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.     

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.     

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.     

Influence of vaccination with CVI988/Rispens on load and replication of a very virulent Marek's disease virus strain in feathers of chickens

Several highly efficacious vaccines are currently available for control of Marek's disease, a lymphoproliferative disease in chickens. However, these vaccines are unable to prevent infection with Marek's disease virus (MDV) in vaccinated birds. This leads to shedding of virulent MDV from feather follicle epithelium and skin epithelial cells of vaccinated and infected chickens. The objective of the present study was to study the interactions between a vaccine strain (CVI988/Rispens) and a very virulent strain of MDV (RB1B) in feathers. We examined genome load and replication of CVI988 and MDV-RB1B strains at various time points post infection. Moreover, we evaluated cytokine expression in feathers as indicators of immunity generated in response to vaccines against MDV. Analysis of feathers collected between 4 and 21 days post infection (d.p.i.) revealed a steady level of CVI988 genome load in the presence or absence of RB1B. Infection with MDV resulted in a significant increase in RB1B genome load peaking at 14 d.p.i. Importantly, vaccination with CVI988 resulted in a significant reduction in accumulation of MDV-RB1B in feathers. RB1B genome accumulation in feather tips was associated with increased expression of interferon-α at 14 d.p.i. and interferon-Sγ at earlier time points, 4 and 7 d.p.i. compared with 10 and 14 d.p.i. Interleukin-10 and interleukin-6 were up-regulated at 14 d.p.i. in the infected groups. This study expands our understanding of the dynamics of replication of vaccine and virulent MDV strains in the feathers and illuminates mechanisms associated with immunity to Marek's disease.     

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.     

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.     

Cytopathology caused by the AC-1 isolate of Marek's disease virus in the feather follicle epidermis

The AC-1 strain of Marek's disease herpesvirus (MDV), recently isolated from an outbreak of Marek's disease in vaccinated chickens, was used for intraperitoneal inoculation of one-day-old Single Comb White Leghorns. Chicks were necropsied 12 to 32 days post inoculation and skin was collected for electron microscopic studies. This paper focuses on early stages of MDHV development in feather follicle epithelial cells and on cytoplasmic changes that occur in formation and release of virus particles. In some nuclei we observed helical threads of DNA which were partially encircled by capsid membranes. This was interpreted to be an early stage of nucleocapsid development. In the infected epidermal cells, the smooth endoplasmic reticulum vesiculated and appeared to be hyperplastic. Immature virus particles released into cytoplasm acquired their outer envelope while budding into cytoplasmic vesicles. Vesicles containing mature viruses were referred to as cytoplasmic vesicular inclusions. It appeared that the degenerative process in cells facilitated the release of virions from the feather follicle epithelium.     

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.     

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.     

Scanning electron microscopy of the bursa of fabricius from normal and testosterone-treated embryos

Scanning electron microscopy (SEM) has revealed the presence of projecting follicles (PF) and button-like follicles (BLF) in the bursa of Fabricius. This study was designed to examine the embryonic bursa with SEM to ascertain which type of follicle appears first and to compare the SEM of the bursa from normal embryos with those having received testosterone propionate (TP) on the 11th day of incubation. The bursa of the latter embryos exhibits an arrested lymphoid development. PF appeared in normal embryos by 16 days and were well developed by 18 days of embryonic development. Inter-follicular epithelium was apparent by 21 days of embryonic development in normal embryos. On the other hand, bursal follicles and interfollicular epithelium failed to form in TP birds. The TP-birds exhibited a characteristic pebble-like epithelium which may attest to the regressive influence of TP on bursal epithelium or to an arrested stage of epithelial development. The PF may lead to the development of BLF or the BLF may be derived independently of PF.     

Marek's disease in broiler chickens: Effect of route of infection and herpesvirus of turkey-vaccination status on detection of virus from blood or spleen by polymerase chain reaction,and on weights of birds,bursa and spleen

The polymerase chain reaction (PCR) has recently emerged as an additional tool for the monitoring and diagnosis of Marek's disease. We investigated a number of factors that may influence the interpretation of PCR results in commercial broiler chickens including the effects of route of infection and herpesvirus of turkeys (HVT)-vaccination status. We also investigated the suitability of peripheral blood lymphocytes (PBL) and spleen as tissues for Marek's disease virus (MDV) detection. HVT-vaccinated and unvaccinated commercial broiler chickens were challenged or not challenged with virulent MDV either by intraperitoneal injection or inhalation of feather dust containing the virus. Blood and spleen samples were collected at weekly intervals to day 35 post-infection for PCR of spleen or PBL. Live weight and lymphoid organ weights were also measured. Spleen and PBL were found to provide similar sensitivity of detection of MDV with a small advantage in favour of spleen. In terms of the timing of detection of MDV, intraperitoneal challenge broadly mimicked natural challenge via inhalation, although infection of birds by inhalation of infective feather dust resulted in slightly later but more complete detection of MDV in challenged birds. Vaccination with HVT delayed the detection of MDV by approximately 10 to 14 days and did not protect against the reduced growth observed in challenged chickens at day 35 post-challenge.     

Molecular and biological characterization of a Marek’s disease virus field strain with reticuloendotheliosis virus LTR insert

A Marek’s disease virus (MDV) field strain designated GX0101 was isolated from a layer flock and confirmed to be a recombinant virus with an insert of a long terminal repeat (LTR) from the reticuloendotheliosis virus (REV). A chimeric molecule containing an REV-LTR insert of 539 bp and its flanking sequences from MDV was amplified and sequenced. An REV-LTR downstream from the Internal Repeat Short (IRS) region has 77.4–98.6% homology to seven REV field strains isolated from different avian species in different parts of the world. The insertion site is located downstream of SORF 1 and upstream of SORF2 in the IRS region near the junction with the Unique Short (US) region in the MDV serotype 1 genome. Chicken experiments were conducted to determine the oncogenicity of the recombinant GX0101 virus and its transmissibility to contact chickens. Dot blot hybridization was used to detect the presence of the pp38 gene in feather tips from GX0101 or Md5 infected and contact birds. The pp38 was detected in GX0101 contact birds about 1–2 weeks earlier than in Md5 birds when both groups were vaccinated with HVT vaccine. Long term pathogenicity tests in specific pathogen free (SPF) chickens reveal that the recombinant GX0101 has a higher virulence than GA, but less virulence than Md5, the very virulent pathotype of MDV. This is the first report on an oncogenic serotype 1 MDV field strain with LTR insert and its pathogenicity.     

Development of a rapid and specific loop-mediated isothermal amplification detection method that targets Marek's disease virus meq gene

A rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) method was developed and evaluated for the detection of Marek's disease virus (MDV) by amplification of conserved MDV meq gene sequences. LAMP is an innovative technique that allows the rapid detection of targeted nucleic acid sequences under isothermal conditions without the need for complex instrumentation. In this study, meq gene sequences were amplified successfully from different MDV strains by LAMP within 60min and no cross-reactivity was observed in a panel of related viruses that were associated with diseases of chickens. The detection limit of LAMP was 3.2 copies/million cells compared with 320 copies/million cells required for conventional PCR. Positive detection rates were assessed using either LAMP or PCR by examination of feather follicles that were collected from chickens infected experimentally with either strain J-1 (n=20) or strain Md5 (n=17), In addition to these samples, three isolates that were suspected to have been infected in the clinic were also tested. Results showed that the positive detection rate for LAMP was 95% (38/40), compared with 87.5% (35/40) and 90% (38/40) for strains J-1 and Md5 by PCR, respectively. These results indicated that the LAMP assay was more sensitive, rapid and specific than conventional PCR for the detection of MDV. This easy-to-perform technique will be useful for the detection of MDV and will aid in the establishment of disease control protocols.     

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.     

Transmission electron microscopic and immunohistochemical observations of resting follicles of feathers in chicken show massive cell degeneration

The molting cycle of feathers includes an anagen (growth) stage, a likely catagen stage where the feather follicles degenerate, and a resting stage where fully grown feathers remain in their follicles and are functional before molting. However, the cytological changes involved in the resting and molting stages are poorly known, so the results of an ultrastructural analysis of these processes in adult chick feathers are presented here. The study showed that the dermal papilla shrinks, and numerous cells present increased heterochromatin and free collagen fibrils in the extracellular matrix. Degeneration of the germinal epithelium of the follicle—the papillary collar—occurs with an initial substantial contraction of cells followed by an increase in heterochromatin, vesicle and lipid accumulation, and membrane and organelle degeneration. Desmosomes are still present between degenerating epithelial cells, but ribosomes and tonofilaments disappear. This suggests that cell necrosis initially proceeds as a major contraction resembling apoptosis—a process termed necroptosis, which was previously also shown to occur during the formation of barbs and barbules in mature down and pennaceous feathers. This study suggests that, aside from apoptosis, the collar epithelium degenerates due to external factors, in particular the retraction of blood vessels supplying the dermal papilla. In contrast, revascularization of the dermal papilla triggers a new phase of feather growth (anagen).     

Lymphocyte depletion in ileal Peyer's patch follicles in lambs infected with Eimeria ovinoidalis.

A total of 14 lambs were experimentally infected with Eimeria ovinoidalis in two separate experiments in two consecutive years. Nine lambs served as uninoculated controls. Material was collected from the ileum 2 weeks after infection in eight lambs and 3 weeks after infection in six lambs. Lambs examined 2 weeks after infection had normal follicles. After three weeks, the follicle-associated epithelium covering the lymphoid follicles of the ileal Peyer's patches showed fusions with adjacent absorptive epithelium, focal hyperplasia, and occasionally necrosis. Macrogametes, microgamonts, and oocysts were often found in the follicle-associated epithelium and the dome region. Various degrees of lymphocyte depletion were present in the ileal lymphoid follicles in all six infected lambs 3 weeks after infection, and four lambs had decreased follicle size. Reduced staining for leukocyte common antigen (CD45), B-cell markers, and the proliferation marker Ki-67 was present in these lambs. Application of the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling method for apoptotic cells revealed decreased staining in the ileal lymphoid follicles 3 weeks after infection. A marker of follicular dendritic cells, 5'- nucleotidase, showed increased reactivity, probably due to condensation of reticular cells following loss of follicle lymphocytes. Reduced staining for carbonic anhydrase in the follicle-associated epithelium and the domes was present in all six lambs examined 3 weeks after infection, indicating decreased production of carbonic anhydrase-reactive 50-nm particles and a decreased lymphoproliferative stimulus. In conclusion, the present study shows that severe E. ovinoidalis infection in lambs causes lesions of the follicle-associated epithelium and may result in lymphocyte depletion and atrophy of the ileal Peyer's patch follicles.