Marek's disease virus-induced transient paralysis in chickens. 3. Differentiation of field cases from classical Marek's disease by central nervous system lesions

Vasculitis with intramural pseudocyst formation primarily in the cerebellar white matter, but also in nuclei of the medulla, resulted in leakage of IgG and albumin and vacuolation of the neuropil (vasogenic oedema) in brains from chickens with clinical signs of Marek's disease virus (MDV)-induced transient paralysis (TP). Demyelination was absent. Chickens that had recovered from TP had a restored blood-brain-barrier, indicated by the rarity of vasculitis and vascular intramural pseudocysts in the cerebellum. In addition, the vacuolation and protein leakage were greatly decreased. The minor vacuolation resulted primarily from intramyelinic (cytotoxic) oedema. The small quantity of extravascular protein was being removed by microglial cells and astrocytes. In one chicken which failed to fully recover from TP (TP-prolonged) there was neither vasogenic oedema, cytotoxic oedema, nor vasculitis in the cerebellum. The medulla of the TP-prolonged chicken had a severe lymphocytosis, swollen axons, neuronal degeneration, secondary demyelination and some associated serum protein leakage. All TP-affected and TP-recovered chickens, and the TP-prolonged chicken, had perivascular mononuclear cell cuffs within all brain sections. Chickens with classical Marek's disease (MD) generally lacked CNS vacuolation, perivascular mononuclear cell cuffs, vasculitis and serum protein leakage. However, in a few cases of MD with severe perivascular mononuclear cell cuffs, focal demyelinating plaques were seen. These plaques had associated vacuolation, serum protein leakage, axonal spheroids and neuronal degeneration.     

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.     

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.     

Sequential skin lesions in chickens experimentally infected with Marek's disease virus

Skin biopsies taken at weekly intervals from the same specific-pathogen free (SPF) chickens inoculated with Md/5 Marek's disease virus revealed two distinctive patterns of perifollicular cutaneous lesions, tumour-associated and non-tumour-associated. The tumour-associated pattern was subdivided into two types. The progressive type was manifested by a continuous increase of lymphoid cell aggregates (LCA) in the skin, resulting in the development of gross skin tumours with or without visceral tumours, and the regressive type showed initially increased and finally regressed LCA in the skin, associated with the development of visceral tumours. The non-tumour-associated pattern was characterized by initial transient small LCA in the skin without evidence of tumour formation. Birds with the tumour-associated pattern, regardless of type, had persistent nuclear inclusions (NI) and positive reactions against MDV1-specific phosphorylated polypeptides in the feather follicle epithelium (FFE) and initial R(1)-type (consisting mainly of small lymphocytes with a few lymphoblasts) to advanced T-type (consisting predominantly of lymphoblasts) feather pulp lesions (FPL). On the other hand, birds with the non-tumour-associated pattern formed transient NI and positive reactions against MDV1-specific phosphorylated polypeptides in the FFE and Ri-type to R(2)-type (consisting mainly of plasma cells with oedema) FPL. Antigen-positive lymphoid cells against MDV1-specific phosphorylated polypeptides were detected in both inflammatory and tumourous lesions, especially in the necrotic tumour lesions in the skin of birds showing the progressive type.     

Expression of cytotoxicity-associated genes in Marek's disease virus-infected chickens

Cytotoxic host responses to Marek's disease virus (MDV) have been attributed to both natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). However, the mechanisms of cell lysis initiated by these cytotoxic responses during MDV infection are not well defined. Therefore, the current study was aimed at elucidating the molecular mechanisms of host cytotoxic responses to MDV infection by investigating the expression of genes in the cell lysis pathway involving granzyme A. Genes encoding cytolytic proteins, NK lysin, and granzyme A were upregulated during early stages of infection, whereas the genes encoding major histocompatibility complex (MHC) class I and the DNA repair and apoptosis protein, poly(ADP-ribose) polymerase (PARP), were downregulated. These findings shed more light on the mechanisms of host response to MDV infection in chickens.     

Marek's disease virus (Kekava strain) replication in chickens, chick embryos and cell cultures.

In the course of 12 passages of Marek's disease virus (MDV) strain Kekava (MDV-Kekava) in chickens, the morbidity varied greatly (from 23 to 50 percent). MDV-Kekava produced plaques in cultures of chick embryo kidney and adult chicken kidney cells and chick embryo fibroblasts (CEF). The virus adaptation to the cultures was very slow. MDV-Kekava induced the formation of pocks on the chorioallantoic membranes (CAM) of chick embryos but the proportion of embryos with CAM lesions did not exceed 24 percent. Serial passaging of the virus in chick embryos beyond the 5th passage was unsuccessful. The results of virus isolation in chickens, cell cultures and chick embryos indicate the possibility of a long-term latent virus carrier state in chickens without development of tumours.     

Development and composition of lymphoid lesions in the spleens of Marek's disease virus-infected chickens: Association with virus spread and the pathogenesis of Marek's disease

Changes in lymphocyte distribution in spleens of Marek's disease virus (MDV) infected White Leghorn chickens of line 72 (MD susceptible) and line 61 (MD resistant) were studied by immunocytochemistry. Lymphocytes expressing the MDV antigen pp38 (predominantly B cells) were detected from 4 to 6 days post-inoculation (d.p.i.) but not at or after 8 d.p.i., and were more numerous in line 72. In line 61, infection resulted in depletion of B lymphocytes and an increase in T lymphocytes from 3 to 6 d.p.i., but no change in distribution of these cells. From 8 d.p.i., the B-dependent tissue began to recover and the T cells decreased in number. In line 72, infection caused a dramatic change in lymphocyte distribution, with formation of 'lymphoid lesions'. Diffuse, irregular patches of B lymphocytes, around the capillaries, became surrounded by large aggregates of TCRαβ1⁺ CD8⁺ and CD4⁺ lymphocytes, bordered by a band of TCRγδ⁺ lymphocytes. From 8 d.p.i., the B-dependent areas partially recovered, while TCRαβ1⁺ CD4⁺ and CD8⁺ lymphocytes, potentially transformed, became extensively scattered throughout the spleen. We conclude that in line 72, replication and spread of MDV is more efficient and T cell responses in early infection are greater, favouring the tumour stage of the disease.     

Relationship between Marek's disease virus load in peripheral blood lymphocytes at various stages of infection and clinical Marek's disease in broiler chickens.

Vaccination with herpesvirus of turkey (HVT) vaccine provides protection against clinical Marek's disease (MD) but does not preclude infection with wild-type MD virus (MDV). The quantity of MDV detected in circulating lymphocytes during the early period after infection may be a useful predictor of subsequent clinical MD later in the life. A study was designed to quantify MDV and HVT copy number in peripheral blood lymphocytes (PBL) using real-time polymerase chain reaction between days 5 and 35 post-challenge and to relate this to subsequent development of gross MD lesions. Female commercial broiler chickens were vaccinated with HVT or were sham-vaccinated at hatch, then challenged with MDV strain MPF-57 at day 2 post-vaccination and reared in positive-pressure isolators up to 56 days post-challenge, when all survivors were euthanized. All dead and euthanized chickens were examined post mortem for gross MD lesions. Birds were scored for MD lesions and mortality. MDV and HVT genome copy numbers were determined for each PBL sample. There was an increase in HVT load in PBL between days 7 and 37 post-vaccination, with marked increases between days 7 and 16 and again between days 30 and 37. There was a steady increase in MDV load to 35 days post-challenge. The mean MDV copy number (log(10)) was greater in chickens subsequently exhibiting gross MD lesions (5.05 +/- 0.21) than in those that did not (2.88 +/- 0.223), with the largest difference at 14 and 21 days post-challenge (P < 0.001). Quantification of MDV during early infection is therefore a potential tool for monitoring MD in broiler flocks.     

Quantitative pathogenicity of Marek's disease virus and vaccine virus strains in chickens: macroscopical aspects

Two virulent strains (JM and K) and one vaccine strain (CVI 988) of Marek's disease virus (MDV), together with two vaccine strains of the herpesvirus of turkeys (HVT) (FC 126 and PB-THV 1), all in the cell-associated state, were administered intramuscularly at 3.7 log TCID50 per dose to day-old SPF White Leghorn chickens. A control group of chicks received uninfected cells. The pathological parameters studied were onset and duration of clinical symptoms, mortality, bird weight and macroscopical lesions of peripheral nerves and visceral organs. Data were obtained from females autopsied at the age of 3, 8 and 20 weeks, and from chickens which died. Virological and serological data were procured mainly from males taken at various ages. The results indicate a clear distinction between virulent and vaccine strains. MD vaccines had no significant influence on bird weight and caused no mortality or macroscopical lesions, whereas the virulent MDV strains produced all these effects. Macroscopical lesions caused by the virulent MDV strains were seen predominantly in nerves (in about 50% of birds succumbing to MD) and gonads (in 0% to 80% of such birds depending on sex and on strain of MDV). Differences between the two virulent strains could be demonstrated. Strain JM induced earlier incidence and shorter duration of clinical disease. With strain JM death occurred earlier in females than in males. Strain K caused significantly more macroscopical lesions in gonads, heart and liver. Under the conditions of the experiment, detection of macroscopical lesions after inoculation with a virulent MDV strain was possible 3 weeks after inoculation.     

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.     

Detection of Marek's disease virus antigens and DNA in feathers from infected chickens

Two novel tests, enzyme-linked immunosorbent assay (ELISA) and dot-blot hybridization, were developed to detect and quantify the antigens and DNA of Marek's disease virus (MDV) in feather tips from infected chickens. In both methods, buffered extracts of the feathers served as the same test material. The ELISA technique was compared to the conventional agar-gel precipitation (AGP) test, using the same convalescent serum from a MDV-infected bird. Of 86 feather samples tested, 34 were negative by both methods, while 6 out of 52 were ELISA positive but AGP negative. Viral antigen detection by the AGP and ELISA methods was compared with the detection of MDV DNA by the dot-blot DNA hybridization technique. At an ELISA reading (OD 405) of 0.3 and above, only 5 out of 48 DNA extracts failed to hybridize with the MDV-DNA probe. The use of the radioactively labelled MDV-DNA probe for hybridization with DNA extracts from feather tips of MDV-infected chickens was both sensitive and specific, and there was good correlation among the different tests.     

Detection of Marek's disease virus antigen in chickens by a novel immunoassay

An immunoassay was developed to detect Marek's disease virus (MDV) antigen on the tips of feathers obtained from MDV-infected chickens. MDV in follicular debris on the feather tip was demonstrated by use of a specific monoclonal antibody. The principle of an indirect ELISA was employed and the feather tip was used as the solid phase. Presence of MDV was reflected by a dark brown precipitate on the feather tip which could be observed by naked eye. This test system proved to be more sensitive than the agar-gel precipitation (AGP) test as all feather tips of MDV-infected animals gave a positive reaction in the feather tip-ELISA whereas about a half yielded a detectable precipitate in the AGP. Advantages of this feather tip-ELISA and applications are discussed.     

Active simian immunodeficiency virus (strain smmPGm) infection in macaque central nervous system correlates with neurologic disease

Simian immunodeficiency virus strain smmPGm can induce neuropathology in macaques and is a model for the development of human HIV-related brain injury. For quantitative studies of proviral presence and expression in the central nervous system (CNS), we inoculated 8 macaques intravenously with the virus. Three animals were necropsied 2 to 4 weeks after development of infection, and we obtained lymphoid tissue biopsies from 5 animals before 5 weeks after infection. Peak plasma viral loads averaged 10 viral RNA Eq/mL at week 2, whereas cerebrospinal fluid viral loads peaked at 10 viral RNA Eq/mL. The proviral DNA loads and viral gag mRNA expression in tissues were quantified by real-time polymerase chain reaction. Two animals developed neurologic disease characterized by meningoencephalitis and meningitis. Proviral DNA levels in CNS tissues of these animals at necropsy revealed 10 and 10 copies/microg of DNA, respectively, whereas viral RNA expression in the CNS reached 100 to 1000 times higher levels than those seen in early necropsies. In sharp contrast, in 2 animals necropsied at later times without CNS disease, virus mRNA expression was not detected in any CNS tissue. Our results are consistent with the hypothesis that active virus expression in the CNS is strongly correlated with neurologic disease and that the event occurs at variable periods after infection.     

Host responses are induced in feathers of chickens infected with Marek's disease virus

Control measures are ineffective in curtailing Marek's disease virus (MDV) infection and replication in the feather follicle epithelium (FFE). Therefore, vaccinated birds which subsequently become infected with MDV, shed the virulent virus although they remain protected against disease. The present study investigated host responses generated against MDV infection in the feather. We observed that in parallel with an increase in viral genome load and viral replication in the feather, there was a gradual but progressive increase in infiltration of CD4+ and CD8+ T cells into the feather pulp of MDV-infected chickens, starting on day 4 and peaking by day 10 post-infection. Concomitant with infiltration of T cells, the expression of interleukin (IL)-18, IL-6, interferon (IFN)-gamma and major histocompatibility complex class I genes was significantly enhanced in the feather pulp of MDV-infected chickens. The finding that host responses are generated in the feather may be exploited for developing strategies to control MDV infection in the FFE, thus preventing horizontal virus transmission.     

Diagnostic utility of squash (smear) technique in the inflammatory lesions of central nervous system

Smears of histologically verified inflammatory lesions of central nervous system (CNS) during 12 years were analysed to determine the diagnostic utility and accuracy of squash (smear) technique. Two hundred and sixteen (216) lesions were analysed. Smears could not be interpreted in 72 lesions (33.33%) as the tissue could not be spread or not representative. Tuberculosis was the commonest etiology (70.83%) followed by bacterial, fungal and parasitic infections. To conclude, smear technique is a useful modality in establishing diagnosis in inflammatory lesions of CNS, but its usefulness is limited by difficulties in spreading tissue.