swelling with more prominent lobular patterns, but visceral urate deposits were not present. The usefulness of the IVPI test in evaluating the pathogenicity potential of nonpathogenic and low-pathogenic strains of avian influenza virus may be limited.     

Nephrotropic properties demonstrated by A/chicken/Alabama/75 (H4N8) following intravenous challenge of chickens.

Tissue tropism properties of A/chicken/Alabama/75 (H4N8) were examined after intravenous inoculation of 5-week-old specific-pathogen-free chickens. From 14 clinically normal chickens euthanatized on days 1-20 postinoculation, the frequencies of virus recovery were highest for cloacal swabs (86%), bursal swabs (64%), and kidney tissues (64%) and lowest for tracheal swabs (14%), thymus tissues (14%), bone-marrow swabs (7%), and brain tissues (0%). Evidence that the high frequency of virus recovery from kidney tissues was associated with virus replication in the kidney tissues was provided by high virus titers, ranging up to 10(9.5) mean embryo infectious dose per gram of kidney tissue, and by identification of intranuclear and intracytoplasmic type A influenza nucleoprotein in kidney cells using immunohistochemistry. Virus-recovery and virus titer results from three chickens that died on days 4 and 5 postinoculation paralleled the results from the clinically normal chickens. These findings indicate that A/chicken/Alabama/75 has nephrotropic properties similar to nephrotropic properties previously reported for waterfowl-origin type A influenza viruses and provide evidence that kidney lesions could be manifestations of systemic influenza infections in commercial laying chickens.     

Experimental selection of virus derivatives with variations in virulence from a single low-pathogenicity H6N1 avian influenza virus field isolate

A mixture of viruses with variations in virulence is likely present within a low-pathogenicity avian influenza virus (LPAIV) population. An H6N1 AIV was isolated from a field case showing 3.8% weekly mortality and a 33% egg production drop in Taiwan. The pathologic lesions included proventricular hemorrhage and urate deposition in the kidneys and on visceral organs. From the field isolate, a done (2838N) that caused no lesions or death was obtained using limit dilution in chicken embryos and a done (2838V) that caused renal lesions and death was obtained using contact infection in chicks. Both clones were inoculated intranasally in 4-week-old specific pathogen free (SPF) chickens to test their virulence. Renal urate deposition was found in chickens inoculated with 2838V but not in chickens inoculated with 2838N. In situ hybridization, polymerase chain reaction, and virus isolation were used to confirm the spread of 2838V from the respiratory tract to the renal tissue. We found that contact infection in chickens is a good method to obtain a more virulent done from a heterogeneous virus population.     

Induction of strong protection by vaccination with partially attenuated serotype 1 Marek's disease viruses

Studies were conducted to better understand the relationship among Marcek's disease (MD) vaccine strains between induction of protective immunity and the degree of attenuation (or virulence). To obtain viruses at different stages of attenuation, very virulent plus MD strains 584A and 648A and selected clones of these strains were serially passaged in chicken and duck cells. These viruses were considered fully attenuated after passage for 70-100 times in chicken embryo cell cultures until they no longer induced gross lesions in susceptible, maternal antibody-negative (ab-) chickens. Lower passages of the same strains were considered partially attenuated, provided their virulence was less than that of the parent strain. Four of five partially attenuated preparations derived from MD virus strains 584A and 648A or the previously attenuated Md11 strain induced 28%-62% higher levels of protection in maternal antibody-positive (ab+) chickens against virulent MD challenge than the fully attenuated counterpart viruses. The partially attenuated 584A/d2/3 strain replicated in chickens but was totally nonprotective. Data from two subsequent trials in ab+ chickens confirmed that protection induced by the partly attenuated (passage 80) preparations was 79% and 118% higher, respectively, than that induced by the fully attenuated (passage 100) preparations of strain 648A. However, in one trial with ab- chickens, no difference in protection between partially and fully attenuated virus was observed. Strong protection (up to 85%) against highly virulent challenge also was provided by preparations of 648A at passages 40-60, which were moderately oncogenic when used alone. Partially attenuated strains tended to replicate to higher titers in both ab+ and ab- chickens compared with fully attenuated vaccines. Also, ab+ and ab- chickens vaccinated with partially attenuated strains developed three- to nine fold more extensive microscopic lesions in peripheral nerves at 14 and 22 days after virulent challenge than chickens vaccinated with fully attenuated strains. When measured in ab+ chickens, loss of lesion induction by 648A was achieved 30 passages earlier (at passage 70) than when measured in ab- chickens. Thus, maternal antibodies appeared to abrogate the pathogenicity of some partially attenuated strains. These studies establish for MD the principle that at least some partially attenuated MD viruses may replicate better and induce stronger immunity against virulent challenge than fully attenuated preparations of the same strain, at least when tested in ab+ chickens. Moreover, depending on passage level, partially attenuated vaccine strains may be relatively innocuous for ab+ chickens, causing few or no lesions.     

A low pathogenic H5N2 influenza virus isolated in Taiwan acquired high pathogenicity by consecutive passages in chickens

H5N2 viruses were isolated from cloacal swab samples of apparently healthy chickens in Taiwan in 2003 and 2008 during surveillance of avian influenza. Each of the viruses was eradicated by stamping out. The official diagnosis report indicated that the Intravenous Pathogenicity Indexes (IVPIs) of the isolates were 0.00 and 0.89, respectively, indicating that these were low pathogenic strains, although the hemagglutinin of the strain isolated in 2008 (Taiwan08) had multibasic amino acid residues at the cleavage site (PQRKKR/G). In the present study, these H5N2 viruses were assessed for their intravenous and intranasal pathogenicity for chickens. It was examined whether Taiwan08 acquires pathogenicity through consecutive passages in chickens. Intravenous pathogenicity of Taiwan08 depended upon the age of the chickens used for the IVPI test; all of the eight-week-old chickens intravenously inoculated with Taiwan08 showed clinical signs but survived for ten days post inoculation (IVPI=0.68), whereas all the six-week-old chickens died (IVPI=1.86). Taiwan08-P8, which were passaged in chickens for eight times, killed all the eight-week-old chickens (IVPI=2.36). The four-week-old chickens died after intranasal inoculation of Taiwan08-P8, indicating that Taiwan08 must have become highly pathogenic during circulation in chicken flocks. These results emphasize the importance of a stamping out policy for avian influenza even if the IVPI of the causal virus is low.     

两株H6N2亚型鸡源禽流感病毒的分离鉴定

本研究利用血凝抑制试验(HI)、反转录-聚合酶链式反应(RT-PCR)、基因测序等方法,对广东省某活禽交易市场进行流行病学调查时获得的两株非H5、H9亚型禽流感病毒65株和C7株进行了亚型鉴定。结果表明这两株病毒均具有血凝活性,且能被抗H6亚型禽流感病毒标准阳性血清特异性抑制。用针对禽流感病毒的M基因、H6亚型禽流感病毒HA基因、N2亚型禽流感病毒NA基因特异性鉴定引物对65株和C7株进行RT-PCR扩增,分别获得特异性目的片段。测序及BLAST分析表明两株分离株与H6N2亚型禽流感广东分离株的HA基因和NA基因核苷酸序列相似性均高达95%以上。将该两分离株鉴定为H6N2亚型禽流感病毒,并命名为A/Chicken/Guangdong/65/2009、A/Chicken/Guangdong/C7/2009。     

H5N1亚型禽流感病毒A/goose/Guangdong/1/96的NS基因对鸡致病能力的影响

为了解H5N1亚型禽流感病毒经自然途径感染SPF鸡后，病毒的致病能力与NS基因的关系，本文主要从病理学角度比较了两株利用反向基因操作技术拯救的病毒RGSGD／1／96和RGSGD／1／2NS的致病能力。虽然只有NS基因不同，但是这两株病毒经鼻腔感染4周龄SPF鸡后表现出完全不同的致病能力，RGSGD／1／96对鸡的致死率为100％，感染鸡只的各组织脏器均可发现严重的病理损伤，该病毒在鸡体内复制能力很强，感染后3d、6d，各组织脏器均可发现大量的病毒抗原；GSGD／1／2NS对鸡的致死率为0，病毒在感染鸡体内只引起肺间质少量淋巴细胞浸润，免疫组织化学检查未发现病毒抗原信号。由于两株病毒只有NS基因不同，说明NS基因决定了H5N1亚型禽流感病毒A／goose／Guangdong／1／96对SPF鸡的致病能力。     

Different infection routes of avian influenza A (H5N1) virus in mice

The continuing outbreaks of avian influenza A H5N1 virus infection in Asia and Africa have caused worldwide concern because of the high mortality rates in poultry, suggesting its potential to become a pandemic influenza virus in humans. The transmission route of the virus among either the same species or different species is not yet clear. Broilers and BABL/c mice were inoculated with the H5N1 strain of influenza A virus isolated from birds. The animals were inoculated with 0.1 mL 10^6.83 TCID₅₀ of H5N1 virus oronasally, intraperitoneally and using eye drops. The viruses were examined by virological and pathological assays. In addition, to detect horizontal transmission, in each group, healthy chicks and mice were mixed with those infected. Viruses were detected in homogenates of the heart, liver, spleen, kidney and blood of the infected mice and chickens. Virus antigen was not detected in the spleen, kidney or gastrointestinal tract, but detected by Plaque Forming Unit (PFU) assay in the brain, liver and lung without degenerative change in these organs (in the group inoculated using eye drops. The detection results for mice inoculated using eye drops suggest that this virus might have a different tissue tropism from other influenza viruses mainly restricted to the respiratory tract in mice. All chicken samples tested positive for the virus, regardless of the method of inoculation. Avian influenza A H5N1 viruses are highly pathogenic to chickens, but its virulence in other animals is not yet known. To sum up, the results suggest that the virus replicates not only in different animal species but also through different routes of infection. In addition, the virus was detection not only in the respiratory tract but also in multiple extra‐respiratory tissues. This study demonstrates that H5N1 virus infection in mice can cause systemic disease and spread through potentially novel routes within and between mammalian hosts.     

Experimental infection of chickens, ducks and quails with the highly pathogenic H5N1 avian influenza virus

Highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype have spread since 2003 in poultry and wild birds in Asia, Europe and Africa. In Korea, the highly pathogenic H5N1 avian influenza outbreaks took place in 2003/2004, 2006/2007 and 2008. As the 2006/2007 isolates differ phylogenetically from the 2003/2004 isolates, we assessed the clinical responses of chickens, ducks and quails to intranasal inoculation of the 2006/2007 index case virus, A/chicken/Korea/IS/06. All the chickens and quails died on 3 days and 3-6 days post-inoculation (DPI), respectively, whilst the ducks only showed signs of mild depression. The uninoculated chickens and quails placed soon after with the inoculated flock died on 5.3 and 7.5 DPI, respectively. Both oropharyngeal and cloacal swabs were taken for all three species during various time intervals after inoculation. It was found that oropharyngeal swabs showed higher viral titers than in cloacal swabs applicable to all three avian species. The chickens and quails shed the virus until they died (up to 3 to 6 days after inoculation, respectively) whilst the ducks shed the virus on 2-4 DPI. The postmortem tissues collected from the chickens and quails on day 3 and days 4-5 and from clinically normal ducks that were euthanized on day 4 contained the virus. However, the ducks had significantly lower viral titers than the chickens or quails. Thus, the three avian species varied significantly in their clinical signs, mortality, tissue virus titers, and duration of virus shedding. Our observations suggest that duck and quail farms should be monitored particularly closely for the presence of HPAIV so that further virus transmission to other avian or mammalian hosts can be prevented.     

Response of Ferrets and Monkeys to Intranasal Infection with Human, Equine and Avian Influenza Viruses

Rhesus monkeys and ferrets were exposed to intranasal inoculation of several strains of egg-adapted avian, equine and human influenza viruses and to strains of mouse-adapted equine influenza viruses. Local replication of virus and seroconversion were observed in the majority of these animals. However, clinical infection was observed only in ferrets.     

Development of a pen-site test kit for the rapid diagnosis of H7 highly pathogenic avian influenza

As well as H5 highly pathogenic avian influenza viruses (HPAIV), H7 HPAIV strains have caused serious damages in poultry industries worldwide. Cases of bird-to-human transmission of H7 HPAIV have also been reported [11]. On the outbreak of avian influenza, rapid diagnosis is critical not only for the control of HPAI but also for human health. In the present study, a rapid diagnosis kit based on immunochromatography for the detection of H7 hemagglutinin (HA) antigen of influenza A virus was developed using 2 monoclonal antibodies that recognize different epitopes on the H7 HAs. The kit detected each of the tested 15 H7 influenza virus strains and did not react with influenza A viruses of the other subtypes than H7 or other avian viral and bacterial pathogens. The kit detected H7 HA antigen in the swabs and tissue homogenates of the chickens experimentally infected with HPAIV strain A/chicken/Netherlands/2586/03 (H7N7). The results indicate that the present kit is specific and sensitive enough for the diagnosis of HPAI caused by H7 viruses, thus, recommended for the field application as a pen-site test kit.     

Highly pathogenic virus recovered from chickens infected with mildly pathogenic 1986 isolates of H5N2 avian influenza virus

A combination of in vitro and in vivo selection procedures was used to examine the possibility that certain mildly pathogenic field isolates of avian influenza (AI) virus may contain minority subpopulations of highly pathogenic virus. Two mildly pathogenic H5N2 isolates, A/chicken/New Jersey/12508/86 (NJ12508) and A/chicken/Florida/27716/86 (FL27716), recovered from chickens epidemiologically associated with urban live-bird markets, were cloned in trypsin-free chicken embryo fibroblast cultures. Selected clones were inoculated intranasally and intratracheally (IN/IT) into specific-pathogen-free laying hens, and virus reisolated from the hens that died was serially passed in hens by IN/IT inoculation. Several highly pathogenic reisolates were recovered from hens infected with the cloned NJ12508 or FL27716 virus. A highly pathogenic NJ12508 reisolate killed 19 of 24 IN/IT-inoculated hens, and a FL27716 reisolate killed all 24 inoculated hens; signs and lesions were typical of fowl plague. In contrast, uncloned NJ12508 stock virus killed 1 of 24 hens and FL27716 stock virus killed 4 of 24 hens, and neither produced the complete spectrum of lesions associated with fowl plague. Recovery of highly pathogenic viruses from these isolates demonstrates the coexistence of pathogenically distinct subpopulations of virus. Competition for dominance among such subpopulations could explain the variable pathogenicity of some AI viruses.