Infectious anemia caused by a parvovirus-like virus in Georgia broilers

Pale chicks with necrotic dermatitis, small bursas of Fabricius (BFs), small thymuses, pale bone marrow, and watery blood were suspected of having parvovirus-like virus- (PVLV) associated disease. Histologic lesions included atrophy or hypoplasia of thymuses and BFs, and septic necrotizing clostridial dermatitis and hepatitis. Clostridium perfringens was cultured from skin and liver. A PVLV was isolated in a Marek's disease tumor cell line (MDCC-MSB1) culture and was identified by physicochemical, immunofluorescent, and morphologic features. This isolate was named GA-1 PVLV. Specific-antibody-negative chicks and embryos infected with heat- or chloroform-treated GA-1 PVLV developed anemia at the same rate. Control chicks never were anemic. This is the first isolation of PVLV from clinically ill chickens in the United States and the first report of PVLV-induced anemia in chickens in the Western Hemisphere.     

Chicken anemia agent: an electron microscopic study

Particles of chicken anemia agent (CAA) negatively stained with uranyl acetate were found to be 26.5 nm in diameter. The surface detail evident on the particles indicated that the virus capsid was composed of 32 structural subunits arranged as in a class P = 3 icosahedron with a triangulation number of 3. Using mouse monoclonal antibodies to CAA and a gold-labeled goat anti-mouse IgG, CAA-specific structures were observed by thin-section electron microscopy in infected MDCC-MSB1 cells and in thymic lymphocytes from experimentally infected chicks. These consisted of electron-dense, granular, non-membrane-bound nuclear inclusions, which were often ring-shaped, and cytoplasmic accumulations of microtubules. Aggregates of virus-like particles were sometimes observed in the nuclei of infected MDCC-MSB1 cells. The nucleolar involvement that is characteristic of the morphogenesis of parvoviruses was not observed with CAA.     

Viral pathogenesis in chicken embryos and tumor induction in chickens after in ovo exposure to serotype 1 Marek's disease virus

We examined the susceptibility of late-stage chicken embryos to infection with oncogenic serotype 1 Marek's disease virus (MDV 1). Intravenous inoculation of MDV 1 at embryonic day (ED) 16 resulted in significant replication of the virus in embryonic tissues. Within 5 days of virus exposure, pp38 viral antigen (pp38) was detected in embryonic bursae and MDV 1 was isolated by plaque assay from the spleens, thymuses, and bursae of embryos. The pathogenesis of MDV 1 after intravenous inoculation at ED 16 was similar to that in chicks exposed to MDV 1 after hatching. In contrast to the response of the embryo to intravenous inoculation, embryos exposed to MDV 1 by the amniotic route did not develop detectable pp38, nor could the virus be isolated from the embryonic tissues by plaque assay. These results show that the route of inoculation of MDV 1 in the embryos is critical for allowing the virus to come in contact with target cells.     

Delayed replication of Marek's disease virus following in ovo inoculation during late stages of embryonal development

Several oncogenic and non-oncogenic isolates of Marek's disease virus (MDV) were inoculated into embryonated eggs on embryonation day (ED) 16 to 18, and embryos or chicks hatching from inoculated eggs were examined for infectious virus and viral internal antigen (VIA) in lymphoid organs. There was no evidence of extensive replication of MDV in any of the embryonic tissues examined. Levels of VIA peaked 4-5 days after chicks hatched. This indicated that MDV remained inactive during embryonation and did not initiate pathogenic events until chicks hatched. Because HVT replicated rapidly in the embryo but MDV did not, in ovo inoculation of HVT simultaneously with oncogenic MDV or several days after MDV resulted in significant protection (P less than 0.025) of hatched chicks against Marek's disease (MD). Little protection was obtained if HVT was given simultaneously with MDV or after MDV to chicks already hatched. The relative susceptibility of the embryo to extensive replication of the vaccine virus but not the challenge virus apparently accounted for protection against MD in chicks hatching from dually infected eggs.     

应用PCR从AA肉种鸡鸡胚和1日龄雏鸡体内检测出CIAV和ALV-J

为了了解鸡传染性贫血病病毒（CIAV）和J亚群禽白血病病毒（ALV-J）对AA肉种鸡鸡胚和1日龄雏鸡的感染情况,试验直接从山东省3个不同AA肉种鸡场的鸡胚和1日龄雏鸡的肝、脾、肾、胸腺、骨髓、法氏囊等组织（器官）提取DNA,进行了PCR扩增及PCR产物的克隆和序列测定。结果显示,被检的3个肉种鸡场的鸡胚和1日龄雏鸡体内均有这2种病毒核酸的检出,其中CIAV的阳性率是20.42%,ALV-J的阳性率是15.83%,二者共感染的阳性率是6.25%;在阳性检出率中弱雏〉死胚〉健康雏〉正常胚。病毒核酸在各感染组织（器官）的含量也有所差异,CIAV以脾的含量最多,ALV-J以肾的含量最多。对肝进行细菌分离鉴定时发现,3个鸡场还存在大肠杆菌、沙门氏菌和霉形体的混合感染。结果提示,在AA肉种鸡鸡胚和雏鸡体内存在CIAV、ALV-J的感染和二者的共感染以及继发性大肠杆菌、沙门氏菌和霉形体的混合感染。     

Relationship of common avian pathogen antibody titers in so-called chicken anemia agent (CAA)-antibody-positive chicks to titers in CAA-antibody-negative chicks.

Antibody titers for infectious bursal disease virus (IBDV), infectious bronchitis virus, Newcastle disease virus, and reovirus from chicks with chicken anemia agent (CAA) antibodies were compared with antibody titers from their CAA-antibody-negative counterparts. These comparisons were made in 396 chickens that were 1 day, 2 weeks, 8-9 weeks, 10 weeks, 17 weeks, or 29-32 weeks old. Only one serum sample was collected from any given chick or chicken. There were no significant differences between the antibody titers at any age for any antigen, with one exception: at 29-32 weeks, the IBDV titers were higher (t = 2.62, df = 142, P less than 0.01) in chickens with CAA antibody. Although not at all likely, we believe that the observation of high IBDV antibody titers in CAA-antibody-positive chicks could have been a spurious one.     

Detection of chicken anemia virus DNA in embryonal tissues and eggshell membranes

Chicken infectious anemia virus (CIAV) is a ubiquitous and highly resistant virus of chickens that causes anemia and death in chicks less than 3 wk of age and immunosuppression in chickens older than 3 wk of age. The production of specific-pathogen-free eggs free of CIAV is essential for research and vaccine production. Currently, flocks are screened for CIAV by antibody tests to ensure freedom from CIAV infection. Recent evidence, however, indicates that chickens may carry and vertically transmit CIAV DNA independently of their antibody status. In this study, we tested embryos and eggshell membrane residues by nested polymerase chain reaction (PCR) as a sensitive method of detecting CIAV DNA. CIAV DNA could be detected in the blastodisks and semen obtained from antibody-positive and -negative chickens. Examination of different tissues between 18 and 20 days of incubation indicated that many but not all organs of individual embryos were positive. The lymphoid organs and gonads had the highest incidence of CIAV DNA, which was significantly different (P < 0.05) from the incidence in the liver. Eggshell membrane samples from embryos or newly hatched chicks were an excellent noninvasive source for the detection of CIAV DNA, identifying significantly more positive embryos than did pooled lymphoid organs. The use of dexamethasone injections as a method to improve the detection of carrier birds did not result in an increase of vertical transmission or cause seroconversion in the treated hens. A combination of testing eggshell membrane residues at hatch and periodic testing of blood DNA by nested PCR can be used to identify chickens carrying CIAV DNA and may be used to eradicate carrier birds.     

Avian leukosis virus subgroup J infection profiles in broiler breeder chickens: association with virus transmission to progeny

Profiles of infection with avian leukosis virus subgroup J (ALV-J) and factors that predict virus transmission to progeny were studied. Eggs from an infected broiler breeder flock were hatched at the laboratory. The flock was reared in a floor pen, transferred to laying cages at 22 wk, and inseminated to produce fertile eggs. A cohort of 139 chickens was tested at frequent intervals over a 62-wk period for virus, viral antigens, or antibodies in plasma, cloacal swabs, egg albumen, and embryos. Virus was detected in 7% of chicks at hatch but spread rapidly so that virtually all chicks became infected between 2 and 8 wk of age. Mortality due to myeloid leukosis and related tumors was 22%. Over 40% of the chicks developed persistent infections, whereas the remainder experienced transient infections. Five types of infection profiles were recognized. Novel responses included hens that were positive for virus intermittently or started late in life to shed viral antigens into the cloaca. ALV-J was isolated from 6% of 1036 embryos evaluated between 26 and 62 wk. However, over 90% of the virus-positive embryos were produced between 29 and 34 wk of age. Of 80 hens that produced embryos, 21 produced at least one infected embryo and were identified as transmitters. All but one transmitter hen would have been detected by a combination of viremia, cloacal swab, and albumen tests conducted between 18 and 26 wk. However, virus was transmitted to embryos from hens that were not persistently viremic or that rarely shed viral group-specific antigen into the albumen of their eggs. Intermittent patterns of both antigen shedding and virus transmission to embryos were observed in some hens. These results validate current screening procedures to identify potential transmitter hens and provide some suggestions for improvement but also show that identification of all transmitter hens by such procedures is unlikely. Thus, eradication programs based solely on dam testing may be less effective than those where dam testing is combined with procedures to mitigate early horizontal transmission in progeny chicks.     

Diagnoses of infections by the so-called chick anemia agent: anemia and direct transmission electron microscopic detection of virus.

Blood from 48 chicks was examined for anemia (packed cell volume), and plasma was examined for virus particles by direct transmission electron microscopy (DTEM). There was agreement between the occurrence of anemia and the presence of CAA virus particles in plasma from anemic chicks (Kappa = 0.2425, Z = 2.096, P = 0.036). Although DTEM is a method that can be used to diagnose CAA in chicks, more sensitive, economical and less laborious diagnostic assays are needed.     

Response of embryonic chicken lymphocytes to in ovo exposure to lymphotropic viruses.

OBJECTIVE: To examine effects of virus exposure on embryonic lymphoid organ structure, apoptosis, and lymphoid cell subpopulations. ANIMALS: Eggs of specific pathogen free (SPF) White Leghorn chickens at embryonation day (ED) 17. PROCEDURES: Eggs were inoculated with 2,000 plaque-forming units (PFU) of serotype 1 herpesvirus (Marek's disease virus [MDV 1]), 2,000 PFU of herpesvirus of turkeys (MDV 3), or 1,000 embryo infectious doses (EID50) of infectious bursal disease virus (IBDV). On post-inoculation days (PID) 3 and 5, lymphoid organ to body weight ratios were determined, and bursa of Fabricius, thymus, and spleen were evaluated for lesions and apoptosis. Proportions of lymphoid cell subpopulations of PID-3 chicken embryos and 7- to 10-day-old chicks were quantitated by flow cytometry. RESULTS: Lymphoid organ weights were similar in virus-free, MDV1, and IBDV groups. Embryos inoculated with 2,000 PFU MDV 3/egg had lower bursal weights than virus-free controls. In a repeated trial, MDV 3 (1,000 PFU to 4,000 PFU) did not reduce bursal weights among groups. Histologic changes were seen in bursae after MDV 1 and IBDV inoculation. Apoptosis was greater in bursae of MDV 1-infected embryos than controls. Lymphoid cell subpopulations were similar among all groups with the exception of CD8+ and IgM+ cells in spleens of IBDV-infected 10-day-old chicks. CONCLUSIONS AND CLINICAL RELEVANCE: Infection with pathogenic strains of MDV 1 and IBDV did not alter lymphocyte subpopulations in embryos or cause complete destruction of lymphoid organs. Changes in lymphoid cell subpopulations exposed as embryos to IBDV were seen only after hatching.     

Effect of Rous associated virus number 7 on lymphoid cells and tissues of the chicken

Infection of chicks or chick embryos with Rous associated virus number 7 (RAV-7) led to a decreased blastogenic response to Concanavalin A (Con A) by lymphocytes isolated from the spleen and thymus. Chicks infected with RAV-7 8 days after hatch manifested decreased Con A blastogenesis 5 weeks postinfection, while chicks infected in ovo at 10 days of incubation showed an unusual pattern of cell density dependent decreased blastogenesis two weeks post-hatch (three weeks post-infection). Histopathological examination of tissues from RAV-7 infected chicks revealed evidence of lymphoid organ involution and widespread lymphoproliferative lesions by 3 weeks of age. The combination of decreased in vitro lymphoid blastogenesis and in vivo lymphoproliferation suggests that RAV-7 interacts with lymphocytes in a fashion that has not previously been described in the chicken.     

Biological characterisation of Australian isolates of chicken anaemia agent

Three Australian isolates of chicken anaemia agent (CAA) resisted treatment at 70 degrees C for 5 min and chloroform treatment. Although minor antigenic differences were detected using monoclonal antibodies to CAA, the Australian isolates were indistinguishable from the reference Cux-1 and Gifu-1 isolates in cross-immunofluorescence and cross-neutralisation tests employing polyclonal chicken antiserums. The Australian viruses were pathogenic for intramuscularly inoculated 1-day-old SPF chicks, but were less pathogenic for 7-day-old chicks. Thus the Australian isolates of CAA did not differ significantly in these properties from previously characterised CAA isolates from other continents.     

Horizontal transmission of a pancreas-passaged avian encephalomyelitis virus in chicks

Pancreas-passaged avian encephalomyelitis (AE) virus was transmitted horizontally in a group of 40 (1-day-old) chicks within 3 weeks after they were intermingled with two orally infected 1-day-old chicks. Viral antigen was detected in the pancreas of these contact-exposed chicks. After 5 weeks, contact-exposed chicks developed high titers against AE virus, but the chicks did not develop clinical signs of AE. The passaged virus could not be recovered from feces of six immunized chicks.     

Clinical epidemiologic and experimental evidence for the transmission of Newcastle disease virus through eggs

Sporadic outbreaks of Newcastle disease (ND) occurred in Taiwan during 1998-2000. In some cases, the disease occurred in broilers less than 2 wk old that originated in a broiler breeder farm, so spread of the ND virus (NDV) from the infected breeder farm to broiler ranches was suspected. The purpose of the present study was to examine the possibility of the transmission of NDV through eggs. Both clinical and experimental evidence were used to prove that this is possible. From epidemiological investigation, the possibility of transmission through eggs was suggested in two separate ND cases from a breeder farm and its progeny because two identical NDVs were isolated from both cases. In order to clarify the possibility of the transmission through eggs, one mean egg lethal dose (ELD50) of NDV was inoculated into the allantoic cavity of 155 9-to-11-day-old specific-pathogen-free (SPF) chicken embryos. Seventy-one hatching chicks from the inoculated embryos were raised for 14 days. The cloacal swabs from those chicks at the ages of 1, 4, and 7 days and the tissues after necropsy at the ages of 14 days were taken for virus isolation. The same NDV was reisolated from three hatching chicks. This experiment confirms that a few chicken embryos infected in ovo with a low titer of NDV can hatch and contain NDV after hatching, which results in NDV spreading through eggs.     

The early pathogenesis in chicken inoculated with non-pathogenic serotype 2 Marek's disease virus.

A newly cloned serotype 2 Marek's disease virus (MDV), strain ML-6, was inoculated via the nasal cavity in specific-pathogen-free chicks to examine early virus replication and the expression of Marek's disease (MD)-related antigens. Following inoculation, viral intracellular antigens (VIAs) were detected in lymphoid organs (bursas and spleens) between 5 and 14 days post inoculation (PI), in feather follicles between 14 and 30 days PI, and in lungs at 3 days PI by the immunohistopathological staining of avidin-biotin-peroxidase complex method. But, very few VIAs were expressed in the thymuses between 5 and 14 days PI. However, MD tumor-associated surface antigens were not detected in any organs. Viruses were isolated from separated spleen cells at 14 and 30 days PI. Fluorescent antibodies of convalescent sera were also detected after 10 days PI. As most of the VIAs were detectable in B-cells in bursas and spleens. B-cells were considered to be the main first target cells for the serotype 2 MDV infection.     

The effects of age, route of exposure, and coinfection with infectious bursal disease virus on the pathogenicity and transmissibility of chicken anemia agent (CAA)

Specific-pathogen-free (SPF) chickens were inoculated with several different concentrations of chicken anemia agent (CAA) by the intra-abdominal, intratracheal, or oral routes. Based on lowered hematocrit values, the birds were most susceptible to CAA introduced by the intra-abdominal route. When SPF chickens were infected with infectious bursal disease virus (IBDV) at 1 day of age, they remained susceptible to CAA up to at least 21 days, whereas birds inoculated with CAA alone were susceptible only at 1 day of age. Infectious bursal disease virus introduced at 1 day of age also increased the susceptibility of birds to contact infection with CAA and resulted in increased mortality rates in CAA inoculates. The response of SPF birds to CAA infection varied following exposure at 1 day of age to two different strains of IBDV (STC and Variant-E). Chicken anemia agent contacts and inoculates infected with the Variant-E strain were affected 1 week earlier by CAA than by STC inoculates, as evidenced by depressed hematocrits. However, the total number of birds affected was similar for both the Variant-E and STC-inoculated chickens. Commercial broiler chickens inoculated at 1, 7, 10, and 14 days of age by non-parenteral routes with CAA or a combination of CAA and IBDV had mean hematocrits that were lower than controls. Several CAA-inoculated birds were considered anemic, with hematocrit values of 25 or less, while uninoculated birds remained within normal ranges.     

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.     

Chicken anemia agent in the United States: isolation of the virus and detection of antibody in broiler breeder flocks

Chicken anemia agent (CAA) was isolated from broiler chickens in Texas with a blue wing or anemia dermatitis-like syndrome. Specific-pathogen-free chicks inoculated with field material developed anemia, and CAA was isolated in MDCC-MSB1 cells from bone marrow and lymphoid tissue from inoculated chicks. One isolate, designated EF88/78/276, was further characterized. Infectivity of EF88/78/276 was resistant to treatment with chloroform and with heat at 70 C for 5 minutes. EF88/78/276 was indistinguishable from the Cux-1 and Gifu-1 isolates of CAA by cross-neutralization tests. Almost all 1-day-old susceptible chicks inoculated intramuscularly with EF88/78/276 developed anemia, but contact-infected chicks did not. Antibody to CAA was detected in broiler breeder flocks from Texas, the Delmarva peninsula, and Alabama.     

Effect of infectious bursal disease on the response of chickens to Mycoplasma synoviae, Newcastle disease virus, and infectious bronchitis virus.

At 35 days of age, chickens which as 1-day-old chicks were inoculated with the infectious bursal disease virus (IBDV) had significantly lower antibody titers against Mycoplasma synoviae, Newcastle disease virus, and infectious bronchitis virus than did those never inoculated with IBDV. The IBDV also had a marked effect on the development of air-sac lesions. Birds infected with IBDV that were later inoculated with M synoviae (day 14), Newcastle disease virus (days 14 and 28) experienced an increased incidence and greater seversity of airsacculitis than did chicks which were not exposed to IBDV.