Marek's disease virus-induced transient paralysis in chickens: demonstration of vasogenic brain oedema by an immunohistochemical method

The presence of vasogenic brain oedema and its distribution in Marek's disease virus (MDV)-induced transient paralysis (TP) were determined in genetically resistant and susceptible inbred White Leghorn chickens. MDV-inoculated TP-susceptible chickens with nervous signs (9 days post-inoculation) had severe vacuolation of cerebellar white matter and associated diffuse leakage of albumin and IgG. The serum protein leakage was associated morphologically with a vasculitis and intramural pseudocyst formation in the walls of blood vessels cuffed by mononuclear cells. This transient vasculitis and resulting vasogenic oedema coincided with the temporary neurological signs seen in TP-susceptible chickens. The vasculitis and vasogenic oedema were not present in brain tissue from recovered MDV-inoculated TP-susceptible chickens, MDV-inoculated TP-resistant chickens, or uninoculated control chickens from either line.     

Marek's disease virus-induced transient paralysis in chickens. 2. Ultrastructure of central nervous system

Ultrastructural changes of the central nervous system (CNS) were compared in inbred White Leghorn chickens that are genetically resistant or susceptible to Marek's disease virus (MDV)-induced transient paralysis (TP). Widened extracellular spaces, containing granular material resembling protein were found in brains of TP-affected chickens. Demyelination was absent in all chickens and intramyelinic oedema was rare, present only in TP-affected chickens. Both MDV-inoculated TP-resistant and TP-susceptible chickens had CNS perivascular mononuclear cell cuffs at all sampling periods, but the TP-affected chickens also had endothelial cell hypertrophy, a greater quantity of intramural phagocytized chromatin debris, and intramural heterophils. Such findings suggest vasculitis as the causative lesion. No open endothelial cell tight junctions, endothelial cells discontinuities, or transendothelial channels were morphologically evident to explain the vasogenic oedema, but an apparent suggestive increase in coated micropinocytotic vesicles in CNS endothelial cells is a possible mechanism to explain the altered blood-brain-barrier.     

Marek's disease virus-induced transient paralysis in chickens. 1. Time course association between clinical signs and histological brain lesions

Association between Marek's disease virus (MDV)-induced clinical signs of transient paralysis (TP) and brain. histological lesions (vasogenic oedema and perivascular mononuclear cell cuffs) were evaluated in TP-susceptible line G-B2 chickens in a time sequence study. The most consistent histological lesions were seen in the cerebellum. Leakage of albumin and vacuolation were parallel in development with clinical signs, but preceded the clinical signs by 6 to 12 h. During resolution of signs, a parallel decline in vacuolation, but not in extra-vascular albumin content was observed. The extravascular albumin shifted from an extracellular to intracellular location. No association was seen between the IgG leakage and vasogenic oedema or clinical signs. Perivascular mononuclear cell cuffing was statistically associated with clinical signs, but evaluation of plotted data indicated the slope for the cuffs was less than the slope for corresponding clinical signs. In addition, cuffing began 2 days prior to clinical signs. Thus, perivascular mononuclear cell cuffing was not causally associated with the TP syndrome.     

Marek's disease virus-induced transient paralysis is associated with cytokine gene expression in the nervous system

Marek's disease (MD)-associated transient paralysis (TP) was experimentally induced in chickens by intraperitoneal inoculation of RB1B strain of Marek's disease virus (MDV). Between 7 and 11 days post-infection (d.p.i.), neck and limb paralysis was observed in 18% of infected chickens, which was associated with various degrees of edema, vacuolation, perivascular cuffing of mononuclear cells, and glial cell infiltration mainly in the cerebrum, cerebellum, and brain stem. The chickens that were infected but did not progress to develop TP until 12 d.p.i. also had similar lesions suggestive of encephalitis in the cerebrum, cerebellum, and brain stem. Chickens infected with MDV had more interleukin (IL)-6, IL-12, and interferon (IFN)-gamma in their brain tissues compared to uninfected chickens. Moreover, IL-18 was significantly increased in brain tissues of birds showing clinical signs of TP compared to uninfected birds. Importantly, the expression of IL-6, IL-18, and IFN- gamma in brain tissues of MDV-infected chickens with signs of TP was significantly increased compared to that in asymptomatic MDV-infected birds. MDV genome load in the brain of chickens showing clinical signs of TP was higher than that in asymptomatic MDV-infected chickens but was not statistically significant. The lesions in the cervical, thoracic, and lumbar spinal cord segments in MDVinfected chickens were characterized mainly by perivascular cuffing of mononuclear cells irrespective of the group. The expression of mRNA for IL-18 and IFN-gamma genes was not significantly different in spinal cord tissues of chickens with TP compared to clinically normal, MDV-infected and noninfected chickens. These results suggest possible underlying immunologic mechanisms for MDV-induced TP.     

The epizootiology of Marek's disease. 3. The interrelationship of virus pathogenicity, antibody and the incidence of Marek's disease

Approximately 1000 day-old White Leghorn chickens were distributed into 5 pens in a brooder house and exposed to natural infection with Marek's disease virus (MDV). Observations were made on the inter-relationship between strains of MDV infecting the chickens, precipitating antibody, incidence of Marek's disease (MD), body weight, egg production, feed consumption and stress through movement. A wide variation in mortality from MD occurred between similar pens in the same brooding house. It was found that the sequence of infection and frequency of isolation of viruses of differing virulence varied from pen to pen and the mortality from MD was closely associated with these differences. Pen location did not affect any of the production parameters examined other than hen housed egg production. Infection with MDV was found to persist throughout the life of the chicken, however, the incidence of viraemia at 72 weeks of age was 60% of that at 10 weeks of age. Precipitating antibody was detected in the sera of most older chickens; however, no apparent differences could be observed in the incidence of precipitating antibody in chickens dying and surviving MDV infection when examined individually or on a pen basis. Chickens dying from MD tended to have an earlier and higher level of viraemia than those surviving infection with MDV. Movement of chickens to a rearing house at 8 weeks of age had little effect on the incidence of MD. It was concluded that this study confirmed the association between a widespread incidence of apathogenic virus and a low incidence of MD, and that the presence of such virus provided a protection against later challenge with pathogenic virus.     

Protection against the JMV Marek's disease-derived transplantable tumour by Marek's disease virus-specific antigens

Chickens immunised with inactivated chicken kidney cells infected with Marek's disease (MD) virus were protected against the lethal effects of inoculation with the MD-derived tumour transplant JMV, which appears not to express viral antigens. It is concluded that the distinction between MD virus-specific and tumour-specific antigens is not as complete as has been thought.     

Anti-viral immunity against Marek's disease virus infected chicken kidney cells

The mechanism of immunity conferred by herpesvirus of turkeys (HVT) was investigated using Marek's disease virus (MDV)-infected chicken kidney cell (CKC) cultures as target cells. Peripheral blood lymphocytes (PBL) from normal or HVT-vaccinated chickens killed MDV-infected target cells in the presence of anti-MDV or anti-HVT serum, whereas normal lymphocytes and HVT-sensitised lymphocytes themselves had no specific cytotoxic effect. Cell-mediated cytotoxicity of PBL from HVT-vaccinated chicken was exhibited specifically against HVT-infected CKC, and that of PBL from chickens inoculated with MDV was exhibited against MDV-infected CKC. From these results, it was concluded that antibody-dependent cellular cytotoxicity to MDV-infected cells may contribute to resistance to Marek's disease induced by HVT vaccination.     

Delayed hypersensitivity and leucocyte migration inhibition in chickens with BCG or Marek's disease infection

Production of Migration Inhibition Factor in vitro by lymphocytes from chickens with delayed hypersensitivity was examined. Chickens were sensitized with Bacille Calmette-Guerin (BCG) and others with Marek's Disease (MD) virus. Peripheral blood was used as a source of cells for in vitro radial migration inhibition. Using purified Band 24 antigen from Mycobacterium bovis and >A< antigen from MD virus, migration inhibition was demonstrated in BCG and MD sensitized chickens respectively, and not in controls. Results correlated well with intra-dermal skin reactions. Old Tuberculin and crude MD antigens inhibited radial migration of leucocytes from control chickens. The use of peripheral blood as a source of lymphocytes facilitates repeated sampling, both prior to and following sensitization. As a result, the role of the bursa and thymus in the induction and maintenance of delayed hypersensitivity can be studied in vitro. Furthermore, the role of cellular immunity and humoral antibodies as contributors to oncogenicity and/or protection in MD can be examined.     

Influence of strain of chickens on the immune response to vaccination against Marek's disease

Field trials were conducted to determine the response of different genetic strains of commercial chickens to vaccination against Marek's disease (MD). Cell-associated and cell-free HVT vaccines conferred the same degree of protection within the same strain of chickens. The incidence of MD in vaccinated birds seemed to be correlated with the natural susceptibility of the chicken strains to the disease. Revaccination did not induce better protection in the most susceptible strain.     

Stimulation of local solid tumour development of the nonproducer Marek's disease tumour transplant JMV by virus-induced immunosuppression

Chickens could be protected against lethal lymphoblastic leukaemia due to the nonproducer JMV Marek's disease (MD) tumour transplant by infection with the herpesvirus of turkeys (HVT) or various strains of MD virus. However, solid JMV tumours developed in MD virus-infected birds at the site of intramuscular or subcutaneous transplantation, but tumours never developed at the site of MD virus inoculation. The incidence and extent of local tumour growth, the development of metastases and the inhibition of tumour regression were related to the pathogenicity of the MD virus strains used for pre-treatment of the chickens. Infection of chickens with reticulo-endotheliosis virus (REV-C) or with chick syncytial virus (CSV), which are nonprotective against MD virus or JMV transplants, stimulated local tumour development of the attenuated JMV-A variant of the JMV transplant. Chickens which did not reject local tumours died of visceral JMV tumour metastases. A direct helper mechanism of viral infection on the oncogenicity of transplants was excluded. The results suggested that virus-induced immunosuppression stimulated the development of local JMV tumours which never occurred in normal chickens. Immunity to the JMV transplant, including resistance to lethal leukaemia and successful regression of local tumours, did not coincide with immunity to MD virus-induced visceral lymphomas or nerve lesions. Vaccinal induced tumour immunity evidently was defective. The significance of these results is discussed with reference to immunological functions of MD tumour-specific antigens.     

Further characterisation of the CVI 988 strain of Marek's disease virus

The CVI 988 strain of Marek's disease virus (MDV) has been studied for markers in vitro and in vivo. It was shown by indirect immunofluorescence assays that this strain belonged to the HPRS-16 serotype of MD viruses. The virus was positive for precipitating 'A' antigen. Medium plaques were produced in cell cultures. Therefore the CVI 988 strain could not be distinguished in vitro from other virus strains of the same serotype. The CVI 988 strain proved to be pathogenic for genetically susceptible Rhode Island Red chickens if inoculated at high doses equivalent to 10 times the field dose of the vaccine. The virus caused symptoms of classical Marek's disease in up to 28.5% of the inoculated chickens with gross lesions restricted to the peripheral nerves. Visceral tumours never occurred. The virus spread to uninoculated control chickens housed in the same pen. The CVI 988 strain can be classified as a mildly pathogenic classical Marek's disease virus of the HPRS-16 serotype.     

Central nervous system lesions induced experimentally by a very virulent strain of Marek's disease virus in Marek's disease-resistant chickens.

The histological lesions of central nervous system (CNS) in Marek's disease-resistant chickens inoculated with a very virulent Marek's disease virus strain (vvMDV), Md/5, were examined. This vvMDV induced high incidences of CNS lesions as well as visceral and peripheral nerve lesions. The principal histopatho-logical changes of CNS consisted of non-suppurative meningoencephalomyelitis and lymphomatous lesion, which were categorized into two types, non-necrotizing and necrotizing. The main changes in the former type were perivascular cuffing of lymphoid cells of variable thickness. Although diffuse lymphomatous lesions were sometimes observed, gliosis, neuronal and axonal degeneration, and satellitosis as well as demyelination in the myelinated fibre paths, were infrequent. The most significant change of the latter type was necrotizing lymphomatous and, sometimes, non-suppurative inflammatory lesions (malacia). In the malacic foci, all the cells, including infiltrating lymphoid cells, neurons and glial cells, were necrotic or degenerated. The malacic lesions were frequently accompanied by fibrinoid necrosis of blood vessels. The present results indicate that necrotizing vasculitis associated with vvMDV may lead to ischaemic damage which in turn induces necrotizing lymphomatous and sometimes, meningoencephalomyelitis lesions. In addition, it is considered that vvMDV can induce a high incidence not only of CNS lesions, but also of visceral and nerve lesions in Marek's disease-resistant chickens.     

Protection of chickens against very virulent infectious bursal disease virus (IBDV) and Marek's disease virus (MDV) with a recombinant MDV expressing IBDV VP2.

To develop a herpes virus vaccine that can induce immunity for an extended period, a recombinant Marek's disease (MD) virus (MDV) CVI-988 strain expressing infectious bursal disease virus (IBDV) host-protective antigen VP2 at the US2 site (rMDV) was developed under the control of an SV40 early promoter. Chickens vaccinated with the rMDV showed no clinical signs and no mortality and 55% of the chickens were considered protected histopathologically after challenge with very virulent IBDV (vvIBDV), whereas all of the chickens vaccinated with the conventional IBDV vaccine showed no clinical signs and were protected. Chickens vaccinated with the CVI-988 or chickens in the challenge control showed severe clinical signs and high mortality (70-75%) and none of them were protected. Also, the rMDV conferred full protection to chickens against vvMDV just as the CVI-988 strain did, whereas 90% of the challenge control chickens died of MD. Antibody levels against IBDV and MDV following the vaccination increased continuously for at least 10 weeks. No histopathological lesions in the rMDV-vaccinated chickens and no contact transmission of the rMDV to their penmates were confirmed. These results demonstrate that an effective and safe recombinant herpesvirus-based IBD vaccine could be constructed by expressing the VP2 antigen at the US2 site of the CVI-988 vaccine strain.     

Propagation and assay of virulent and attenuated JMV Marek's disease-associated tumour cells

JMV tumour cells were shown to cause a lethal lymphoblastic leukaemia in young chickens as well as in chicken embryos. The incubation period was very short but dose-dependent. Chickens died in 4 to 12 days, embryos in 7 to 14 days, after inoculation. Embryo-passaged attenuated JMV (JMV-A) caused the same lesions in embryos as virulent JMV. The dose-response relationship depended on the route of inoculation and on the quality of the tumour cell preparation. Intramuscular (i.m.) inoculation of leukaemic blood or embryo lymphoblasts provided the most satisfactory response. Intraperitoneal (i.p.) inoculation and lymphoblastic chicken spleens as a source of JMV were definitely less suitable. The dose-response curves obtained in yolk sac-inoculated embryos were similar to the curves obtained by i.m. inoculation of chickens. Only 4 to 10 lymphoblasts were needed per lethal dose (50%) in chickens and 50 to 80 in embryos. The pathogenicity and antigenicity of JMV and JMV-A were strictly cell-associated. No Marek's disease (MD) virus or any other avian virus could be detected, either by various virus isolation procedures, or by serological methods. Contact transmission of JMV to other chickens did not occur. Antibodies against surface antigens on JMV lymphoblasts were detected in JMV and JMV-A chicken hyperimmune sera. These sera reacted against MD lymphoblastoid cell lines (HPRS-1 & 2, MSB-1) as well as MSB-1 anti-serum, but all sera reacted also against thymus lymphocytes from normal chickens. The results of absorption tests suggested that the surface antigens of JMV lymphoblasts and of the tested cell lines were not identical. The majority of tumour cell surface antigens appeared to represent genetically specific histocompatibility or lymphocyte antigens. A common MD tumour-associated surface antigen (MATSA) could not be identified serologically (FA test) on the tumour cells studied.     

Depression of vaccinal immunity to Marek's disease by infection with reticuloendotheliosis virus.

The effect of infection with low-virulence, tissue culture-propagated strains of reticuloendotheliosis virus (REV) on protective vaccinal immunity against Marek's disease (MD) lymphomas was investigated. Vaccinated chickens inoculated at hatching with greater than 10(4) focus-forming units of REV and challenged with MD virus were poorly protected against MD lesion development as indicated by protective indices of 53 to 79% for strain CS (P less than 0.05) and 42 to 49% for strain T (P less than 0.01) compared to 78 to 100% for REV-free controls. Furthermore, the response of blood lymphocytes to mitogen stimulation and the antibody response to sheep erythrocytes and Brucella abortus were less in REV-inoculated chickens than in controls. The REV-induced depression of immune responses was more severe in chickens infected with mildly pathogenic strain T than in chickens infected with the apathogenic strain CS and was generally transient with both virus strains. Little or no depression of immune responses was observed in chickens inoculated with less than 10(3) focus-forming units of REV. These studies extend knowledge on the immunodepressive ability of low-virulence REV strains and establish that infection with these viruses depresses certain parameters of MD vaccinal immunity, an important model for cellular immunity against virus-induced neoplasia in the chicken.     

Virologic and serologic studies of zoo birds for Marek's disease virus infection.

One hundred and eleven zoo birds representing 49 species in 14 orders were examined for Marek's disease (MD) herpesvirus (MDHV) infection. MDHV was isolated from 10 birds, all belonging to genus Gallus. The precipitating antibodies against MDHV were demonstrated only in the Gallus birds, when 51 selected birds including 34 Galliformes and 17 other birds representing 12 species from nine orders were examined. The 10 MDHV isolates all induced morphologically similar plaques in cell cultures closely resembling those of HN strain, a low pathogenic isolate of MDHV. Six of the 10 isolates, when inoculated into an experimental line of chickens highly susceptible to MD, caused only a minimal degree of histologic lesions without causing clinical MD, gross MD lesions, or deaths from MD. Natural hosts of MD are probably Galliformes, primarily affecting Gallus and less often other genera of Galliformes.     

Titers of precipitins in strains of chickens exposed to Marek's disease

Titers of precipitating antibody against Marek's disease (MD) herpesvirus were determined on serum collected from five strains of chickens at intervals throughout the 411 day test period. The average titers for the genetically resistant Cornell K strain were consistently higher (P <.01) than for the genetically susceptible Cornell S strain. This relationship between disease resistance and antibody titers may have been fortuitous because the titers of three other strains varied in relationship to each other and did not appear to be related to resistance against MD. However, when strain effect was removed there was some relationship between antibody titer and resistance to the disease since chickens that died from MD had lower titers up to 139 days (P <.05) than did others.     

Very Virulent Plus Strains of MDV Induce an Acute Form of Transient Paralysis in Both Susceptible and Resistant Chicken Lines

Abstract Marek's disease (MD) is a lymphoproliferative disease of domestic chickens caused by a highly cell-associated alpha herpesvirus, Marek's disease virus (MDV). Clinical signs of MD include depression, crippling, weight loss, and transient paralysis (TP). TP is a disease of the central nervous system that affects MD-susceptible chickens 8-11 days post-infection (dpi), normally resulting in recovery 1-3?d after the onset of clinical signs. In this study we inoculated chickens from lines 7(2) (MD-susceptible) and 6(3) (MD-resistant) with a very virulent plus strain of MDV at 2?wk of age, and collected brain samples from birds with and without TP at 5, 11, and 21?dpi for gene expression profiling and histological analysis. Data revealed that chickens inoculated with MDV had higher levels of IL-6, IL-10, IL-18, IFN-α, IFN-β, IFN-γ, MHC I, and CD18 in their brains at 11?dpi compared to the uninfected control birds. In addition, the expression levels of IL-6, IL-10, IFN-α, IFN-β, and IFN-γ were significantly higher in the brains of the birds showing clinical signs of TP than in asymptomatic chickens. Comparative analysis between the two chicken lines showed that the expression levels of IL-6, IL-10, IFN-β, IFN-γ, IL-18, CD18, and MHC I were significantly higher in the brains of the birds from line 6(3) with TP than those of line 7(2) exhibiting neurological disorders. A differential expression pattern was observed for some of the tested genes at different time points post-inoculation. Histological analysis showed lymphocytic meningitis, perivascular cuffing, and neuronal degeneration within the brains of birds from both susceptible and resistant lines exhibiting TP at 11?dpi. Vaccination prevented development of TP and other MD-associated clinical symptoms. These observations are suggestive of an underlying immunological mechanism for viral-induced neurological dysfunction, and the differential responses of the two chicken lines to MDV infection.     

Cross-protection between JMV Marek's disease-derived tumour transplant, Marek's disease and turkey herpesviruses

Cross-protection tests were conducted using attenuated JMV (JMV-A) Marek's disease-derived lymphoblasts, glutaraldehyde-treated JMV tumour cells, attenuated Marek's disease virus (MDV, strain HPRS-16/att) and turkey herpesvirus (HVT, strain FC126) as vaccines, and virulent JMV and MDV (HPRS-16) for challenge. The JMV and JMV-A preparations were free of MDV, leukosis and reticuloendotheliosis viruses. Vaccination of chickens with attenuated MDV or with HVT provided good protection against both JMV lymphoblastosis and Marek's disease (MD). In one experiment HVT (cell-free) caused a better resistance to JMV than to MD. Inoculation of JMV-A always resulted in a 100% resistance to virulent JMV. However, JMV-A did not induce any appreciable resistance to MD, even when the birds were challenged with MDV by contact exposure. Control experiments revealed that high doses of normal lymphocytes from uninfected chickens also had a protective effect against JMV. The 50% protective dose varied from 10(7) to 10(8) lymphocytes. JMV tumour cells inactivated by glutaraldehyde were used in different experiments but rarely caused a clear-cut protection against virulent JMV. The results of this study suggested that a one-way relationship exists in vivo between HVT or MDV and JMV lymphoblastic leukaemia. However, resistance induced against JMV tumour cells appeared to be related to histocompatibility antigens at least as much as to tumour-specific cell surface antigens. The results obtained failed to provide clear evidence for or against vaccinal resistance to MDV being dependent on the action of a common Marek's disease tumour-associated surface antigen (MATSA) additional to the immune response to viral antigens.