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.     

Immunopathologic investigations with an attenuated chicken anemia virus in day-old chickens

The immunopathologic effects induced by two attenuated chicken anemia virus (CAV) isolates, known as cloned isolate 34 (CI 34) and cloned revertant isolate 18 (CRI 18), that were derived from highly passaged pools of Cux-1 CAV isolate, were compared with those induced by a pathogenic, molecularly cloned, low-passage Cux-1 isolate (CI Cux). This comparison involved the intramuscular inoculation of 1-day-old specific-pathogen-free chicks with each of the viruses and investigation of birds at selected days postinoculation for gross pathology and depletions in the thymic T-cell populations as determined by flow cytometry. Whereas infection with the pathogenic CI Cux produced severe anemia and pronounced bone marrow and thymus lesions, infections with the attenuated CRI 18 and CI 34 isolates produced no anemia, no or mild lesions, respectively, and moderate T-cell depletion. The results suggest that, with CAV, reduced pathogenicity for 1-day-old chicks correlates with reduced depletion of T-cell populations in the thymus and with reduced severity of lesions in the thymus and bone marrow.     

Identification and virulence characterization of fowl adenoviruses in Korea

Since 2007, 55 adenovirus strains have been isolated from commercial chicken flocks in Korea and have been identified and the pathogenicity of these isolates was confirmed in specific-pathogen-free chickens of different age. Based on sequencing analysis of the hexon gene, 55 FAdV isolates were genetically related to the IBH-2A strain of FAdV3 (4 isolates, 99.2% to 100%), the KR5 strain of FAdV4 (22 isolates, 97.9% to 99.2%), the 764 strain of FAdV9 (11 isolates, 99.1% to 99.3%), and the 1047 strain of FAdV11 (18 isolates, > 99%). Experimental infections with four serotypes of FAdV resulted in high mortality of 18-day-old chicken embryos and 1-day-old chicks with marked liver necrosis similar to those observed in the natural outbreaks. Notably, specific hydropericardium was observed in chicks challenged with the K531 strain (serotype 4). However, 3-wk-old chickens challenged with FAdVs, regardless of serotype, did not show any clinical signs or mortality except histologic lesions of focal hepatocytic necrosis with mild lymphocytic infiltration. The results indicate that four FAdV serotypes (3, 4, 9, and 11) are the dominant serotypes of FAdVs in the Korea and are pathogenic enough to cause clinical disease in young chicks. The present investigation provides important information on the epidemiology and pathogenesis of FAdVs and highlights the importance of control strategies against FAdV infection in Korea.     

Isolation and identification of chicken infectious anemia virus in Brazil.

Seven chicken infectious anemia virus (CIAV) isolates were obtained from seven broiler flocks with poor performance in two states of Brazil. All isolates induced thymus atrophy, bone-marrow aplasia, and low hematocrit values when inoculated into 1-day-old susceptible chicks. The CIAV isolates were resistant to treatment with chloroform and were able to pass through 50-nm-pore-size filters. CIAV-specific antigens could be demonstrated in tissues of experimentally infected chicks using a monoclonal antibody specific for CIAV. These characteristics of the virus and the virus-induced lesions demonstrate that CIAV is present in Brazil and that the virus is associated with production problems.     

A paramyxovirus of serotype 3 isolated from African and Australian finches

Two virus isolates were obtained from exotic finches (Ortygospiza atricollis and Poephila cincta) suffering from apathy, diarrhea, conjunctivitis, and dysphagia. The isolates were identified as paramyxoviruses based on their multiplication characteristics in embryonating chicken eggs, chicken embryo fibroblasts, and chicken embryo kidney cell cultures, on morphology upon electron microscopy, and on other biological properties. Both isolates were serologically related to the reference strain of the paramyxovirus serotype 3. Intravenous infection of 42-day-old chicks resulted in no clinical signs, but intracerebral infection of 1-day-old chicks resulted in mortality and intracerebral pathogenicity indices of 0.25 to 0.35. Of five finches from various species inoculated with isolate 840/85, three remained clinically healthy through 6 weeks, but two died: one (Poephila cincta) 5 days postinoculation after showing nervous distress, and the other (Amandava amandava) suddenly 42 days postinoculation.     

Pathogenicity of CL-1 chicken anemia agent.

Twelve-day-old broiler-type chickens had hemorrhagic necrotic wing tips. After 10 blind subcultures in an MDCC-MSB1 cell line, a virus (so-called chick anemia agent [CAA]) was isolated and designated CL-1 CAA. Five-day-old specific-pathogen-free chicken embryos from a commercial breeder flock that were found not to possess antibody against CAA were infected with CL-1 virus via yolk-sac injection. Many (49%) infected embryos were small and apparently had died from severe systemic hemorrhage. Hatched chicks were small and had pale feathers, skin, skeletal muscles, bone marrow, and viscera. All infected chicks had small thymuses. These thymuses often were so small that they could not be found grossly (P = 0.002). Anemia occurred within 4 days post-hatch. Microscopically, all hematopoietic organs were markedly atrophic. Septic necrotizing lesions were seen only in organs from CL-1-injected chicks. Physicochemical and pathological characteristics of this virus indicate that it is similar to other isolates of CAA found in Europe and Japan.     

Virulence factors of avian Escherichia coli

A total of 45 strains of Escherichia coli isolates from chickens with colisepticemia were examined for virulence factors commonly found in pathogenic groups of E. coli. These strains were studied for the following: pathogenicity in 1-day-old chicks; toxin, hemolysin, and colicin production; cell invasiveness and adherence; hemagglutination for fimbriae detection; serum resistance; aerobactin production in iron-limited conditions; and plasmid content. The characteristics exhibited by virulent strains were invasion for HeLa and chicken fibroblast cells, serum resistance, colicin V, and aerobactin production. None of the isolates were toxigenic or positive in hemagglutination tests. The molecular genetic studies of the virulence factors by agarose electrophoresis showed that the plasmids of these strains are of high molecular weight.     

Comparisons of packed cell volumes (PCVs) from so-called chicken anemia agent (CAA; a virus)-free broilers to PCVs from CAA-free specific-pathogen-free leghorns.

Packed cell volumes (PCVs) from 3-, 7-, 14-, 21-, 28-, and 35-day-old clinically healthy chicken anemia agent (CAA)-free and specific-pathogen-free (SPF) leghorn chicks were compared with PCVs from age-matched clinically healthy CAA-free broiler chicks. The PCVs of the SPF chicks regressed significantly (F = 20.6, df = 2/3, P < 0.025) on age in a linear fashion. The PCVs of the broiler chicks regressed significantly (F = 9.56, df = 2/3, P < 0.05) on age in a cubic parabola. The mean PCVs of the broiler chicks were significantly (P < 0.05) higher than PCVs of SPF chicks at every tested time interval. Results indicate that PCV values are higher in broiler chicks than in SPF leghorn chicks, and that PCVs increase as chicks age. Clinicians, diagnosticians, and investigators who intend to work with chicken blood must be aware of these differences.     

Vaccination against infectious anemia of poultry (CAA)--results of field studies]

Infectious anemia of poultry is a disease of high economical significance. Connatal infection of chicks with the chicken anemia agent (CAA) via the embryonated egg causes anemia along with severe immunosuppression, thus rendering the chicken susceptible for secondary infections. In order to prevent infection of young chicks, it is necessary to induce immunity against CAA in parent flocks, with the aim to prevent connatal spread of the infection and provide maternal protection for baby chicks. In this publication, the efficacy and use of a live CAA vaccine is reported. From autumn 1986 until summer 1990, 3 experimental vaccine charges were applied in 85 broiler parent flocks with totally 3.1 million chickens. In addition, totally 293,000 broiler breeder and 171,000 layer breeder chicken were vaccinated in 1989/90. The vaccine was administered between the 13th and 19th week of life by drinking water without adverse effect to the birds. Chicken anemia symptoms were observed only at the begin of laying period in two parent flocks. These flocks had been vaccinated in the 17th and 19th week, respectively. The offsprings of all other vaccinated parent flocks remained free of chicken anemia. Day-old chicks derived from vaccinated parent flocks were protected against CAA challenge infection. It is emphasized, that vaccination should be performed within the 13th to 15th week of life, because according to our observations, this will lead to an immediate seroconversion.     

Genetic variations in maternal transfer and immune responsiveness to infectious bursal disease virus

The immune responsiveness to infectious bursal disease virus (IBDV) in four native and crossbred chicken lines was compared. ELISA IBDV antibody titers in hen serum samples, yolk from matched eggs and sera from matched 1-day-old chicks from each chicken line with an identical vaccination program were measured, and plotted. There was considerable variation between lines in the measured IBDV specific antibodies, in vaccinated parent hens and in the amounts of inherited maternally derived antibodies in both yolk and progeny chicks. Differences in ratios of the inherited antibody level from hen to 1-day-old chicks were also found among different chicken lines. Breed differences in regressions of IBDV antibody levels in yolk to that of hen or progeny chicks' sera were also found, so prediction of serum titer of hen and/or progeny chicks from yolk are varied among chicken lines.     

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.     

Production and preliminary characterization of monoclonal antibodies to chicken anemia agent

Mice were immunized with partially purified preparations of the Cux-1 isolate of chicken anemia agent (CAA), and their splenocytes were fused with NSO myeloma cells. Three patterns of staining of CAA-infected cells were recognized when the resulting hybridomas were screened by indirect immunofluorescence (IIF). Hybridomas representative of each staining pattern were cloned, and the monoclonal antibodies (MAbs) were characterized. Type 1 staining was indistinguishable from that produced by polyclonal chicken antisera to CAA. Type 2 staining was confined to large nuclear inclusions. Type 3 staining was predominantly nuclear and granular, and differed from type 1 in being more intense and occurring in a higher proportion of nuclei. Three MAbs producing type 1 staining were predominantly Cux-1-specific by IIF; they also reacted to lower titers with the Gifu-1 isolate but not at all with three other CAA isolates. These MAbs had very slight neutralizing activity against Cux-1. Another MAb giving type 1 staining reacted with all CAA isolates tested to high titers in IIF and neutralization tests. MAbs with type 2 and type 3 staining reacted by IIF with all CAA isolates tested but possessed no neutralizing activity. The availability of MABs to CAA should facilitate development of diagnostic tests for the virus.     

Serologic and pathogenetic studies on avian reoviruses isolated in Japan

Eighty-nine avian reoviruses isolated from diseased and clinically normal chickens were classified serologically using antisera against five prototype strains. Eighty-three strains were classified into five serotypes; six strains were untypable. Most of the cytopathogenic strains that produced a clear cytopathic effect (CPE) in chicken embryo fibroblasts (CEFs) were highly pathogenic for chicken embryos (80% or more mortality via the allantoic sac) and for chicks (severe footpad swellings and tenosynovitis). These strains were classified into a single serotype represented by the TS-142 prototype strain. However, 10 strains that could not produce a clear CPE in CEFs showed very low pathogenicity for embryos and chicks, and these strains were serologically different from the TS-142 prototype strain. There was a strong relationship between pathogenicity and serotype. About 17% of the isolates were considered highly pathogenic.     

鸭源新城疫病毒的分离鉴定

从病死肉鸭肝脏中分离到2株病毒(ZH1、ZH2),均能够凝集鸡、肉鸭、绵羊、山羊、猪、人、兔、牛等的红细胞,且这种血凝性可被NDV标准阳性血清所抑制.参照NDV毒力判定标准及其方法对分离毒株ZH1、ZH2进行了鸡胚最小致死量平均死亡时间(MDT)、鸡胚半数感染量(EID50)以及1日龄鸡脑内接种致病指数(ICPI)测定,结果ZH1、ZH2株的MDT为52 h和44 h,EID50为106.4/0.1mL和108.64/0.1mL,ICPI为1.93和1.975.表明这2株分离病毒均为NDV强毒株.     

Experimental infection in specific-pathogen-free chicks with avian reovirus and avian nephritis virus isolated from broiler chicks showing runting syndrome

Avian reovirus (ARV) and avian nephritis virus (ANV) were individually isolated from runty 10-day-old broiler chicks. The ARV isolate, IR-R, the ANV isolate, IR-N, and the reference strain of ANV, G-4260, were inoculated orally into 1-day-old chicks of two specific-pathogen-free (SPF) chicken lines, 151 and PDL-1. Growth retardation without the presence of gross lesions was clearly observed at 7 and 14 days postinoculation (PI) in chicks of both lines inoculated with the IR-R strain. On the other hand, in chicks inoculated with IR-N strain, growth retardation was observed only in the chicks of line 151 at 7 and 14 days PI. Microscopically, nephritis was observed in both chicken lines at 7 and 14 days PI. When chicks that were inoculated with the IR-N strain at 1 day of age were inoculated with the IR-R strain at 3 days of age, growth retardation was observed in the chicks of line PDL-1 at 10 and 17 days PI. However, the growth retardation was less severe than in the group receiving a single inoculation of the IR-R strain.     

Pathogenicity of Campylobacter jejuni for turkeys and chickens.

A Campylobacter jejuni isolate obtained from a turkey liver, designated C101, and a C. jejuni isolate obtained from the feces of a chicken, designated C111, were used to inoculate their respective hosts. Isolate C101 depressed weight gain by 20% when inoculated into newly hatched poults or 4-day-old poults. It also caused death, hepatic necrosis, and generalized hemorrhages in turkey embryos. The chicken-derived isolate, C111, did not reduce weight gain in newly hatched chicks, but it did induce mortality in chicken embryos. The supernatant of the cultures of both C. jejuni isolates also caused mortality in embryos.     

Characterization of Pseudomonas aeruginosa isolates associated with mortality in broiler chicks

In mid-2000, a broiler chicken company in Alabama experienced high early mortality rates in chicks from two different hatcheries. Five isolates of Pseudomonas aeruginosa, obtained from these contaminated hatcheries and resulting broiler chicks with omphalitis, were selected to determine virulence of the bacteria. One-day-old specific-pathogen-free white leghorn chicks were placed into positive pressure isolation units (10 chicks per unit); feed and water were provided ad libitum. The five isolates of P. aeruginosa (1 x 10(1) or 1 x 10(1) colony-forming units/bird) were used to challenge two replicates of 10 chicks via yolk sac inoculation. Two control groups were injected with 0.1 ml of phosphate-buffered saline, and two groups received no treatment. Mortality was recorded daily, and the chicks that died were necropsied and liver and yolk sacs were cultured. After 14 days, the remaining chickens were euthanatized and necropsied. Bacterial isolates retrieved from liver and yolk sacs were identified by the API 20 NE typing system to confirm that they were the same as the challenge isolate. Virulence varied greatly among the isolates, resulting in mortality rates from 0 to 90%. The challenge isolates produced different and often distinctive postmortem lesion patterns. Antibiotic sensitivity tests showed that all five isolates were resistant to sulfisoxazole, ceftiofur, penicillin, lincomycin, bacitracin, oxytetracycline, erythromycin, naladixic acid, and tetracycline. The isolates varied in sensitivity to other antibiotics, but all isolates were sensitive to gentamicin.     

Pathogenicity of infectious laryngotracheitis virus as measured by chicken embryo inoculation

A comparison was made between pathogenicities for chicken embryos of unattenuated and attenuated strains or isolates of infectious laryngotracheitis (ILT) virus. All 11-day-old chicken embryos inoculated with 10(3.0) or 10(4.0) TCID50 of unattenuated strain NS175 via allantoic cavity died within 6 days. On the contrary, no chicken embryos of the same age died when inoculated with the same amount of cell-culture-attenuated isolate C7 in a like manner. The mortality index for chicken embryos (MICE) was obtained by dividing the cumulative number of embryos dying within 7 days by the cumulative number of embryos surviving 7 days. The reliability of the MICE test was confirmed by duplicate and triplicate experiments with strain NS175 and isolate C7. MICE obtained in the experiments with 9 different strains or isolates of ILT virus ranged from 0 to 1, and the values were well correlated with the pathogenicities for chickens. The results from the present work suggest that strains or isolates with MICE less than 0.16 would have low or no pathogenicity for chickens, and those with MICE more than 0.27 would be highly pathogenic. Further studies are needed using additional isolates of ILT virus with varied pathogenicities.     

Isolation and characterization of attenuated plaque variants of infectious bursal disease virus

Attenuated plaque variants were obtained from infectious bursal disease virus adapted to chick embryo cell cultures. The large plaque (Lp) clone and the small plaque (Sp) clone formed homogeneous plaques about 5 and 1 mm in diameter, respectively. Neutralization tests indicated that these clones differed little from their parent strain in antigenicity. Sp clones showed a retarded growth rate in chick embryo cell cultures as compared with Lp clones. The clones were significantly less pathogenic for chick embryos than the parent strain, although Lp clones were more pathogenic than Sp clones, and they were much less pathogenic for 1-day-old chicks and 28-day-old chickens. Both clones had immunizing potency in 28-day-old chickens, although the Lp clone had a somewhat higher potency than the Sp clone. These findings suggest the Lp and Sp clones, in particular the Lp clones, to be useful as live virus vaccine strains.