Isolation and preliminary characterization of chicken anemia virus from chickens in Nigeria

Chicken anemia virus (CAV) was isolated for the first time from the Nigerian chicken population. The virus was recovered from necropsied birds from broiler and pullet flocks that suffered disease outbreaks tentatively diagnosed as infectious bursal disease. A sensitive polymerase chain reaction (PCR) assay detected CAV DNA in tissues of necropsied birds. Restriction endonuclease analysis performed with the 733-bp PCR product and the Cfo I enzyme indicated at least two different CAVs were circulating among the Nigerian chicken population. Four isolates were obtained from pooled liver and thymus tissues using the MDCC-MSB1 cell line. These isolates were found to be antigenically closely related to the Cuxhaven-1 (Cux-1) reference strain of CAV when reacted with four monoclonal antibodies prepared against the Cux-1 virus. One of the isolates (isolate A) induced thymus atrophy, bone marrow aplasia, and low hematocrit values when inoculated into 1-day-old specific-pathogen-free chickens. These findings not only demonstrate that CAV is present in Nigeria, but they also likely represent the first cell culture isolation of the virus in Africa.     

一株鸡传染性贫血病毒对不同日龄SPF鸡的致病性研究

为评价鸡传染性贫血病毒AV1550株的致病性,取1日龄、7日龄和14日龄SPF鸡分别经胸部肌肉注射不同病毒含量的病毒液,同时设置正常对照,隔离饲养观察21日。感染后14日采血测定红细胞压积,21日统计死亡率、体重变化以及胸腺、骨髓、法氏囊病变情况并测定1日龄SPF鸡感染后不同组织中的病毒载量。结果表明,1日龄SPF鸡感染AV1550株后,表现出精神沉郁、增重减缓、贫血等明显的临床症状,死亡率为53.9%;死亡鸡或观察期结束时存活鸡剖检,可见胸腺萎缩,骨髓变成淡黄色;不同剂量感染后14日,均能引起红细胞压积显著下降;21日时,胸腺病毒载量最高,可达106.7copies/mg 。7日龄SPF鸡感染后,出现增重减缓,高感染剂量（100000EID50）出现贫血,部分鸡出现胸腺萎缩和骨髓病变,但病变率低于30%。14日龄SPF鸡感染后,不引起明显临床症状。研究证实,CAV对SPF鸡的致病性具有明显的日龄依赖性,红细胞压积降低、骨髓病变、胸腺萎缩以及胸腺病毒载量测定可作为评价CAV致病的指标。     

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.     

Biological and molecular characterization of chicken anemia virus isolates from Slovenia

The presence of chicken anemia virus (CAV) in Slovenia was confirmed by inoculation of 1-day-old chickens without antibodies against CAV and isolation of the virus on the Marek's disease chicken cell-MSB1 line and by polymerase chain reaction (PCR). Experimental inoculation of 1-day-old chickens resulted in lower hematocrit values, atrophy of the thymus, and atrophy of bone marrow. CAV was confirmed by PCR in the thymus, bone marrow, bursa of Fabricius, liver, spleen, ileocecal tonsils, duodenum, and proventriculus. The nucleotide sequence of the whole viral protein (VP)1 gene was determined by direct sequencing. Alignment of VP1 nucleotide sequences of Slovenian CAV isolates (CAV-69/00, CAV-469/01, and CAV-130/03) showed 99.4% to 99.9% homology. The VP1 nucleotide sequence alignment of Slovenian isolates with 19 other CAV strains demonstrated 94.4% to 99.4% homology. Slovenian isolates shared highest homology with the BD-3 isolate from Bangladesh. Alignment of the deduced VP1 amino acids showed that the Slovenian isolates shared 100% homology and had an amino acid sequence most similar to the BD-3 strain from Bangladesh (99.6%) and were 99.1% similar to the G6 strain from Japan and the L-028 strain from the United States. The Slovenian isolates were least similar (96.6%) to the 82-2 strain from Japan. A phylogeneric analysis on the basis of the alignment of the VP1 amino acids showed that CAV isolates used in the study formed three groups that indicated the possible existence of genetic groups among CAV strains. The CAV isolates were grouped together independent of their geographic origin and pathogenicity.     

Chicken anemia virus in broilers: dynamics of the infection in two commercial broiler flocks

Chicken anemia virus (CAV) can cause a disease syndrome characterized by severe anemia, bone marrow atrophy, and severe immunosuppression in young chicks. Maternal antibodies prevent these clinical signs but do not prevent infection, transmission of the virus, or immunosuppression. The clinical disease is rare today because of the widespread practice of vaccinating breeders, but the subclinical form of the disease is ubiquitous. The dynamics of CAV infection, CAV antibody responses, relative lymphoid organ weights, and associated lesions were studied in two broiler flocks from a commercial producer. Both groups had detectable CAV antibodies at hatch, which waned over the first 3 wk of life. Both groups had detectable CAV DNA in both thymi and bursae over the same period. At 35 days of age, virus was detectable by polymerase chain reaction in 16 of 20 chickens, and 7 of 20 had detectable antibodies. By 42 days of age, virus was detectable in 18 of 20 chickens, and 18 of 20 had antibodies to CAV. We observed a decrease in relative thymic weights beginning at 35 days of age, coincidental withthe detection of CAV in the thymus. Bursal sizes began to decrease at 28 days of age, coincidental with a rise in antibody titers to infectious bursal disease virus. In this study, we demonstrated that under typical field conditions CAV infections in broilers have unique dynamics unlike those reported in egg laying strains of chickens managed under specific-pathogen-free conditions.     

Pathological and immunohistochemical studies of subclinical infection of chicken anemia virus in 4-week-old chickens

Subclinical infection of chicken anemia virus (CAV) at 4 to 6 weeks of age, after maternal antibodies have waned, is implicated in several field problems in broiler flocks. In order to understand the pathogenesis of subclinical infection with CAV, an immunopathological study of CAV-inoculated 4-week-old SPF chickens was performed. Sixty 4-week-old SPF chickens were equally divided into CAV and control groups. The CAV group was inoculated intramuscularly with the MSB1-TK5803 strain of CAV. Neither mortality nor anemia was detected in the CAV and control groups. In the CAV group, no signs were observed, except that some chickens were grossly smaller compared with the control group. Sporadic thymus lobes appeared to be reddening and atrophied. Within the first two weeks p.i. of CAV, there was a mild to moderate depletion of lymphocytes in the thymus cortex and spleen in some chickens. Moreover, lymphoid depletion of the bursa of Fabricius, proventriculus and cecal tonsils was observed. Hyperplastic lymphoid foci were observed in the liver, lungs, kidneys and heart at the 4th week p.i. of CAV. Immunohistochemically, a moderate lymphoid depletion of CD4(+)and CD8(+) T cells in the thymus cortex and spleen was observed in some chickens within two weeks p.i. of CAV. CAV inclusions and antigens were detected infrequently in the thymus cortex and spleen. It could be concluded that the immunosuppression in subclinical infection with CAV occurs as a result of reduction of cellular immunity.     

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.     

PCR和斑点杂交检测鸡传染性贫血病毒

通过聚合酶链反应（ｐｃＲ）扩增鸡传染性贫血病毒（ｃＡＶ）ＤＮＡ特定片段，将此ｐｃＲ产物用Ｄｉｇｏｘｉｇｅｎｉｎ标记后作为探针进行斑点杂交，以此检测ＣＡＶ并作流行病学调查。对从江苏省不同地市随机采集的２月龄左右不同疾病的病鸡ＤＮＡ样品进行检测，在被检的５２个不同鸡群来源的病料中，有３６个鸡群来源的病料ＤＮＡ显示ＣＡＶ阳性（群阳性率为６９．２％）；备组织中ＣＡＶＤＮＡ含量有所差异，依次为胸腺＞脾、骨髓、肝＞肾、法氏囊、脑。用ＰＣＲ检测得到的阳性鸡的组织病料感染ＳＰＦ鸡，在感染后第２４天检查，出现贫血症状。初步调查表明，在江苏一些地区ＣＡＶ感染已相当普遍，但它与发病的关系还有待于进一步研究。     

Oral infection with chicken anemia virus in 4-wk broiler breeders: lack of effect of major histocompatibility B complex genotype

The pathologic consequences of chicken anemia virus (CAV) oral inoculation in 4-wk-old broiler breeders of different major histocompatibility B complex (MHC) genotypes were evaluated. MHC B complex was determined by hemagglutination and sequence-based typing. Clinical signs, serology, gross lesions, histopathologic analysis, and CAV genome quantification were used to evaluate disease progression. Clinical disease was not apparent in the inoculated broilers throughout the experimental period. At 14 days postinoculation, antibodies against CAV were detected in 26.4% (29/110) of the inoculated birds. The distribution of percent positive was 34.6% (9/26) and 32.3% (10/31) of the chickens with B A9/A9 and B A9/A4 MHC genotypes, respectively, and seroconversion in six other genotypes was 19% (10/53). These differences among MHC genotypes for specific seroconversion rate were not statistically significant. CAV genomes were detected in the thymus of 87.7% (93/110) of the inoculated birds with no statistically significant differences between MHC genotypes. Mild thymic lymphocytolysis, lymphedema, and medullary hemorrhage were observed in the inoculated chickens. Histomorphometric analysis showed that cortical lymphocyte-to-parenchyma ratios did not differ between inoculated and uninoculated groups or among MHC genotypes. Similar findings have been reported previously in white-leghorn chickens of similar age, suggesting that broilers show a similar resistance to the effects of CAV infection at this age. The absence of significant clinical and pathological changes in the orally inoculated broilers at this age contrasts with CAV-associated thymus damage seen frequently in condemned commercial broilers at harvest.     

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.     

鸡贫血病病毒感染雏鸡细胞周期的研究

用流式细胞仪对雏鸡感染鸡贫血病毒后不同时期胸腺和骨髓细胞的细胞周期进行了分析，结果显示感染鸡骨髓和胸腺发生了Ｇ１阻滞，这表明ＤＮＡ合成抑制，Ｇ２／Ｍ期细胞数量明显减少，这说明增殖性细胞在减少。本实验结果提示增殖性细胞减少是鸡传染性贫血病（ＣＩＡ）中胸腺淋巴细胞和骨髓造血细胞大量减少的重要原因之一，这为阐明ＣＡＶ的发病机理提供了新资料     

我国白羽肉用型鸡群中CAV、REV和REOV感染状况的血清学调查

为了解鸡传染性贫血病毒（CAV）、禽网状内皮增生病病毒（REV）和呼肠孤病毒（REOV）在我国白羽肉用型鸡中的感染状态，在2003—2004年，检测了来自5省市8个公司不同年龄鸡群血清样品中3种病毒抗体的存在状况。结果表明，在送检的75个鸡群中，对CAV、REV和REOV呈现抗体阳性的鸡群分别有64个（85．3％）、36个（48％）和74个（96％）。在总共检测的1764份血清样品中，对这3种病毒的平均抗体阳性率分别为51．4％、9．8％和75．1％。在1日龄雏鸡，对CAV和REOV的平均母源抗体阳性率可达100％和81．1％，但对REV只有7．4％。抗体阳性率随年龄变化的动态分析表明，对REV和REOV的母源抗体在出壳后2～3周内消失，而对CAV的母源抗体则可持续3～4周。对CAV和REOV的抗体从5周龄起再次出现，到20周龄时，所有送检鸡群全部阳性，平均阳性率在90％以上。有近一半送检鸡群对REV呈现抗体阳性，抗体阳性率普遍较低，即使在达到开产年龄后，仍还有很高比例鸡为抗体阴性，即对REV仍为易感鸡。研究表明，我国多数鸡群中都同时存在着这3种病毒的感染，但它们在感染的程度和动态等流行病学特点上显著不同，应根据鸡群中抗体的阳性率分别采取不同的措施。     

鸡传染性贫血病毒海安分离株感染雏鸡的病原动态分析

雏鸡是鸡传染性贫血病毒（Chickenanemiavirus,CAV）的最易感宿主。为系统了解中国国内CAV海安分离株（HA9805）感染雏鸡后的病原动态及与血清学和致病性的关系,本研究对1日龄SPF鸡进行了人工感染实验,感染后不同时间进行样品采集,并用病毒学、血清学以及组织病理学方法进行了检测与分析。研究结果发现,CAV海安分离株（HA9805）通过胸肌接种可引发雏鸡CAV病毒血症,引起CAV特征性临床症状及病理变化,并导致雏鸡高达13．3％的死亡率;血清中CAV核酸以及中和抗体的产生与组织病理变化以及疾病进程密切相关。该结果为CAV的早期诊断,制定有效的预防与控制措施提供了科学依据。     

鸡传染性贫血病病毒TaqMan实时荧光定量PCR检测方法的建立

根据鸡传染性贫血病病毒(chicken infectious anemia virus,CAV)基因组保守区域设计1对特异性引物和探针,通过优化反应条件,建立了一种快速检测CAV的TaqMan实时荧光定量PCR方法,同时验证其特异性、灵敏性和重复性.本试验建立的TaqMan实时荧光定量PCR方法灵敏度可达1.8×10¹拷贝/μL,远高于常规PCR方法;并与禽类其他病毒性疾病无交叉反应,具有高特异性.用分离的病毒人工感染1日龄SPF雏鸡,14日龄剖检,对感染鸡体内各器官的病毒分布及载量进行检测,结果表明,在脑、心脏、肝脏、脾脏、肺脏、肾脏、胸腺、法氏囊和血清中均可检测到病毒,肝脏、胸腺病毒载量明显高于其他组织.本研究建立的TaqMan实时荧光定量PCR方法特异性强、灵敏度高、重复性好,可同时检测大量临床样品,适用于CAV的诊断与流行病学分析.     

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。     

Hematology and serum chemistry values of the lesser rhea (Pterocnemia pennata) raised in Patagonian farms (Argentina).

Hematologic and serum chemistry values were determined for chicks, juveniles, and adult farmed lesser rheas (Prerocnemia pennata). Blood samples were taken during March-April 1998 from the brachial veins of lesser rhea, including 64 chicks, 24 juveniles, and 36 adults, raised in Patagonian farms (Argentina). Lesser rhea chicks had significantly lower erythrocyte counts, hematocrit, hemoglobin, red cell indices, and copper than did the juveniles and adult individuals. No significant differences were observed between females and males. Most values were similar to those reported for other ratites. The data obtained provide hematologic and serum chemistry values for lesser rhea from farms.     

Genetic characterization of chicken anemia virus from commercial broiler chickens in Alabama.

Chicken anemia virus (CAV) isolates show extremely limited genetic variability worldwide. We determined the nucleotide sequence of an 823-nucleotide portion of the 2.3-kb CAV genome found in 10 liver and/or spleen specimens of Alabama 29-to-49-day-old commercial broiler chickens exhibiting lymphocyte depletion of the thymus submitted to the state diagnostic laboratory because of problems unrelated to anemia. We determined the nucleotide sequence directly from DNA isolated from tissues, without isolation of virus in culture. This procedure enabled us to characterize CAV that might not have replicated in culture and avoided the potential for changes during passage. Results confirmed the limited genetic variability of CAV. All sequences were identical in 93% of nucleotide positions. The sequences encoded only two distinct VP1 hypervariable regions, and both had been found previously in other CAV isolates. A novel amino acid, glutamine, was found at VP1 position 22 in half the sequences, replacing the histidine residue encoded by most previously characterized CAV genomes. We were able to distinguish among CAV genomes with different codons at VP1 amino acid 22 and different hypervariable regions by restriction endonuclease analysis of polymerase chain reaction products.     

用地高辛探针对江苏省鸡传染性贫血病进行流行病学调查

用地高辛标记的核酸（ＤＮＡ）探针对的扬州、江都、连云港、苏州、无锡等５蛭９个养鸡场（户）随机取样１０６份（分别为１２￣６０日龄的肉鸡、蛋鸡和草鸡,采集胸腺、法氏囊和脾脏组织,抽提ＤＮＡ）进行检测ＣＡＶ感染情况,ＣＡＶ阳性鸡２２份阳必率为２０．７％,各鸡场阳性率人５￣６０％不等。结果表明,在处的一些地区鸡群中ＣＡＶ的感染已非常普遍,有的地区甚至十分严重,控制该病的发生已刻不容缓。     

Polymerase chain reaction amplification for direct detection of chicken anemia virus DNA in tissues and sera.

Direct detection of chicken anemia virus (CAV) DNA in tissues and sera was investigated by a polymerase chain reaction (PCR) assay. Using a pair of primers constructed to amplify the coding sequence of the CAV DNA genome, the PCR assay was shown to be extremely sensitive, being able to detect 1 fg of CAV replicative form DNA. The oligonucleotide primers used for the PCR yielded 583 base-pair (bp) amplified product, which was sized by ethidium bromide-agarose gel electrophoresis. Tissue samples from seven cases of suspected chicken infectious anemia were obtained for CAV isolation. DNA extracted from the homogenized suspension of pooled tissues of each case was analyzed by the PCR assay. Results showed that five of the seven cases were positive for CAV DNA by PCR, whereas CAV was isolated from four cases only. The PCR assay also detected CAV DNA in two of 37 serum samples from disease-free chickens. The specificity of PCR was confirmed by chemiluminescence dot-blot analysis of the amplified products.