B. W. Calnek

Characterisation of two highly oncogenic strains of Marek's disease virus 1 2

The RB-1B and ALA-8 strains of Mareks disease (MD) virus, which were isolated from chickens with MD and which had been vaccinated with the herpesvirus of turkeys (HVT), were evaluated for their oncogenic potential in genetically susceptible (P-line) and resistant (N-line, PDRC) chickens. RB-1B and ALA-8 were both highly oncogenic, causing a high incidence of MD in both susceptible and resistant birds. Vaccination of P-line birds with SB-1 or HVT did not protect satisfactorily against RB-1B. However, a bivalent vaccine consisting of SB-1 and HVT enhanced protection significantly. HVT alone, and the bivalent vaccine, protected PDRC and N-line chickens well against RB-1B, but SB-1 was less protective in PDRC birds. HVT protected equally well against challenge with ALA-8 and the standard JM-10 strain. Differences in the pathogenesis of viral infection could not be detected among ALA-8, RB-1B and JM-10 between 4-7 days post-infection (d.p.i.). However, after d.p.i. 12 RB-1B caused significantly higher levels of viral internal antigen and virus isolation rates than did JM-10 in the same genetic strain. Prior vaccination prevented the expression of ALA-8 at 5 and 20 d.p.i., but not that of RB-1B. Pathogenetic events such as expression of VIA or level of virus infection appeared to be directly related to the level of protection observed in challenged birds.

Classification of Marek's disease viruses according to pathotype: philosophy and methodology

The concept of pathotype in Mareks disease (MD) probably dates from the recognition of a more virulent form of the disease in the late 1950s (Benton & Cover, 1957). Distinctions between MD virus strains were further expanded with the description of the vv pathotype in the early 1980s and of the vv+ pathotype in the 1990s. Pathotype designations reflect important biological properties that correlate with the break-through of vaccinal immunity in the field. However, pathotyping methods applied by various laboratories have not been uniform, preventing critical comparison of results. Better uniformity of pathotyping procedures is desirable. The Avian Disease and Oncology Laboratory (ADOL) method is based on induction of lymphoproliferative lesions in vaccinated chickens. This method has been used to pathotype more than 45 isolates and is the basis for the current pathotype classification of MD virus strains. Its limitations include requirements for a specific type of chickens (15×7 ab+), large numbers of animals, and a statistical method to compare lesion responses to those of JM/102W and Md5 control strains. Because of these limitations, it has not been and is not likely to be used in other laboratories. Comparability in pathotyping can be improved by the comparison of field isolates with standard prototype strains such as JM/102W, Md5 and 648A (American Type Culture Collection) or their equivalents. Data may be generated by different in vivo procedures that measure tumour induction, neurological disease (both neoplastic and non-neoplastic lesions), or solely non-neoplastic criteria (such as lymphoid organ weights or virus replication). Methods based on neoplastic criteria, especially when generated in MD-immunized chickens, will probably correlate most closely with that of the ADOL method and be most relevant to evolution of MD virus in the field. Based on data from several trials, a modification of the ADOL method that utilizes fewer chickens and can be conducted with commercial specific pathogen free strains is proposed. The modified method is based on “best fit” comparisons with prototype strains, and is expected to provide results generally comparable with the original method. A variety of other alternative criteria (see earlier) are also evaluated both for primary pathotyping and as adjuncts to other pathotyping methods. Advantages and disadvantages of alternative methods are presented. Le concept de pathotype pour la maladie de Marek (MD) date probablement de lidentification dune forme plus virulente de la maladie à la fin des années 1950 (Benton & Cover, 1957). Les distinctions entre les souches de virus de la MD (MDV) ont été développées plus tardivement avec la description des pathotypes très virulents (vv) au début des années 1980 et des hypervirulents (vv+) dans les années 1990. Les désignations de pathotype reflètent des propriétés biologiques importantes qui correspondent au franchissement de limmunité vaccinale sur le terrain. Cependant les méthodes de pathotypage, mises en pratique dans différents laboratoires, nont pas été les mêmes empêchant la comparaison critique des résultats. Une meilleure harmonisation des procédures de pathotypage est souhaitable. La méthode du Laboratoire des Maladies Aviaires et dOncologie (ADOL) est basée sur linduction des lésions lymphoprolifératives chez les poulets vaccinés. Cette méthode a été utilisée pour pathotyper plus de 45 souches et est la base de la classification actuelle par pathotype des souches de MDV. Les limites de cette méthode incluent les exigences en ce qui concerne le type de poulets (15x7 ab+), un nombre important danimaux, et une méthode statistique pour comparer les réponses lésionnelles à celles des souches témoins JM/102W et Md5. Du fait de ces limites, cette méthode na pas été utilisée dans dautres laboratoires, et ne sera probablement pas. La comparabilité du pathotypage peut être améliorée par la comparaison des souches du terrain à des souches prototype standard, telles la JM/102W, la Md5 et la 648A (American Type Culture Collection) ou à des souches équivalentes. Les données peuvent être générées par différentes procédures in vivo qui mesurent linduction des tumeurs, la maladie neurologique (les lésions néoplastiques et non-néoplastiques), ou seulement les critères non-néoplastiques (tel les poids des organes lymphoïdes ou la réplication virale). Les méthodes basées sur les critères néoplastiques, particulièrement quand ils apparaissent chez des poulets immunisés MD, devraient probablement correspondre plus étroitement à celles de la méthode dADOL et être plus en rapport avec lévolution des virus sur le terrain. A partir des données de plusieurs essais, une modification de la méthode de lADOL est proposée. Elle utilise moins de poulets et peut être réalisée avec des variétés commerciales de poulets SPF. Cette méthode modifiée est basée sur de meilleures comparaisons des souches prototypes, et on peut sattendre à fournir des résultats généralement comparables à ceux de la méthode originale. Dautres critères alternatifs (Cf.supra) sont également évalués aussi bien pour le premier pathotypage que comme compléments à dautres méthodes de pathotypage. Les avantages et les inconvénients de ces méthodes alternatives sont présentés. Der Begriff des Pathotyps bei der Marekschen Krankheit (MK) entstand wahrscheinlich im Zusammenhang mit dem Auftreten einer virulenteren Form der Erkrankung in den späten 1950iger Jahren (Benton &Cover, 1957). Die Unterscheidung zwischen verschiedenen MK-Virus (MKV)-Stämmen wurde mit der Beschreibung des vv-Pathotyps in den frühen 1980iger Jahren und des vv+-Pathotyps in den 1990iger Jahren weiter ausgedehnt. Diese Pathotypbezeichnungen reflektieren wichtige biologische Eigenschaften, die mit Impfdurchbrüchen im Feld im Zusammenhang stehen. Die Pathotypisierungsmethoden in verschiedenen Laboratorien waren jedoch nicht einheitlich, was einen tatsächlichen Vergleich der Ergebnisse bislang verhinderte. Aus diesem Grund ist die Vereinheitlichung der Pathotypisierungsverfahren wünschenwert. Die Methode des ,Laboratoriums für Vogelkrankheiten und –onkologie‘ (Avian Disease and Oncology Laboratory (ADOL)) basiert auf der Induktion lymphoproliferativer Läsionen in vakzinierten Hühnern.. Diese Methode wurde für die Pathotypisierung von mehr als 45 Isolaten verwendet und ist die Basis für die derzeitige Pathotyp-Klassifizierung von MKV-Stämmen. Ihre Anwendung ist eingeschränkt aufgrund des Erfordernis eines bestimmten Hühnertyps (15x7 ab+), einer großen Anzahl von Versuchstieren und einer statistischen Methode, die den Vergleich der auftretenden Läsionen mit denjenigen durch die Kontrollstämme JM/102W und Md5 ermöglicht. Aufgrund dieser Einschränkungen war und ist diese Methode nicht für die Anwendung in anderen Laboratorien geeignet. Die Vergleichbarkeit der Pathotypisierung kann durch den Vergleich von Feldisolaten mit Standardprototypstämmen wie JM/102W, Md5 und 648A (American Type Culture Collection) oder ihrer Äqivalente verbessert werden. Die entsprechenden Daten können durch zwei verschiedene in vivo-Verfahren gewonnen werden, die entweder die Tumorinduktion und die neurologische Erkrankung (sowohl neoplastische als auch nicht-neoplastische Veränderungen) oder nur nicht-neoplastische Kriterien (wie Gewicht der lymphatischen Organe oder Virusreplikation) ermitteln. Ergebnisse, die auf der Bestimmung neoplastischer Kriterien basieren, insbesondere wenn sie in MK-immunisierten Hühnern durchgeführt werden, werden wahrscheinlich am ehesten mit den nach der ADOL-Methode erhobenen Daten korrelieren und für die Beurteilung der Evolution des MKV im Feld von größter Bedeutung sein. Basierend auf den Daten von verschiedenen Untersuchungen wird eine Modifizierung der ADOL-Methode, bei der weniger Hühner verwendet werden, die außerdem aus kommerziellen SPF-Stämmen sein können, vorgeschlagen. Die modifizierte Methode basiert auf ”Best Fit“-Vergleichen mit den Prototypstämmen, d.h. mit welchem Prototyp gibt es die größte Übereinstimmung in den Befunden, und es wird erwartet, dass sie Ergebnisse erbringt, die mit denen der Originalmethode generell vergleichbar sind. Eine Vielzahl von anderen Alternativkriterien (siehe oben) wurden ebenfalls auf ihre Eignung sowohl für die Primärpathotypisierung als auch als Ergänzung zu anderen Pathotypisierungsmethoden beurteilt. Vor- und Nachteile dieser Alternativmethoden werden erläutert. El concepto de patotipo en la enfermedad de Marek (MD) data probablemente de finales de los 1950s cuando se reconoció una forma más virulenta de enfermedad (Benton y Cover, 1957). Las distinciones entre las diferentes cepas de virus de MD (MDV) fueron aún mayores al describirse el patotipo vv a principios de los ochenta y el vv+ en los noventa. La designación de patotipo refleja propiedades biológicas importantes que se correlacionan con la capacidad de romper la inmunidad maternal en el campo. A pesar de ello, los métodos de clasificación de los diferentes patotipos en varios laboratorios no han sido uniformes, lo cual ha impedido una comparación crítica de los resultados. El método utilizado en el Avian Disease and Oncology Laboratory (ADOL) se basa en la inducción de lesiones linfoproliferativas en pollos vacunados. Este método ha sido utilizado para clasificar más de 45 aislados y es la base para la clasificación actual de los patotipos de cepas de MDV. Las limitaciones de este método son varias: necesidad de un tipo específico de pollos (15x7 ab+), uso de un gran número de animales y de un método estadístico para comparar las respuestas lesionales con las de las cepas control JM/102W y Md5. Debido a estas limitaciones no ha sido y no es probablemente usado en otros laboratorios. La comparación en el patotipado puede ser mejorada mediante la comparación de aislados de campo con cepas prototipo como las JM/102W, Md5 y 648A (American Type Culture Collection) o sus equivalentes. Los datos pueden

RELATIONSHIP BETWEEN THE IMMUNOSUPPRESSIVE POTENTIAL AND THE PATHOTYPE OF MAREK'S DISEASE VIRUS ISOLATES

Isolates of Mareks disease virus (MDV) representing three pathotypes of differing virulence were compared for relative immunosuppressive properties in genetically susceptible P2a-strain and genetically resistant N2a-strain chickens. Criteria of immunosuppression were 1) persistence of early cytolytic infection (i.e., a delay or failure to enter latency) in lymphoid organs, 2) atrophy of the bursa of Fabricius and thymus as measured by organ weight proportional to body weight at 8 and 14 days postinfection (DPI), and 3) histopathologic evidence of necrosis and atrophy in lymphoid organs. No significant differences in infection level were observed among the pathotypes during the early (4-5 DPI) period of infection. However, the extent of persistent cytolytic infection at 7-8 DPI, based on numbers of tissues positive and mean scores in immunofluorescence tests, was greater (P < 0.05) for three isolates (RK1, 584A, 648A) in the highest virulence pathotype (very virulent-plus MDV [vv + MDV]) than for two isolates (JM16, GA5) in a lower virulence (virulent MDV [vMDV]) pathotype. Results from two isolates (RB1B, Md5) classified in the intermediate very virulent pathotype (very virulent MDV [vvMDV]) fell between those from the other two pathotypes. Similarly, there was a stepwise effect of viral pathotype in which the vv + MDV isolates caused the most severe damage to lymphoid organs in terms of atrophy (relative organ weights) and histopathologic changes. Organs from chickens infected with vv + MDVs showed little recovery between 8 and 14 DPI. The vMDV isolates caused the least severe damage, and lymphoid organs showed a significant return toward normal by 14 DPI; vvMDV isolates induced intermediate degrees of atrophy and recovery. The same pattern of relationship between virulence pathotype and degree of bursal and thymic atrophy was also observed in genetically resistant N2a chickens. These results suggest that the degree of immunosuppression is linked to virulence and that a simple measure of atrophic changes (relative organ weights) in the bursa of Fabricius and thymus might be useful in determining the pathotype classification of new MDV isolates. The basis for differences in immunosuppressive potential of MDV isolates needs further clarification.

Field trials with a bivalent vaccine (HVT and SB-1) against Marek's disease.

White leghorn chickens on five farms were given a bivalent Mareks disease (MD) vaccine consisting of turkey herpesvirus (HVT) and SB-1 (a nononcogenic MD virus); other chickens received only HVT. The farms had histories of vaccination failures, presumably owing to an exceptionally virulent challenge MD virus. The bivalent vaccine uniformly protected chickens better than HVT alone between 12 and 16-20 weeks of age, when serious MD losses occurred. During that period, total mortality in groups given both viruses ranged from 0.39 to 1.26% (mean 0.86%), whereas that in HVT-vaccinated groups not exposed to SB-1 varied from 1.92 to 7.44% (mean 3.43%). Chickens in pens or rows with close contact to those given bivalent vaccine also had low MD mortality rates (0.46-1.06%, mean 0.77%), probably from the spread of SB-1.

Stable Transfection of Reticuloendotheliosis Virus-Transformed Lymphoblastoid Cell Lines

Lymphoblastoid T cell lines were established by infection of chicken splenocytes with reticuloendotheliosis virus (REV). The target cells first were cultured in interleukin-containing conditioned medium or were stimulated by concanavalin A, or both. Most cell lines were T cells expressing CD3 and one of the T cell receptors, and all cell lines were positive for major histocompatibility complex (MHC) class II antigens. Several REV-transformed cell lines were stably transfected using electroporation with a selectable plasmid, pNL1, containing the neor gene. Transfected cell lines were selected using G418 and were maintained for periods up to 137 days. Transfected cell lines were susceptible to MHC class-I restricted lysis by cytotoxic T lymphocytes from REV-infected chickens.

In vitro isolation, propagation, and characterization of duck hepatitis virus type III.

The in vitro isolation, propagation, and characterization of duck hepatitis virus Type III (DHV-III), is described. This virus, which is serologically distinct from the classical (Type I) DHV, replicated in liver and kidney cell cultures of duck origin. Replication was limited in chicken and quail kidney and duck embryo fibroblast cultures. It did not replicate in a variety of other cell cultures of avian or mammalian origin. The virus was grown successfully in embryonating eggs of ducks, but not of chickens. DHV-III passed through a 50-nm membrane filter, was stable at pH 3.0 and resisted treatment with 5% chloroform. Virus growth was not inhibited by treatment with 5-iodo-2-deoxyuridine. Electron-microscope examination revealed crystalline arrays in the cytoplasm; virus particles had cubic symmetry, and were about 30 nm in diameter. By these properties, this virus can be classified as a member of the picornavirus group.

Papovavirus infection in hand-fed parrots : virus isolation and pathology

Papovavirus infection was diagnosed in 44 parrots of at least 18 species exclusive of the budgerigar (Melopsittacus undulatus). The birds were 14 days to 4 months old and had been removed from parental care and hand-fed as nestlings. The birds had been unexpectedly found dead after having evidenced no premonitory signs of illness, or they died following a short (12-to-48-hour) period of lassitude and anorexia. In most cases, necropsies revealed pallor, multiple hemorrhages, splenomegaly, and hepatomegaly with multifocal necrosis. Histological lesions included multifocal to diffuse hepatic necrosis that spared the periportal hepatocytes, karyomegaly of splenic reticuloendothelial cells and cells in other tissues, membranous glomerulopathy, and necrosis of bursal medullary lymphocytes. Papovaviruses were isolated from two cases, and papovavirus infection was confirmed in 27 of the birds by the fluorescent-antibody test using a conjugate against a papovavirus isolated from a budgerigar.

Effect of immunocompetence on the establishment and maintenance of latency with Marek's disease herpesvirus.

Mareks disease virus (MDV) infections normally have an early cytolytic phase in lymphoid organs at 3 to 6 days post-infection followed by a period of latent infection. Little is known about the mechanisms that govern latency with herpesvirus infections, including Mareks disease (MD). To investigate the importance of immunocompetence for either the establishment or the maintenance of latency in MD, immunosuppressive treatments were applied prior to infection with MDV or after latency was established. These included cyclosporin (Cs) or betamethasone (BM) treatments, neonatal thymectomy plus cyclophosphamide treatment (Tx/Cy), and infection at a young age before full competence. The effect of all the treatments was determined by examining tissues and spleen cells for evidence of virus replication before and after cultivation in vitro. Induced (Cs or Tx/Cy treatments) or natural (infection at a young age) incompetence resulted in prolonged and more widespread early cytolytic infection. Immunosuppression by Cs after latency had developed resulted in a reappearance of cytolytic infection in the spleen and it enhanced the cytolytic infection in the thymus and the bursa of Fabricius. After immunosuppression with Cs, cytolytic infection was found mostly in T cells, although many of the virus-positive cells did not have markers for either T cells or B cells. Immunosuppression by BM after latency had developed also resulted in the reappearance of cytolytic infection in the spleen but only at a very low level. These results suggest that immunocompetence is required for the establishment and maintenance of latency.

Pathogenesis of infection with attenuated Marek's disease virus strains.

Recently, attenuated Mareks disease virus (MDV) became of renewed interest as a component in bi- or polyvalent vaccines. The effect of attenuation on the pathogenesis of infection was investigated. Cloned preparations of the JM-16, BC-1A and RB-1B strains of MDV were attenuated by serial passage in chick kidney cells or chicken embryo fibroblasts. Subclones were obtained from the JM-16 strain at passage (p) 26 (JM-16d) and 50 (JM-16a, b and c). The passage level at which each virus became attenuated was dependent on the virus strain. The highly oncogenic RB-1B strain was still oncogenic after 37 passages, while JM-was already attenuated at p. 27. In ovo infection of high passage JM-16 and RB-1B (p 54 and 55) demonstrated the presence of residual pathogenicity. Attenuated virus failed to induce the early cytolytic infection which is characteristic for the pathogenesis of infection with oncogenic MDV. Low levels of lymphocyte-associated viraemia could be detected after infection with all attenuated viruses except with the subclone JM-16a. This virus was, however, able to induce moderate protection against challenge and antibodies were detectable, suggesting that cells other than lymphocytes became infected. The pathogenesis after in ovo infection with attenuated virus was similar to that after infection of chicks. The in vivo data suggested that attenuation reduced the efficiency of infection of, or virus replication in, lymphocytes. A markedly reduced ability to establish in vitro infection of lymphocytes by exposure to heavily infected lymphocytes was observed, and this supports this hypothesis. The altered characteristic of attenuated virus to infect lymphocytes in vivo or in vitro was not caused by the selection of temperature sensitive or thymidine kinase negative mutants.

Cultivation and characterisation of avian lymphocytes with natural killer cell activity.

Conditioned medium (CM), as a presumed source of lymphokines including interleukin-2, was prepared from chicken spleen cell cultures stimulated with concanavalin A (con A). When CM was used to cultivate spleen cells from 6- to 8-week-old P-2 chickens, eight of nine spleens yielded cell lines which grew continuously for at least 50 days. Six of the cultures were tested for natural killer (NK) cell activity against LSCC-RP9, a lymphoblastoid cell known to be susceptible to NK cells, and against several Mareks disease lymphoblastoid cell lines (MDCC-CU2, -CU36 and -MSB-1). All six cultures lysed the RP9 cells in a chromium release assay with high levels of specific release (30 to 50%) at effector cell to target cell ratios of 5:1 or 10:1. CU2 and CU36, which are NK-cell resistant, were not lysed, while there was a low level of specific activity against MSB-1. The cells were characterised for surface and internal antigens with monoclonal antibodies and were negative for thymocyte antigen, IgM, a T cell antigen also present on granulocytes and red blood cells, and two antigens found in macrophages. Two of the six lines examined had a low number of cells expressing la antigen, while the other four were negative. An antigen present on circulating T cells and a macrophage (sub)population was present on all lines. The majority of the cells had the morphological appearance of mammalian large granular lymphocytes (LGL) with a rather small kidney-shaped nucleus. Granules and vacuoles were present in the cytoplasm. However, there was a variable percentage of lymphoblastoid (LB) cells also present. Cell lines established with CM from con A-stimulated spleen lymphocytes in other studies were also shown to have high levels of NK activity regardless of the relative proportions of cells with the morphological appearance of LGLs or LBs, or the relative frequency of expression of the two T cell markers or la antigen, all of which varied markedly.