Cutaneous lesions in broiler chickens spontaneously affected with Marek's disease

A total of 91 8- to 9-week-old broiler chickens with Marek's disease (MD) skin tumours ("skin leukosis"), collected from 15 farms at the processing plants, were examined pathologically. Grossly, the skin lesions comprised various sizes and numbers of feather follicular nodules, which tended to fuse with each other as their size increased. Histologically, the lesions were classified into five types: type A was small lymphoid cell aggregates (LCA) consisting mostly of small lymphocytes with a few lymphoblasts and very rare mitotic figures; type B had large LCA consisting mainly of small lymphocytes with considerable numbers of lymphoblasts and very rare mitotic figures; type C was characterized by large coalesced LCA consisting almost equally of small lymphocytes and lymphoblasts with infrequent mitotic figures; type D exhibited very large coalesced LCA consisting mainly of lymphoblasts with some small lymphocytes and occasional mitotic figures; type E had very large coalesced LCA consisting almost completely of lymphoblasts with frequent mitotic figures. The histological MD cutaneous lesions were related to the size of gross skin nodules, the small feather follicular nodules consisted mainly of types B and C with type A, whereas large fused feather follicular nodules were composed mainly of types D and E. Nuclear inclusions were frequently found in the feather follicular epithelium in all skin nodules except for the largest fused ones. MD visceral lesions were more pronounced in birds having marked skin leukotic lesions. Feather pulp lesions (FPL) were more related to the visceral than the skin lesions; the constituent cells of FPL were compatible with those of the former lesions.     

Demonstration of a marek's disease virus-specific antigen in tumour lesions of chickens with marek's disease using monoclonal antibody against a virus phosphorylated protein

By use of monoclonal antibody against a Marek's disease virus (MDV) serotype 1-specific phosphorylated protein, MDV antigen-positive cells were demonstrated in tumour lesions of various visceral organs of chickens with Marek's disease. However, these tumour lesions did not appear to have the MDV glycoproteins gA and gB, which are considered to be late gene products of the virus genome gA and gB as well as the phosphorylated protein were detected in the feather follicle epithelium, which is a permissive site for MDV replication.     

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.     

Direct detection of Marek’s disease virus in poultry dust by loop-mediated isothermal amplification

Marek’s disease virus (MDV) is a serious concern for poultry production and represents a unique herpesvirus model. MDV can be shed by doubly infected chickens despite vaccination. The fully infectious MDV particles are produced in the feather follicle epithelium (FFE), and MDV remains infectious for many months in fine skin particles and feather debris. Molecular biology methods including PCR and real-time PCR have been shown to be valuable for the detection of MDV DNA in farm dust. Recently, loop-mediated isothermal amplification (LAMP) was found to be useful in the detection of MDV in feathers and internal organs of infected chickens. LAMP is also less affected by the inhibitors present in DNA samples. Taking into account the advantages of LAMP, direct detection of MDV DNA in poultry dust has been conducted in this research. The detection of MDV DNA was possible in 11 out of the 12 examined dust samples without DNA extraction. The DNA was retrieved from dust samples by dilution and incubation at 95 °C for 5 min. The direct detection of MDV DNA in the dust was possible within 30 min using a water bath and UV light. The results were confirmed by electrophoresis and melting curve analysis of the LAMP products. Our results show that LAMP may be used to test for the presence of virulent MDV in poultry farm dust without DNA extraction.     

The use of serotype 1- and serotype 3-specific polymerase chain reaction for the detection of Marek's disease virus in chickens

A serotype 1- and serotype 3-specific detection of Marek's disease virus (MDV) by polymerase chain reaction (PCR) was developed. The sensitivity of the method when applied to cell culture grown virus was comparable with that of cultivation. The method was applied to various tissue samples from chickens experimentally inoculated with serotype 1 or serotype 3 MDV.The serotype 1 strains CVI988 and RB-1B could be detected in feather follicle epithelium up to 56 and 84 days post-inoculation (p.i.), respectively, while the MDV-3 serotype was detected until 42 days p.i. The purpose of this study was to develop and evaluate a reliable and easy-to-handle method for surveillance of the occurrence of MDV in chicken flocks. We emphasize the development of a method, which can be applied to types of samples conveniently collected in the field, e.g. feather tips and blood samples. In addition, the PCR was applied to samples collected from four commercial table egg layer flocks of young stock or pullets vaccinated with either serotype 1 (CVI988) or serotype 3 (HVT) vaccine. These flocks had various clinical signs of Marek's disease. MDV-1 was detected in buffy-coat cells, spleen, liver, skin, feather tips and ovaries. The detection of MDV in feather tips appeared to be as sensitive as co-cultivation of buffy-coat cells, although an inhibiting factor was observed in extracts from feather tips of non-white chickens. This inhibition could be overcome in most extracts by applying a bovine serum albumen pretreatment. The PCR proved to be a convenient tool for the monitoring of MDV in the poultry population, and feather tips were the most convenient and sensitive samples.     

Mustererkennung zur Differenzialdiagnose lymphoepithelialer Läsionen der Speicheldrüsen

The prototype of a salivary lymphoepithelial lesion is the autoimmune disease Sjögren’s syndrome with the characteristic lymphoepithelial duct lesions (LEL). The distinction of Sjögren’s syndrome from cases with initial transformation into associated marginal zone B-cell lymphoma (MALT type) can be very challenging, whereby the presence of small “halos” can lead to over-diagnosis. The HIV-associated cystic lymphoepithelial lesion can be histologically almost identical to Sjögren’s syndrome and often needs clinical correlation. The sporadic lymphoepithelial cyst of the parotid gland is a frequent finding and has no clinical consequence; however, this entity needs to be identified and distinguished from the above-mentioned entities. The most frequent diagnosis in resected submandibular glands is chronic-fibrosing sialadenitis, so-called Küttner’s tumour. Altogether, there is a wide spectrum of lymphoepithelial interaction in the area of salivary glands, including biphasic lymphoepithelial tumours with an obligate lymphoid component, epithelial tumours with facultative tumour-associated lymphoid proliferation, and different processes of intraparotid lymph nodes. The immunohistological reaction for pan-keratin can be very helpful for a thorough pattern analysis of the different lymphoepithelial lesions. The relative frequency of the lesions in different salivary glands can also be diagnostically helpful.     

Absence of Marek's disease virus antigen in the feather follicle epithelium of vaccinated chicks demonstrated by immunofluorescence

In chickens vaccinated with MDV strain CVI 988, HVT strain FC 126 or PB-THV 1, no MDV-specific antigens could be demonstrated in the feather follicle epithelium by immunofluorescence (IF). In chickens given virulent MDV strains, the epithelium of the feather follicle was positive in IF. In an experiment where chickens were vaccinated with strain CVI 988, positive IF was observed in the lung, bursa and pancreas, but not in the feather follicle epithelium, kidney, cloaca, or caecal tonsils. Absence of IF antigen in the skin may be indicative of avirulence of the strain of MDV.     

Comparative studies on pathogenical, virological and serological properties of Marek's disease virus isolated from Japanese quail and chicken

The QM-1 strain of Marek's disease virus (MDV) isolated from Japanese quail (JQ) induced higher mortality (96%) with MD lesions in quail than the JM strain of MDV isolated from chickens (C) (21%). In contrast both viruses induced MD in 90% of chickens. Pathological changes produced in quail inoculated with JQ-MDV or C-MDV were similar. However, the incidence of tumour lesions in the duodenum of quail inoculated with JQ-MDV was significantly higher. Neither virus induced tumour lesions in the duodenum of chickens. Appearance of viraemia in quail inoculated with MDV was delayed and its level was low as compared with chicken cases. MDV antigens in feather tips appeared in quail inoculated with JQ-MDV but not with C-MDV. No MDV was recovered from the MDV-inoculated quail by direct kidney cell cultures, although MDV was recovered by subpassage of the cultures into chicken embryo fibroblasts. No antibodies were detected in the affected quail by agar gel precipitation test.     

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.     

Replication of Marek's disease virus in chicken feather tips containing vaccinal turkey herpesvirus DNA

The presence of herpesvirus of turkeys (HVT) DNA in the feather tips of chickens vaccinated with HVT was assessed by dot blot hybridisation with a probe specific for HVT and lacking homology to MDV DNA. Only small amounts of HVT DNA were detected in the feather tips of chickens that were vaccinated or left in contact with HVT vaccinated chickens. However when chickens were challenged with virulent MDV, HVT DNA was detected in the feather tips of vaccinated chickens and the largest amount was detected 35 days after vaccination. HVT DNA was recovered in significantly higher quantities from some of the MDV-infected chickens than from those infected by contact. This suggests that MDV infection may provide helper functions for HVT. MDV DNA was identified in the feather tips of MDV-challenged chickens from 25 to 45 days after challenge. Thus, immunisation of chickens with HVT did not prevent the replication of MDV in the feather tips but only diminished it.     

Differential attenuation of the induction by Marek's disease virus of transient paralysis and persistent neurological disease: A model for pathogenesis studies

Since different biological characteristics of Marek's disease virus (MDV) are attenuated at different passage levels in cell culture, an analysis of attenuation times provides, in theory, a model for establishing the presence or absence of relationships between characteristics, thus providing a basis to link them to genetic changes in the causative virus. We have used this model to better understand the pathogenesis of the central nervous system infection as well as to evaluate the relationship of clinical neurological disease to various other parameters of MDV infection. Inoculation of 15 2 7 crossbred chickens with strain 648A of very virulent plus MDV at different passage levels (between 10 and 100) showed that two neurological syndromes (transient paralysis (TP) and persistent neurological disease), were attenuated at different passage levels. While strain 648A lost the ability to induce TP between 30 and 40 passages in chicken embryo fibroblast cultures, an event closely related with all parameters of MDV infection involving viral replication (early cytolytic infection in lymphoid organs and viral replication in the feather follicle epithelium), the ability to induce persistent neurological disease was lost between 80 and 90 passages in chicken embryo fibroblasts, coincident with the loss of neoplastic lesions in peripheral nerves and other visceral organs. These data strongly suggest that transient paralysis and persistent neurological disease are unrelated and differently regulated. Moreover, comparison of brain changes induced by strain 648A at passage level 30 (TP) and at passage level 40 (no TP) also contributed to a better understanding of which brain alterations are associated with the onset of TP. The use of viruses at different passage levels with varying degrees of attenuation is presented as a useful tool for studying pathogenesis of MDV infection.     

Deletion of the Marek’s disease virus UL41 gene (vhs) has no measurable effect on latency or pathogenesis

Marek’s disease is an economically important disease of poultry and is caused by an alphaherpesvirus, Marek’s disease virus (MDV). The predicted protein product of the MDV UL41 open reading frame has significant protein sequence identity with the virion host shutoff (vhs) protein gene in other alphaherpesviruses. To determine whether the MDV UL41 gene functions as a vhs protein, we utilized a transient co-transfection assay and demonstrated that the MDV UL41 protein was as active in degrading RNA as the vhs protein of herpes simplex virus type 1. To evaluate whether the MDV UL41 gene was involved in pathogenesis, we deleted the MDV UL41 gene. The UL41 deletion mutant replicated in cell culture as well as the parental MDV. The deletion mutant was inoculated into susceptible day-old chicks. The pattern and degree of tumor lesions and neurovirulence produced by the deletion mutant was same as the pattern of lesions induced by the parental virus. The only observable difference between the inoculation of MDV and the MDV deletion mutant was that the early in vivo cytolytic infection with the deletion mutant was of a longer duration than in the non-mutant MDV.     

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.     

Isolation of Marek's disease virus from Japanese quail with lymphoproliferative disease

Sixty-one Japanese quail from eight flocks with problems of recurring outbreaks of lymphoproliferative diseases resembling Marek's disease (MD), were examined aetiologically. Gross lymphomatous lesions were seen in 17 of the quail and 11 out of 56 quail had MD virus (MDV) feather tips antigens. MDV antibody was detectable in only one of 22 quail. None of 9 quail had antibodies against reticuloendotheliosis virus (REV). No MDV was isolated from the total 42 materials of quail using cell culture technique. No REV and avian leukosis virus (ALV) were isolated from some of them. However, specific-pathogen-free chicks inoculated with the blood materials revealed MD. and four MDVs were recovered from them. The isolates proved free from REV and ALV. The isolates were placed into serotype 1 by the indirect immunofluorescent antibody test. These results indicate that MDV is aetiologically involved in the present outbreaks of lymphoproliferative disease in Japanese quail.     

Marek's disease virus gene clones encoding virus-specific phosphorylated polypeptides and serological characterization of fusion proteins

Marek's disease virus (MDV) gene clones, RA2 and GA8, constructed inE. coli bacteriophage lambda-gt11 (gt11) were identified by a monoclonal antibody (MAb), H19.47, against a putative transformation-related viral antigen consisting of a complex of three phosphorylated polypeptides, pp41, pp38, and pp24. Both recombinants have a MDV-DNA insert of about 0.5 kb and are mapped to the region ofBamHI-H orEcoRI-X fragments of the MDV genome by Southern blot hybridization. Immunoblot and immunoprecipitation with H19.47 identified a recombinant beta-galactosidase-MDV 140-kD fusion protein for RA2 and a 127-kD fusion protein for GA8.Immunoprecipitation of³⁵S-methionine-labeled, MDV-infected chicken embryo fibroblasts (CEF) with antisera against RA2 and GA8 fusion proteins recognized five polypeptides, of which three (p41, p38, and p24) are specified by H19.47 and the remaining two, p135 and p20, have not been previously identified. Immunoprecipitation of³²P-phosphate-labeled or³H-glucosamine-labeled, GA-MDV-infected CEF with the antiserum against RA2 fusion protein identified a phosphorylated polypeptide of 38 kD and two glycoproteins of 60 and 49 kD, respectively. The antisera against recombinant fusion proteins thus revealed the existence of epitopes common to the phosphorylated polypeptides and other MDV-specific polypeptides.Sera from chickens or mice hyperimmunized with the purified fusion proteins reacted with serotype 1, MDV-infected CEF in the fluorescent antibody (FA) test to significant titers. These immune sera did not react with either serotype II or III, indicating the serotype specificity of the phosphorylated polypeptides.Requests for reprints should be addressed to Lucy F. Lee, USDA, Agricultural Research Service, Regional Research Laboratory, 3606 East Mount Hope Road, East Lansing, MI 48823, USA.