Pathologic changes in the organs in chickens after infection with the turkey herpesvirus THV-BIO-I]

The objective of our work was to investigate the dynamics of pathological lesions of chicken organs after infection with high doses of turkey herpesvirus THV-BIO-I. This virus strain is commonly used in form of the Marvak vaccine against Marek's disease of poultry in Czechoslovakia. High doses of the vaccine are used in practice with respect to the epizootological situation. The incidence of pathological lesions in the organs of Brown Leghorn chickens was investigated in a five-week experiment. One-day chickens were infected intramuscularly with the HVT strain at the doses of approximately 10(2), 10(3) and 10(4) PFU in 0.2 ml of infective inoculum per chick. The body weights of ten chickens of each group were recorded at intervals of 1, 2, 3 and 5 weeks after infection, serological examination was performed for precipitating antibodies to MDV and the feather was examined for MDV-antigen. Bursae Fabricii and spleens were weighed. Thymus, bursae Fabricii, spleens, peripheral nerves (n. ischiadicus and pl. brachialis) and gonads were sampled for histopathological examination. Neither maternal nor post-infection antibodies were found in any chick. Cytolytic lesion severity of lymphoid organs was scored using the scale of immunosuppression degrees (0-4). Morphological criteria were published in a previous paper (Halouzka and Jurajda, 1991b). The differences observed in the weights of bursa Fabricii and spleen between the infected and control chickens were not statistically significant. The observed lymphoid infiltrations in the skin, gonads, nerves and other tissues following the HVT infection are well-known and correlate with the infection dose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and study of biological properties of non-oncogenic Marek's disease herpesviruses in chickens. 1. Characterization in vitro]

The present paper deals with isolation of nonpathogenic chicken herpesviruses of Marek's disease (MD) and their in vitro characterization. Two strains of herpesviruses (denoted as VUB-M and -K isolates) were isolated from leucocytes separated from the blood of fattening chickens without clinical and pathomorphological symptoms of Marek's disease and from the blood of laying hens of a laboratory nonvaccinated flock without clinical symptoms, but with almost 100% incidence of precipitating MD antibodies. After in vitro adaptation, they were cytopathic for chicken embryonal fibroblasts in three days after infection. Isolates were identified as chicken herpesviruses of Marek's disease on the basis of the origin and nature of cytopathic effect in CEF (Biggs and Milne, 1972), lack of infectivity of culture medium and loss of infectivity of cells after their destruction through repeated cycles of freezing and thawing (Biggs and Payne, 1967), then serologically by agar gel immunodiffusion against specific serum (Chubb and Churchill, 1968) and electronoptically.     

Immunological aspects of Marek's disease in chickens with maternal antibodies]

The dynamics of the production of immunoprecipitation antibodies to Marek's disease virus was studied in the serum of chickens with maternal antibodies in relation to the occurrence of the immunoprecipitation antigens of Marek's disease virus in feather follicles. One-day-old chickens were infected by the contact method with Marek's disease virus. The first occurrence of immunoprecipitation antigen was detected on the 14th day after infection and this occurrence persisted throughout the experiment, i. e. until the 112th day after infection. The antibodies were first detected the 28th day after infection and their titre kept rising until the 98th day after infection. Immunoprecipitation antibodies and antigens of Marek's disease virus were detected in some tumorously changed kidneys. Immunoelectrophoretic examination revealed in the same kidneys immunoglobulins of the class IgY, IgA and beta-globulin. The slowest-migrating fraction of IgY, together with IgA, beta-globulin and C-reactive protein were detected in the skin extracts from infected poultry. Indirect haemagglutination enabled the detection of the presence of haemagglutination antibodies in rabbit immunoglobulin to the skin antigen of Marek's disease virus, and in avian immunoglobulin to the same virus. Haemagglutination antigen was revealed in the extract from tumorously changed kidneys.     

Postvaccination viremia in breeding flocks of layers and broilers after vaccination against Marek's disease of fowl

The immunity state after vaccination against Marek's disease (MD) was studied in three multiplier flocks of laying fowl (MFL), five multiplier flocks of broiler fowl (MFB), and one commercial layer flock (CLF). The occurrence and average titres of post-vaccination viraemia in the selected sets of chickens from these flocks, examined at the age of three or twenty weeks, were used as the immunity criterion. The development of post-vaccination viraemia, following the administration of the MARVAK vaccine at the doses of 100 and 1000 PFU per bird in the HX-SL and Shaver layer hybrids, was examined under laboratory conditions at the same time. In the group of birds examined in the third week of age and coming from the MFL vaccinated with the recommended MARVAK vaccine (dose (100 PFU per bird), 64.3% of the chicks were viraemic, the average viraemia titre being 12 PFU/10(7) leucocytes. After the administration of a four-fold vaccine dose, 57.1% of the chicks were viraemic, the average titre being 3.2 PFU/10(7) leucocytes. After the administration of a ten-fold dose of MARVAK vaccine, 80% of the chicks were viraemic and the average titre of viraemia was 8 PFU/10(7) leucocytes. In the MFB vaccinated with the recommended dose of the MARVAK vaccine, the percentage of viraemic chickens was 48.3% and the average titre of viraemia was 6.5 PFU/10(7) leucocytes. In the pullets examined at the age of 20 weeks the number of viraemic birds ranged from 20 to 50% and the average viraemia titres were from 3.8 to 13.1 PFU/10(7) leucocytes. In the flock affected by acute MD, no post-vaccination viraemia was found in the clinically diseases pullets. In the chickens of the HX-SL line vaccinated with the recommended MARVAK vaccine dose, viraemia culminated in the third week after vaccination (31.5 PFU/10(7) leucocytes), and after the use of the dose ten times as high as the recommended one the culmination came a week later (47 PFU/10(7) leucocytes). In the Shaver chicks vaccinated with the recommended dose or with the ten-fold dose of the MARVAK vaccine, the post-vaccination viraemia culminated in the fourth week after vaccination (94.9 and 116.8 PFU/10(7) leucocytes). The post-vaccination precipitation antibodies were first detected in the eighth week after vaccination.     

Marek's disease in turkeys. II. Characterization of the viral glycoprotein B gene and antigen of a turkey strain of Marek's disease virus

Marek's disease virus (MDV) causes immunosuppression and tumors in chickens. As sporadic cases of Marek's disease (MD) were recorded in turkeys, the antigenic and genomic characteristics of the MDV glycoprotein B (gB) gene and antigen of turkeys were compared to the chicken MDV gB. The whole chicken and turkey gB genes were sequenced and found identical. By immunoblotting of infected-cell culture lysates using chicken convalescent and gB monoclonal antibodies, the antigenic epitopes of the chicken and turkey viruses were found to differ. The turkey MDV had a unique epitope, compared to the chicken MDV and compared with our previous findings. While the chicken MDV had two epitope types, heat-labile but dithiothreitol (DTT)-stable and heat-stable but DTT-labile, the turkey MDV gB epitope is both heat and DTT-labile.     

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.     

Dose-dependent inhibition of virus rescue from lymphocytes latently infected with turkey herpesvirus or Marek's disease virus

The number of plaque-forming units (PFU) of turkey herpesvirus (HVT) isolated per 10(6) latently infected splenic lymphocytes was determined by co-cultivation on permissive monolayer cultures in 35-mm-diameter Petri dishes. Doses of 1 x 10(6) spleen cells or less per culture gave uniform dose-related titers, whereas doses of 8 x 10(6) cells often yielded less than 1-2% of the expected number of PFU. Intermediate doses gave proportionally reduced virus yields. This dose-dependent inhibition was observed with spleen cells from birds within a week after infection and became more marked with time. A similar phenomenon occurred with a non-oncogenic Marek's disease virus (MDV) isolate (SB-1) but not with oncogenic MDV isolates (CU-2, JM-10, GA-5), except in genetically resistant birds. High numbers of uninfected spleen cells mixed with low numbers of HVT-infected cells during assay reduced titers only slightly. Immunosuppression by combined neonatal thymectomy and cyclophosphamide treatment before HVT infection prevented the inhibition, but embryonal bursectomy had no effect.     

Immunopathology of Marek's disease in quails: presence of antinuclear antibody and immune complex.

Antinuclear antibody (ANA), a marker for autoimmune reactions, was detected in the sera of quails with Marek's disease (MD). The autoantibody was detected 3 weeks after infection in quails infected with chicken Marek's disease virus and 4 weeks after infection in quails infected with quail Marek's disease virus. The ANA titers were low and ranged from 10 to 40. A speckled type of nuclear fluorescence was the characteristic staining feature. In addition to the presence of ANA, immune complexes (IC) were also detected in the kidney glomeruli of quail infected with Marek's disease virus. Initially about 25-30% of the glomeruli in the kidneys of infected quails had IC deposits. In subsequent periods, the amount of IC deposit and the number of glomeruli showing IC also increased considerably. The findings of the present study suggested autoimmunity may play a pathogenic role in MD.     

Characterisation of Newcastle disease viruses isolated in India.

Eleven Newcastle disease virus (NDV) isolates obtained from outbreaks of disease in chickens (9) and Japanese quail (2) in Tamil Nadu, India were characterised in pathogenicity tests, antigenically, using mouse monoclonal antibodies (MAbs), and other established tests devised to distinguish between different strains. All 11 isolates were shown to be highly virulent for chickens. In indirect immunoperoxidase tests used to assess the ability of a panel of 28 MAbs to bind to infected cell cultures, 10 of the isolates showed an identical reaction pattern, the other isolate (No. 4) failed to react with one MAb which bound to cells infected with the other isolates. Isolates 9 was unstable at pH 3 while the other 10 were stable. All other properties were shared by the 11 isolates.     

禽脑脊髓炎的调查及病毒分离

对陕西省宝鸡、渭南等地的24个鸡群进行了禽脑脊髓炎（AE）的调查,所调查鸡群都有产蛋下降史或产蛋正在下降,下降幅度平均为17.2%。用琼扩试验（AGP）检测所采集的256份血清,AE阳性率平均高达85.6%。有10个鸡群阳性率达100%。跟踪调查2个种鸡群,发现孵出的雏鸡从3日龄开始发病,而且有AE的症状,采发病雏鸡的脑组织,接种6龄鸡胚分离病毒,连传3代,琼扩检测分离毒,呈AE抗原阳性,人工感染1日龄雏鸡复制出与原发病鸡群相同的疾例,表明分离毒是AE病毒。     

Testing the immunogenicity of the dermal antigen in Marek's disease virus]

An experiment was performed to study the immunogenicity of the dermal antigen of Marek's disease virus, extracted from the skin of 30-day-old chickens, infected with Marek's disease virus on the first day of life. Three kinds of samples were tested: (1) dermal antigen centrifuged at 10 000 g per 0.5 h, (2) dermal antigen centrifugated at 10 000 g per 0.5 h and 100 000 g per 1 h, (3) dermal antigen treated like sample (2) and partly purified by DEAE-cellulose chromatography. Samples (1) and (2) were inoculated to two-day-old chickens and the vaccination was repeated, using complete Freund's adjuvant, 21 days later. Sample (3) was inoculated to two-day-old chickens with DEAE-dextran. All the three groups were challenged together with the controls (non-vaccinated chickens) on the seventh day after the first vaccination. A reduction of mortality was observed in the chickens vaccinated with and re-vaccinated with sample (1) (23.07%) and in the chickens vaccinated with sample (3) (30.76%). The chickens of the latter group were the last to start dying from Marek's disease--only after the 10th week of life. In the chickens which had been vaccinated and revaccinated with sample (2) the mortality was not reduced. The study is continued, with particular emphasis on the relationship of DEAE-dextran to protection against Marek's disease.     

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.     

Protecting chickens against Marek's disease under laboratory conditions by the administration of Marvak, a Czechoslovak produced vaccine and vaccine imported from East Germany

Laboratory trials were conducted to compare the efficiency of two vaccines against Marek's disease (MD) on the basis of the dynamics of post-vaccination viraemia and by means of a challenge test. Two groups of the HX-SL hybrid layers, each containing 40 birds, were vaccinated with the MARVAK cellular vaccine manufactured in Czechoslovakia (100 PFU per chick) or with the Insel Riems vaccine manufactured in the GDR (1000 PFU per chick). In weekly intervals, half the birds of each group were tested for post-vaccination viraemia and the other half, including 20 non-vaccinated birds, were infected by contact with the pathogenic virus of MD from the second day of age. The viraemia in the birds vaccinated with MARVAK culminated in the third week of age when it reached the average value of 25 PFU/10(7) leucocytes; later on, by the eighth week of age, it decreased to 0.62 PFU/10(7) leucocytes. The maximum number of viraemic chicks was found in the third and fourth week of age (80%); on an average it ranged from 60%. The average viraemia in the chickens treated with the vaccine from the GDR ranged from 1.6 to 3.6 PFU/10(7) leucocytes in the second to eighth week of age. A 100% positivity of the tested birds was found in the sixth week of age; it ranged around 70% on an average. In all the challenged groups of birds the precipitation antigen of Marek's disease was detected in feather follicles already 14 weeks from contact infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

鸡马立克氏病三价活疫苗剂量配比和免疫剂量的研究

用马立克氏病病毒BJMDV-1株分别攻击由CVI988/B5、HCV2/B5和FC126/B5毒株组成的,剂量配比不相同的马立克氏病(MD)三价活疫苗免疫的鸡群,结果表明MD三价活疫苗中CVI988/B5、HCV2/B5及FC126/B5毒株的合适配比为2:1:2。按照此剂量配比制备的MD三价活疫苗,用RB1B超强毒株进行攻毒,当RB1B攻毒对照组MD阳性率为85.71%时,MD三价活疫苗的半数保护剂量(PD50)均数为111PFU/只,95%置信区间为585～53PFU/只的范围内,并确定MD三价活疫苗的免疫剂量为2500PFU/只。用该剂量对鸡进行MD三价活疫苗免疫,可使鸡体产生抗RB1B超强毒株攻击的坚强免疫力。     

Studies on the pathogenesis of chicken infectious anaemia virus infection in six-week-old SPF chickens

The pathogenesis of chicken infectious anaemia virus (CAV) infection was studied in 6-week-old and one-day-old SPF chickens inoculated intramuscularly with graded doses of Cux-1 strain (10(6)-10(2) TCID50/chicken). Viraemia, virus shedding, development of virus neutralizing (VN) antibodies and CAV distribution in the thymus were studied by virus isolation, polymerase chain reaction (PCR), immunocytochemistry (IP) and in situ hybridization until postinfection day (PID) 28. In 6-week-old chickens infected with high doses of CAV, viraemia and VN antibodies could be detected 4 PID and onward without virus shedding or contact transmission to sentinel birds. However, virus shedding and contact transmission were demonstrated in one-day-old infected chickens. In the 6-week-old groups infected with lower doses, VN antibodies developed by PID 14, transient viraemia and virus shedding were detected. The thymus cortex of all 1-day-old inoculated chickens stained with VP3-specific mAb. Cells with positive in situ hybridization signal were fewer and scattered throughout the thymus tissue of the one-day-old inoculated chickens as compared to IP-positive cells. These results suggest that early immune response induced by high doses of CAV in 6-week-old chickens curtails viral replication and prevents virus shedding.     

An investigation of the disease status of village poultry in Mauritania

Three contagious poultry diseases in Mauritania were studied. Serum samples and tracheal swabs were taken from 80 chickens in village poultry flocks in each of three different regions. Antibodies against Newcastle disease virus (NDV) were detected in 4.8% of chickens, antibodies against Gumboro disease virus in 15.8% of chickens and antibodies against Salmonella pullorum in 6.2% of chickens. Six isolates of NDV were made, of which four formed plaques on chicken embryo fibroblast monolayers. Seropositivity was highest in the Trarza region, bordering the River Senegal, and four out of six of the NDV isolates were made from chickens in this region.     

An enzyme-linked immunosorbent assay (ELISA) for detection of Marek's disease virus-specific antibodies and its application in an experimental vaccine trial

An enzyme-linked immunosorbent assay (ELISA) for the detection of Marek's disease virus (MDV)-specific antibodies was developed. Chicken embryo cells (CEC) or chicken kidney cells (CKC) were infected with MDV vaccine strain CVI988/Rispens, and infected-cell lysates were prepared at day 5 post-infection by freeze-thawing. Uninfected-cell lysates served as negative controls. Sera were used at a 1 : 100 dilution and were added in parallel to wells containing the infected and uninfected cell lysates. The optical densities at 492 nm (OD(492 nm)) were measured after detection of bound chicken antibodies with anti-chicken IgG peroxidase conjugate and colour reactions using o-phenylenediamine (OPD) as a substrate. The best results concerning the signal-to-noise ratio were obtained by using CKC cells rather than CEC for antigen preparation. The OD(492 nm) of plasma or serum samples with infected CKC was <0.02 when samples of unvaccinated and unchallenged maternal antibody-negative white leghorn chickens were tested. Sera and plasma samples of positive control birds exhibited OD(492 nm) of <0.01 when tested with uninfected CKC. The assay was used to monitor a trial that compared experimental BAC DNA vaccines and a commercial vaccine. Sustained seroconversion and antibody titers that were constantly rising until day 84 after vaccination (71 days after challenge) was observed only when chickens did not develop Marek's disease. In contrast, chickens developing the disease mounted marginal and short-lived antibody titers only. We conclude that the developed ELISA may be a valuable tool for the evaluation of the efficacy of MDV vaccination under experimental but possibly also under field conditions.     

The occurrence of Marek's disease in genetically different groups of chickens

The occurrence of Marek's disease was studied under laboratory conditions in four genetically different groups of chickens (Brown Leghorn, F1 hybrids of the CB x IA inbred lines, pullets of the paternal branch of the grandparent stock of the Hybro meat type, and final hybrids of the White Hisex layer type) after infection with the Georgia strain of Marek's disease (MD) virus, used in two doses (1600 and 16,000 PFU per one bird). MD was diagnosed on the basis of the occurrence of macroscopic tumours; when these tumours were absent in birds which had died within 105 days from infection, the dead bodies were subjected to microscopic examination (peripheral nerves, gonads, skin, bursa of Fabricius, thymus, spleen). The size of the parenterally administered dose of the virus had no significant effect on mortality, occurrence of tumours and the MD virus in any of the groups of chickens tested. However, there was a time shift in the mortality curve in chickens infected with a lower dose. The significantly highest occurrence of MD was recorded in BrL chickens (100%). A somewhat lower occurrence of MD was recorded in the Hisex White chickens (87.8%) and in the CB x IA hybrids (73.8%). However, the dead CB x IA chickens had a higher occurrence of tumours (96.6%) than the Hisex White chickens (77.1%). The lowest MD occurrence was recorded in the pullets of the Hybro meat type (25.5%). The organ most frequently affected by tumours after infection of the birds with the Georgia strain was the liver (24.1%).     

免疫剂量和母源抗体对禽流感重组鸡痘病毒活载体疫苗免疫效力的影响

利用表达 H 5亚型禽流感病毒 (AIV)血凝素基因的重组鸡痘病毒 (r FPV- HA)以不同剂量免疫 1日龄 SPF鸡、有或无母源抗体 (FPV、AIV H5)的商品鸡 ,并于免疫后 2 1d利用同亚型 AIV通过肌肉注射进行致死性攻击 ,通过检测免疫后 HI抗体应答、比较攻毒后发病率和死亡率评价免疫剂量和母源抗体对 r FPV- HA免疫效力的影响。结果发现 ,免疫后 2 1d,15 %～ 2 0 %的 SPF鸡和无母源抗体商品鸡可检出 HI抗体 ,而含母源抗体商品鸡检测不到 HI抗体。利用H5亚型 AIV致死性攻击后 ,10 3～ 10 6 PFU的 r FPV- HA可保护 95 %～ 10 0 %的 SPF鸡和无母源抗体商品鸡抵御强毒攻击 ,使之免于发病和死亡 ;而不同剂量 r FPV- HA接种的含母源抗体商品鸡有 80 %～ 90 %发病和死亡。结果表明 ,在较宽的免疫剂量范围内 ,r FPV- HA对 SPF鸡和无母源抗体商品鸡可提供良好的保护 ,显示出一定的应用前景 ;母源抗体影响 r FPV- HA诱导的免疫应答 ,且提高免疫剂量亦不能克服其干扰作用 ,这提示在实际应用中需优化免疫程序 ,避免母源抗体干扰。     

Comparative pathogenesis studies with oncogenic and nononcogenic Marek's disease viruses and turkey herpesvirus.

An apparently nononcogenic Marek's disease virus (SB-1) and turkey herpesvirus could be readily isolated from spleen, bursa of Fabricius, thymus, and peripheral blood lymphocytes of chickens beginning 4 to 6 days after inoculation, but unlike infections with two isolates of oncogenic Marek's disease virus (JM-10 and CU-2), virus replication in these cells was rare, and necrosis in the organs was essentially absent. Splenic enlargement was observed regularly during the first 4 to 11 days after inoculation, and Marek's disease tumor-associated surface antigen was observed on splenic and other lymphocytes in the four viral inoculation groups. Cellular cytotoxicity of splenic lymphocytes was demonstrated in vitro with cultured Marek's disease tumor cells (MSB-1 lymphoblastoid cell line) as the target in a chromium-release assay. The four viral infections induced sensitized lymphocytes.