Pathogenicity of West Nile virus for turkeys

In the fall of 1999, West Nile virus (WNV) was isolated during an outbreak of neurologic disease in humans, horses, and wild and zoological birds in New York, Connecticut, and New Jersey. Turkeys could potentially be a large reservoir for WNV because of the high-density turkey farming and the presence of large wild turkey populations in the eastern seaboard of the United States. Little is known about the pathogenicity of WNV in domestic or wild turkeys. Specific-pathogen-free 3-wk-old turkeys were inoculated subcutaneously with 10(3.3) mean tissue culture infective doses of a WNV strain isolated fromthe index case in a New York crow. No clinical signs were observed in the turkeys over the 21 days of the experiment. One turkey died abruptly at 8 days postinoculation (DPI). Many turkeys developed viremia between 2 and 10 DPI, but the average level of virus was very low, less than needed to efficiently infect mosquitos. Low levels of WNV were detected in feces on 4 and 7 DPI, but no virus was isolated from oropharyngeal swabs. WNV wasnot transmitted from WNV-inoculated to contact-exposed turkeys. All WNV-inoculated poults seroconverted on 7 DPI. In the turkey that died, WNV was not isolated from intestine, myocardium, brain, kidney, or cloacal and oropharyngeal swabs, but sparse viral antigen was demonstrated by immunohistochemistry in the heart and spleen. Turkeys in contact with WNV-inoculated turkeys and sham-inoculated controls lacked WNV specific antibodies,and WNV was not isolated from plasma and cloacal and oropharyngeal swabs. These data suggest that WNV lacks the potential to be a major new disease of turkeys and that turkeys will not be a significant amplifying host for infecting mosquitos.     

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.     

Experimental West Nile virus infection in Eastern Screech Owls (Megascops asio)

Eastern Screech Owls (EASOs) were experimentally infected with the pathogenic New York 1999 strain of West Nile virus (WNV) by subcutaneous injection or per os. Two of nine subcutaneously inoculated birds died or were euthanatized on 8 or 9 days postinfection (DPI) after <24 hr of lethargy and recumbency. All subcutaneously inoculated birds developed levels of viremia that are likely infectious to mosquitoes, with peak viremia levels ranging from 10(5.0) to 10(9.6) plaque-forming units/ml. Despite the viremia, the remaining seven birds did not display signs of illness. All birds alive beyond 5 DPI seroconverted, although the morbid birds demonstrated significantly lower antibody titers than the clinically normal birds. Cagemates of infected birds did not become infected. One of five orally exposed EASOs became viremic and seroconverted, whereas WNV infection in the remaining four birds was not evident. All infected birds shed virus via the oral and cloacal route. Early during infection, WNV targeted skin, spleen, esophagus, and skeletal muscle. The two morbid owls had myocardial and skeletal muscle necrosis and mild encephalitis and nephritis, whereas some of the clinically healthy birds that were sacrificed on 14 DPI had myocardial arteritis and renal phlebitis. WNV is a significant pathogen of EASOs, causing pathologic lesions with varying clinical outcomes.     

Experimental and serologic observations on avian pneumovirus (APV/turkey/Colorado/97) infection in turkeys

An avian pneumovirus (APV) was isolated from commercial turkeys in Colorado (APV/Colorado) showing clinical signs of a respiratory disease. The results of virus neutralization and indirect fluorescent antibody tests showed that the APV/Colorado was partially related to APV subgroup A but was unrelated to APV subgroup B. Turkeys experimentally inoculated with the APV/Colorado were observed for signs, lesions, seroconversion, and virus shedding. Thirty-six 7-wk-old turkeys were distributed into three groups. Eighteen turkeys were inoculated oculonasally with APV/Colorado, six were placed in contact at 1 day postinoculation (DPI), and 12 served as noninoculated controls. Tracheal swabs and blood samples were collected at 3, 5, 7, 10, 14, and 21 DPI. Tissues were collected from three inoculated and two control turkeys on aforementioned days for pathologic examination and APV isolation. Inoculated turkeys developed respiratory disease, yielded APV at 3, 5, and 7 DPI, and seroconverted at 10 DPI. Contact turkeys yielded APV at 7 and 10 DPI. No gross lesions were observed in the turbinates, infraorbital sinuses, and trachea. However, microscopic examination revealed acute rhinitis, sinusitis, and tracheitis manifested by congestion, edema, lymphocytic and heterophilic infiltration, and loss of ciliated epithelia. The inflammatory lesions were seen at 3 DPI and became extensive at 5 and 7 DPI. Active regenerative changes in the epithelia were seen at 10 and 14 DPI. Serologic survey for the presence of antibodies in commercial turkeys (24,504 sera from 18 states) and chickens (3,517 sera from 12 states) to APV/Colorado showed seropositive turkeys in Minnesota, North Dakota, and South Dakota and no seropositive chickens. This report is the first on the isolation of an APV and APV infection in the United States.     

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.     

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.     

Pathogenesis of chicken anemia virus: comparison of the oral and the intramuscular routes of infection

The events during the pathogenesis of chicken anemia virus (CAV) infection following intramuscular (IM) and oral inoculation were further elucidated and compared by sequential clinical, pathologic, and morphometric histopathologic evaluations, and by sequential determination of CAV genome concentrations in different organs. Specific-pathogen-free chickens were inoculated by IM or oral routes with the same dose (2 x 10(6) mean tissue culture infective dose [TCID50]) of CAV isolate 03-4876 at 1 day of age. Weights and hematocrits were obtained at 7, 10, 14, 18, 21, 25, and 28 days postinoculation (DPI). Seven birds from each group were necropsied at 7, 10, 14, and 28 DPI, and samples of thymus, Harderian gland, and cecal tonsils (CT) were obtained for histopathologic examination and CAV genome quantification by real-time polymerase chain reaction. Peak CAV genome concentrations were detected in the thymus at 10 and 14 DPI in the IM and orally infected chickens, respectively. High CAV DNA concentrations were maintained throughout the experimental period until 28 DPI, despite specific seroconversion occurring by 14 DPI in the IM-inoculated chickens. CAV was isolated from both orally and IM-infected chickens 28 DPI. Peak CAV genomes in the thymuses of IM and orally infected chickens coincided with peak lymphocyte depletion in these organs. Lymphocyte repopulation of the thymus occurred by 28 DPI in spite of the presence of the virus in the organs of both infected chicken groups. CAV genomes were detected in the CT, but histopathologic changes were not observed. Compared with the IM route of infection, orally infected chickens did not show apparent signs of illness. Clinical parameters, including reduction of weight gains and hematocrits, and gross and histopathologic changes were delayed and less severe in the orally inoculated chickens. This was concurrent with a delay in accumulation of CAV genomes in the thymus of these chickens.     

Experimental infection with low and high pathogenicity H7N3 Chilean avian influenza viruses in Chiloe wigeon (Anas sibilatrix) and cinnamon teal (Anas cyanoptera)

Two different wild duck species common in Chile and neighboring countries, Chiloe wigeon (Anas sibilatrix) and cinnamon teal (Anas cyanoptera), were intranasally inoculated with 10(6) mean embryo infective dose (EID50) of the H7N3 low pathogenicity (LP) avian influenza virus (AIV) (A/chicken/Chile/176822/02) or high pathogenicity (HP) AIV (A/chicken/Chile/ 184240-1/02), in order to study the infectivity and pathobiology of these viruses. None of the virus-inoculated ducks had clinical signs or died, but most seroconverted by 14 days postinoculation (DPI), indicating a productive virus infection. Both LPAIV and HPAIV were isolated from oral swabs from two of six Chiloe wigeons and from oral and/or cloacal swabs from all five of the cinnamon teal at 2 DPI. Both LPAIV and HPAIV were efficiently transmitted to cinnamon teal contacts but not to Chiloe wigeon contacts. This study demonstrates that the cinnamon teal and Chiloe wigeons were susceptible to infection with both Chilean H7N3 LPAIV and HPAIV, but only the cinnamon teal showed contact transmission of the virus between birds, suggesting that the cinnamon teal has the potential to be a reservoir for these viruses, especially the LPAIV, as was demonstrated in 2001 with isolation of a genetically related H7N3 LPAIV strain in a cinnamon teal in Bolivia. However, the definitive source of the H7N3 Chilean LPAIV still remains unknown.     

Pathogenicity and transmission of H5N1 avian influenza viruses in different birds

In this study, we selected three H5N1 highly pathogenic avian influenza viruses (HPAIVs), A/Goose/Guangdong/1/1996 (clades 0), A/Duck/Guangdong/E35/2012 (clade 2.3.2.1) and A/Chicken/Henan/B30/2012 (clade 7.2) isolated from different birds in China, to investigate the pathogenicity and transmission of the viruses in terrestrial birds and waterfowl. To observe the replication and shedding of the H5N1 HPAIVs in birds, the chickens were inoculated intranasally with 10⁶ EID₅₀ of GSGD/1/96, 10³ EID₅₀ of DkE35 and CkB30, and the ducks and geese were inoculated intranasally with 10⁶ EID₅₀ of each virus. Meanwhile, the naive contact groups were set up to detect the transmission of the viruses in tested birds. Our results showed that DkE35 was highly pathogenic to chickens and geese, but not fatal to ducks. It could be detected from all the tested organs, oropharyngeal and cloacal swabs, and could transmit to the naive contact birds. GSGD/1/96 could infect chickens, ducks and geese, but only caused death in chickens. It could transmit to the chickens and ducks, but was not transmittable to geese. CkB30 was highly pathogenic to chickens, low pathogenic to ducks and not pathogenic to geese. It could be transmitted to the naive contact chickens, but not to the ducks or geese. Our findings suggested that H5N1 HPAIVs from different birds show different host ranges and tissue tropisms. Therefore, we should enhance serological and virological surveillance of H5N1 HPAIVs, and pay more attention to the pathogenic and antigenic evolution of these viruses.     

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.     

Persistence of the V4 strain of Newcastle disease virus in an open-range flock of chickens

The V4 strain of Newcastle disease virus was introduced into a small open range flock of bantam chickens, by dosing half the birds directly into the crop. As indicated by rises in titres of haemagglutination inhibition antibody, the virus spread to the uninoculated birds and persisted in the flock for two years, infecting chickens that were introduced by natural brooding and rearing. All new clutches of chicks seroconverted by 80 days of age, and the titres of adult birds showed a concurrent rise, suggesting that the chicks were amplifying the virus. The modes of spread and of persistence of the virus were not determined; although cloacal swabs were taken regularly, only one yielded virus. Antibody titres of the inoculated birds remained above the presumptive protective level of 3 (log2) for over a year, whereas the titres of birds infected by contact were generally less than 3.     

Effects of chemically or virus-induced immunodepression on response of chickens to avian leukosis virus

The effects of chemically or virus-induced immunodepression on the infection profile (development of viremia and antibody) and shedding of avian leukosis virus (ALV) were studied in progeny chickens of experimental or commercial breeder flocks. Chickens were infected with ALV subgroup A by contact at hatching and by oral inoculation at 4-5 weeks of age. In the first experiment, chickens were inoculated with a virulent strain of infectious bursal disease virus (IBDV) at 1 day or 6 weeks of age. In the second experiment, chickens were neonatally treated with cyclophosphamide (CY), or were inoculated with strain T of reticuloendotheliosis virus (REV) at hatching, or were inoculated with strain JM of Marek's disease virus (MDV) at 2 weeks of age. The infection profile and cloacal shedding of ALV in chickens exposed to ALV and inoculated with immunodepressive viruses or CY were compared with those in hatchmates exposed only to ALV. In two of four chicken lines tested in the first experiment, shedding of ALV, as determined by virological assays of cloacal swabs at 22 weeks of age, was significantly higher in chickens infected with IBDV at 1 day of age than in uninfected hatchmates. The rate of shedding of ALV in one of these two lines was also significantly higher in chickens infected with IBDV at 6 weeks of age than in uninfected chickens. Further, the frequency of ALV-antibody detection at 22 weeks of age was significantly lower in chickens of these two lines infected with IBDV at 1 day of age than in uninfected chickens. In the second experiment, neonatal treatment with CY significantly increased the frequency of viremic chickens of both experimental and commercial flocks. The frequency of ALV-viremic chickens at 22 weeks of age was considerably higher in the REV- and MDV-inoculated groups (54% and 44%, respectively) than in control hatchmates (29%), but only in chickens of the commercial line. These findings suggest that chemically or virus-induced immunodepression may lead to an increase in rates of viremia and shedding of ALV in chickens infected with virus after hatching, especially in certain genetic lines.     

新城疫LaSota活疫苗对基因Ⅶ型分离株的免疫保护性试验

从山东省发病鸡群分离鉴定了一株新城疫病毒(NDV),命名为SDLY01。经蚀斑纯化后进行毒力测定和序列分析表明分离株SDLY01属于基因Ⅶ型NDV强毒。20只7日龄SPF鸡免疫新城疫活疫苗LaSot a后14 d分别用NDV标准强毒F48E8和分离株SDLY01攻毒,同时设同日龄SPF鸡为对照组,未免疫任何疫苗。攻毒后观察10 d,免疫组在攻毒后食欲、精神均正常;对照组在攻毒后2～4d发病死亡,并表现ND典型的临床症状和病理变化。攻毒后第3、5、7、9 d对免疫组试验鸡取喉头、泄殖腔棉拭进行病毒分离,F48E8攻毒组病毒分离均为NDV阴性,SDLYO1攻毒组第5 d病毒分离NDV阳性,第3、7和9d病毒分离阴性。本研究结果表明LaSot a活疫苗对F48E8和SDLY01均能提供100%免疫保护,但不能完全抑制基因Ⅶ NDV分离株在体内的复制和排毒。     

Pathogenesis of West Nile virus lineage 1 and 2 in experimentally infected large falcons

West Nile virus (WNV) is a zoonotic flavivirus that is transmitted by blood-suckling mosquitoes with birds serving as the primary vertebrate reservoir hosts (enzootic cycle). Some bird species like ravens, raptors and jays are highly susceptible and develop deadly encephalitis while others are infected subclinically only. Birds of prey are highly susceptible and show substantial mortality rates following infection. To investigate the WNV pathogenesis in falcons we inoculated twelve large falcons, 6 birds per group, subcutaneously with viruses belonging to two different lineages (lineage 1 strain NY 99 and lineage 2 strain Austria). Three different infection doses were utilized: low (approx. 500 TCID50), intermediate (approx. 4 log10 TCID50) and high (approx. 6 log10 TCID50). Clinical signs were monitored during the course of the experiments lasting 14 and 21 days. All falcons developed viremia for two weeks and shed virus for almost the same period of time. Using quantitative real-time RT-PCR WNV was detected in blood, in cloacal and oropharyngeal swabs and following euthanasia and necropsy of the animals in a variety of neuronal and extraneuronal organs. Antibodies to WNV were first time detected by ELISA and neutralization assay after 6 days post infection (dpi). Pathological findings consistently included splenomegaly, non-suppurative myocarditis, meningoencephalitis and vasculitis. By immunohistochemistry WNV-antigens were demonstrated intralesionally. These results impressively illustrate the devastating and possibly deadly effects of WNV infection in falcons, independent of the genetic lineage and dose of the challenge virus used. Due to the relatively high virus load and long duration of viremia falcons may also be considered competent WNV amplifying hosts, and thus may play a role in the transmission cycle of this zoonotic virus.     

Sequential mitogen stimulation of peripheral blood lymphocytes from chickens inoculated with infectious bursal disease virus

Chickens were inoculated wih the pathogenic Edgar strain of infectious bursal disease virus at 1 week, 2 weeks, or 1 day of age. In the 3 experiments, phytohemagglutinin stimulation of peripheral blood lymphocytes was significantly decreased on day 3 or 4 after inoculation. Subsequently, on days 7 through 21, stimulations were similar between lymphocytes from inoculated birds and those from control birds. Pokeweed mitogen stimulation was affected minimally in virus-inoculated chickens. In each experiment, on day 7, the spontaneous [3H]thymidine uptake was greater in nonstimulated lymphocyte cultures from inoculated chickens than in such cultures from control chickens. In an additional experiment, chickens 1 week of age were exposed to a pathogenic vaccinal virus given in their water. The vaccinal virus exposure resulted in significant decrease of phytohemagglutinin stimulation of lymphocytes on days 3 and 7 of the experiment. A significant decrease in pokeweed mitogen stimulation was observed on day 10 after inoculation.     

Characterization of Newcastle disease virus (avian paramyxovirus-1) isolated from pigeons

Newcastle disease virus (avian paramyxovirus-1) was isolated from pigeons in 12 states between May 1984 and December 1985. One of the isolates was from a feral pigeon; the remainder were from privately owned pigeon lofts. Use of monoclonal antibodies showed seven of the eight isolates tested to be indistinguishable from the 1982 and 1983 Great Britain and European isolates. Clinical signs were paralysis, torticollis, tremors, incoordination, and death. Pigeons inoculated with the paramyxovirus-1 isolates intravenously or intramuscularly developed clinical disease identical to that described for natural infection; however, only one pigeon inoculated intranasally developed clinical signs. The mean death time for inoculated pigeons was 9.5 days, with a range of 4 to 25. Virus was shed for up to 20 days. Primary lesions observed on necropsy were gastroenterocolitis and pancreatic necrosis. Chickens experimentally infected by the cloacal, intranasal, or caudal thoracic air-sac route remained healthy. However, the intracerebral pathogenicity index (ICPI) in day-old chickens was similar to that observed with velogenic Newcastle disease virus isolates. Four of six isolates inoculated intravenously into 6-week-old chickens induced neurotropic disease.