Coinfection of specific-pathogen-free chickens with Marek's disease virus (MDV) and chicken infectious anemia virus: effect of MDV pathotype

Both Marek's disease virus (MDV) and chicken infectious anemia virus (CIAV) infections are prevalent in chickens throughout the world. In the past decade, MDV strains with increased virulence (very virulent plus MDV pathotype [vv+MDV]) have been isolated. The purpose of this experiment was to determine the effects of coinfection of chickens with CIAV and a vv+MDV isolate. Specific-pathogen-free chickens were inoculated at 1 day posthatch with RB1B (very virulent MDV pathotype [vvMDV]) only, 584A (vv+MDV) only, CIAV only, RB1B + CIAV, 584A + CIAV, or nothing. Samples of spleen, thymus, and bursa of Fabricius were collected at 4, 7, 10, and 13 days postinoculation (DPI). Thymic and bursal atrophy at 13 DPI and final mortality at 30 DPI were significantly greater in chickens inoculated with 584A with or without added CIAV, or with RB1B plus CIAV, compared with birds inoculated with RB1B alone. Both amounts of virus reisolated and levels of virus detected by quantitative-competitive polymerase chain reaction were greater at 4 DPI in 584A inoculates compared with RB1B inoculates. To monitor the early cytolytic infection, northern analysis was done with a probe for the MDV immediate early gene ICP4 (infected cell protein 4). In the absence of CIAV, ICP4 expression was more apparent in chickens inoculated with 584A than in those inoculated with RB1B. CIAV coinfection increased ICP4 expression in the spleens of chickens infected with RB1B. These results indicated that inoculation of chickens with the 584A isolate caused a more robust early cytolytic infection compared with inoculation with RB1B alone and support the classification of 584A as a vv+MDV strain. Coinfection with CIAV exacerbated vvMDV strain RB1B infection. The extent of this exacerbation was less evident when birds were coinfected with 584A and CIAV.     

A comparative evaluation of the protective efficacy of rMd5deltaMeq and CVI988/ Rispens against a vv+ strain of Marek's disease virus infection in a series of recombinant congenic strains of White Leghorn chickens

Marek's disease (MD) is a lymphoproliferative disease of domestic chickens caused by a highly infectious, oncogenic alpha-herpesvirus known as Marek's disease virus (MDV). MD is presently controlled by vaccination. Current MD vaccines include attenuated serotype 1 strains (e.g., CVI988/Rispens), avirulent serotype 2 (SB-1), and serotype 3 (HVT) MDV strains. In addition, recombinant MDV strains have been developed as potential new and more efficient vaccines to sustain the success of MD control in poultry. One of the candidate recombinant MDV strains, named rMd5deltaMeq, was derived from Md5, a very virulent strain of MDV lacking the MDV oncogene Meq. Our earlier reports suggest that rMd5deltaMeq provided protection equally well or better than commonly used MD vaccines in experimental and commercial lines of chickens challenged with very virulent plus (vv+) strains of MDV. In this study, maternal antibody-positive (trial 1) and negative (trial 2) chickens from a series of relatively MD resistant lines were either vaccinated with the rMd5deltaMeq or CVI988/Rispens followed by infection of a vv+ strain of MDV, 648A, passage 10. This report presents experimental evidence that the rMd5deltaMeq protected significantly better than the CVI988/Rispens (P < 0.01) in the relatively resistant experimental lines of chickens challenged with the vv+ strain of MDV. Together with early reports, the rMd5deltaMeq appeared to provide better protection, comparing with the most efficacious commercially available vaccine, CVI988/Rispens, for control of MD in lines of chickens regardless of their genetic background.     

Similar pattern of iNOS expression, NO production and cytokine response in genetic and vaccination-acquired resistance to Marek's disease

NO is produced by macrophages through activation of the inducible enzyme NOS and its production is triggered as an antiviral and antitumoral immune mechanism. Replication of Marek's disease herpes virus (MDV) is inhibited by NO in vitro. MDV induces T-lymphomas in the chicken and a genetic resistance to tumor development has been linked to the B21 major histocompatibility complex. During the first initial week of viral replication after inoculation of the highly virulent RB-1B MDV strain, histocompatible B21/B21 chickens developed strong iNOS expression and NO production capacity in the spleen, in parallel with strong systemic NO production in the serum. Comparable NO response was not seen with the vaccinal strain HVT. In contrast, reduction in spleen macrophage number and delay in iNOS gene expression was observed in genetically susceptible B13/B13 chickens after MDV infection, in addition to suppression of IFN-gamma-inducible NO production. However, vaccination with HVT 3 days before RB-1B inoculation restored strong iNOS gene expression in the spleen 1 week later and inducible NO production 3 weeks later. Following the pattern of iNOS gene expression, early strong expression of cytokines with powerful iNOS-inducing activity such as IFN-gamma and CC chemokines from the MIP family (MIP-1beta, K203) was observed in genetic resistance and resistance acquired after vaccination with HVT. In conclusion, resistance to MDV appeared preferentially linked in both types of resistance to the early establishment of cytokine induction characteristic of a Th1 immune response, thus favoring the development of an early and strong NO response.     

Protective efficacy of Marek's disease virus (MDV) CVI-988 CEF65 clone C against challenge infection with three very virulent MDV strains

Comparative 50% protective dose (PD50) assays were performed using a plaque-purified preparation of Marek's disease virus (MDV) strain CVI-988 at the 65th chicken embryo fibroblast (CEF) passage level (MDV CVI-988 CEF65 clone C) and three commercial MD vaccines: herpesvirus of turkeys (HVT) FC126, MDV CVI-988 CEF35, and a bivalent vaccine composed of HVT FC126 and MDV SB-1. In addition, comparative PD50 assays were performed in groups of chickens with maternal antibody to each of the three vaccines. Three representatives of the newly emerged biovariant very virulent (vv) MDV strains-RB/1B, Tun, and Md5-were employed as challenge virus. The experiments made feasible the differentiation between virulent MDV and vvMDV strains, within serotype 1. Vaccination with CVI-988 clone C vaccine resulted in PD50 estimates of about 5 plaque-forming units (PFUs) against challenge infection with each of the three vvMDV strains. The PD50 estimate of CVI-988 clone C vaccine was 12-fold below the PD50 of HVT FC126. The protective synergism of bivalent vaccine, composed of HVT and SB-1, was confirmed by groups given the lowest vaccine doses. The bivalent vaccine, however, resulted in incomplete protection in groups given the highest vaccine doses. Homologous maternal antibodies to serotype 1 caused a fivefold increase in the PD50 estimate of CVI-988 clone C. Heterologous maternal antibodies against HVT did not interfere with efficacy of CVI-988 clone C vaccination. However, the combination of maternal antibodies against both HVT and SB-1 (serotypes 2 and 3) showed a strong adverse effect on CVI-988 clone C vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative analysis of Marek's disease virus (MDV) glycoprotein-, lytic antigen pp38- and transformation antigen Meq-encoding genes: association of meq mutations with MDVs of high virulence

Marek's disease (MD) is a highly contagious lymphoproliferative and demyelinating disorder of chickens. MD is caused by Marek's disease virus (MDV), a cell-associated, acute-transforming alphaherpesvirus. For three decades, losses to the poultry industry due to MD have been greatly limited through the use of live vaccines. MDV vaccine strains are comprised of antigenically related, apathogenic MDVs originally isolated from chickens (MDV-2), turkeys (herpesvirus of turkeys, HVT) or attenuated-oncogenic strains of MDV-1 (CVI-988). Since the inception of high-density poultry production and MD vaccination, there have been two discernible increases in the virulence of MDV field strains. Our objectives were to determine if common mutations in the major glycoprotein genes, a major lytic antigen phosphoprotein 38 (pp38) or a major latency/transformation antigen Meq (Marek's EcoRI-Q-encoded protein) were associated with enhanced MDV virulence. To address this, we cloned and sequenced the major surface glycoprotein genes (gB, gC, gD, gE, gH, gI, and gL) of five MDV strains that were representative of the virulent (v), very virulent (vv) and very virulent plus (vv+) pathotypes of MDV. We found no consistent mutations in these genes that correlated strictly with virulence level. The glycoprotein genes most similar among MDV-1, MDV-2 and HVT (gB and gC, approximately 81 and 75%, respectively) were among the most conserved across pathotype. We found mutations mapping to the putative signal cleavage site in the gL genes in four out of eleven vv+MDVs, but this mutation was also identified in one vvMDV (643P) indicating that it did not correlate with enhanced virulence. In further analysis of an additional 12 MDV strains, we found no gross polymorphism in any of the glycoprotein genes. Likewise, by PCR and RFLP analysis, we found no polymorphism at the locus encoding the pp38 gene, an early lytic-phase gene associated with MDV replication. In contrast, we found distinct mutations in the latency and transformation-associated Marek's EcoRI-Q-encoded protein, Meq. In examination of the DNA and deduced amino acid sequence of meq genes from 26 MDV strains (9 m/vMDV, 5 vvMDV and 12 vv+MDVs), we found distinct polymorphism and point mutations that appeared to correlate with virulence. Although a complex trait like MDV virulence is likely to be multigenic, these data describe the first sets of mutations that appear to correlate with MDV virulence. Our conclusion is that since Meq is expressed primarily in the latent/transforming phase of MDV infection, and is not encoded by MDV-2 or HVT vaccine viruses, the evolution of MDV virulence may be due to selection on MDV-host cell interactions during latency and may not be mediated by the immune selection against virus lytic antigens such as the surface glycoproteins.     

Susceptibility of adult chickens, with and without prior vaccination, to challenge with Marek's disease virus

Marek's disease (MD) outbreaks can occur in previously healthy adult layer or breeder flocks. However, it is not clear whether such outbreaks are caused by recent challenge with highly virulent (vv and vv+) strains of MD virus (MDV; i. e., new infection hypothesis) or by exacerbation of an earlier MDV infection (i. e., old infection hypothesis). To discriminate between these hypotheses, adult White Leghorn chickens of laboratory strains or commercial crosses with or without prior vaccination or MDV exposure were challenged at 18-102 wk of age with highly virulent MDVs, and lesion responses were measured. Horizontal transmission was studied in one trial. Challenge of adult chickens, which were free from prior MDV vaccination or exposure, with highly virulent MDV strains induced transient paralysis or tumors in 60%-100% of 29 groups (mean = 91%), and horizontal spread of virus was detected. The magnitude of the response was similar to that induced by challenge at 3 wk of age. In contrast, comparable challenge of adult chickens, which had been vaccinated or exposed to MDV early in life, induced transient paralysis or tumors in 0%-6% of 12 groups (mean = 0. 5%), although some birds showed limited virologic evidence of infection and transmission of the virus to contacts. The MD responses were influenced by the virulence of the challenge virus strain, and to a lesser extent by virus dose and route of exposure. Strong inflammatory lesions were induced in the brain and nerves of adult specific pathogen-free (SPF) chickens at 9-15 days after infection. The low susceptibility of previously vaccinated and exposed groups to challenge at > or =18 wk of age suggests that late outbreaks of MD in commercial flocks are not likely a result of recent challenge alone and that additional factors could be involved.     

Cytotoxic T lymphocyte responses to Marek's disease herpesvirus-encoded glycoproteins

Cell-mediated immune responses are important for protective immunity to Marek’s disease (MD), especially because MD herpesvirus (MDV) infection is strictly cell-associated in chickens with the exception of the feather follicle epithelium. A system previously developed using reticuloendotheliosis (REV)-transformed cell lines stably expressing individual MDV genes allows the determination of relevant MDV proteins for the induction of cytotoxic T lymphocyte (CTL) responses. To examine the importance of glycoproteins for the induction of CTL, the MDV genes coding for glycoproteins (g) C, D, E, H, I, K, L, and M were stably transfected into the REV-transformed chicken cell lines RECC-CU205 (major histocompatibility complex (MHC): B²¹B²¹) and RECC-CU91 (MHC: B¹⁹B¹⁹). All transfected cell lines were lysed by REV-sensitized, syngeneic splenocytes obtained from MD-resistant N2a (MHC: B²¹B²¹) and MD-susceptible P2a (MHC: B¹⁹B¹⁹) chickens, indicating that the expression of individual MDV glycoproteins did not interfere with antigen processing pathways. Only cell lines expressing gI were recognized by CTL from both N2a and P2a MDV-infected chickens. Cell lines expressing glycoproteins gC and gK, and to a lesser extent, gH, gL, and gM were lysed by syngeneic MDV-sensitized splenocytes from N2a birds but not P2a birds. In contrast, gE was recognized by MDV-sensitized effector cells from the P2a line and not the N2a line. Glycoprotein D was not recognized by either line, with the exception of one marginally significant P2a assay. These results indicate that late viral glycoproteins are relevant for the induction of cell-mediated immunity during MDV infection.     

The efficacy of recombinant fowlpox vaccine protection against Marek's disease: its dependence on chicken line and B haplotype

Earlier studies have shown that the B haplotype has a significant influence on the protective efficacy of vaccines against Marek's disease (MD) and that the level of protection varies dependent on the serotype of MD virus (MDV) used in the vaccine. To determine if the protective glycoprotein gene gB is a basis for this association, we compared recombinant fowlpox virus (rFPV) containing a single gB gene from three serotypes of MDV. The rFPV were used to vaccinate 15.B congenic lines. Nonvaccinated chickens from all three haplotypes had 84%-97% MD after challenge. The rFPV containing gB1 provides better protection than rFPV containing gB2 or gB3 in all three B genotypes. Moreover, the gB proteins were critical, since the B*21/*21 chickens had better protection than chickens with B*13/*13 or B*5/*5 using rFPV with gB1, gB2, or gB3. A newly described combined rFPV/gB1gEgIUL32 + HVT vaccine was analyzed in chickens of lines 15 x 7 (B*2/*15) and N (B*21/*21) challenged with two vv+ strains of MDV. There were line differences in protection by the vaccines and line N had better protection with the rFPV/gB1gEgIUL32 + HVT vaccines (92%-100%) following either MDV challenge, but protection was significantly lower in 15 X 7 chickens (35%) when compared with the vaccine CVI988/Rispens (94%) and 301B1 + HVT (65%). Another experiment used four lines of chickens receiving the new rFPV + HVT vaccine or CVI988/Rispens and challenge with 648A MDV. The CVI 988/Rispens generally provided better protection in lines P and 15 X 7 and in one replicate with line TK. The combined rFPV/gB1gEgIUL32 + HVT vaccines protected line N chickens (90%) better than did CVI988/Rispens (73%). These data indicate that rFPV + HVT vaccines may provide protection against MD that is equivalent to or superior to CVI988/ Rispens in some chicken strains. It is not clear whether the rFPV/gB1gEgIUL32 + HVT vaccine will offer high levels of protection to commercial strains, but this vaccine, when used in line N chickens, may be a useful model to study interactions between vaccines and chicken genotypes and may thereby improve future MD vaccines.     

Early cell-mediated immune responses to Marek's disease virus in two chicken lines with defined major histocompatibility complex antigens

N2a and P2a chickens, resistant and susceptible to Marek's disease (MD), respectively, were used to examine relationships between major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) and natural killer (NK)-like cell activity with resistance to infection with Marek's disease virus (MDV). Ten-day-old chickens were infected with MDV and euthanatized at selected times to evaluate for NK cell and MHC-restricted cytotoxicity. The N2a MDV-infected chickens had an early cell-mediated immune response characterized by a sustained NK-like cytotoxicity that coincided with a measurable MHC-cytotoxicity that was lower than controls. Although MHC-restricted and NK cell cytotoxicity was demonstrated in P2a MDV-infected chickens at 8 dpi, both abruptly decreased and remained low for the remainder of the 20-day experiment. The critical time point that may determine the resistance to MD appears to be within the first 2 weeks post-infection. Improvement of the chicken NK cell activity may be a good candidate for both selection and immunomodulation MD control programs.     

Four distinct neurologic syndromes in Marek's disease: effect of viral strain and pathotype

A chronological study of central nervous system disorders induced by Marek's disease virus (MDV) has been conducted. Neurologic clinical signs were recorded daily for individual chickens of two genetic lines after inoculation of 13 serotype 1 MDV strains representing all three pathotypes. In addition to classical transient paralysis (TP) previously described by many workers, and acute TP, described in the companion paper, we have identified for the first time two other neurologic syndromes, persistent neurologic disease (PND) and late paralysis (LP). PND designates birds that showed a variety of neurologic signs (ataxia, torticollis, and nervous tics) after recovery from paralysis (12-15 days postin-oculation [DPI]) that either persisted through the observation period or presented a cyclic pattern. LP was a rare syndrome characterized by the late onset of the paralytic stage (about 20 DPI), perhaps indicating occasional failure of the initial intraabdominal inoculation to induce infection. Clinical signs and histopathologic alterations of the brain were also evaluated sequentially in chickens of two genetic lines after inoculation with two MDV strains (virulent MDV and very virulent plus MDV). Although clinical response differed greatly among treatment groups, types of lesions (endotheliosis, mononuclear perivascular cuffing, vasculitis, vacuolization, and increase in cellularity of the neuropil) were similar. However, early onset of lesions (by 6 days) appeared to be associated with a greater severity of clinical signs. We also found that neurologic response was greatly influenced by viral pathotype (virulence). This study thus confirms that the central nervous system is an important target organ for MDV resulting in several distinct clinical manifestations and suggests that neurologic responses in antibody-free chickens might be a useful criterion for virus pathotyping.     

Marek's disease virus infection in the brain: virus replication, cellular infiltration, and major histocompatibility complex antigen expression

Marek's disease virus (MDV) infection in the brain was studied chronologically after inoculating 3-week-old chickens of two genetic lines with two strains of serotype I MDV representing two pathotypes (v and vv+). Viral replication in the brain was strongly associated with the development of lesions. Three viral antigens (pp38, gB, and meq) were detected in the brain of infected chickens. Marked differences between v and vv+ pathotypes of MDV were identified for level of virus replication, time course of brain lesions, and expression of major histocompatibility complex (MHC) antigens. Two pathologic phenomena (inflammatory and proliferative) were detected in the brain of chickens inoculated with vv+MDV, but only inflammatory lesions were observed in those inoculated with vMDV. Inflammatory lesions, mainly composed of macrophages, CD4+ T cells, and CD8+ T cells, started at 6-10 days postinoculation (dpi) and were transient. Proliferative lesions, characterized by severe infiltrates of CD4+CD8- T cells (blasts), started at 19-26 dpi and persisted. Expression of MHC antigens in endothelial cells and infiltrating cells within the brain was influenced by MDV infection. Upregulation of MHC class II antigen occurred in all treatment groups, although it was more severe in those inoculated with vv+MDV. MHC class I antigen was downregulated only in those groups inoculated with vv+MDV. These results enhance our understanding of the nature and pattern of MDV infection in the brain and help to explain the neurovirulence associated with highly virulent MDV.     

Effect of diluting Marek's disease vaccines on the outcomes of Marek's disease virus infection when challenged with highly virulent Marek's disease viruses

Dilution of Marek's disease (MD) vaccines is a common practice in the field to reduce the cost associated with vaccination. In this study we have evaluated the effect of diluting MD vaccines on the protection against MD, vaccine and challenge MD virus (MDV) kinetics, and body weight when challenged with strains Md5 (very virulent MDV) and 648A (very virulent plus MDV) by contact at day of age. The following four vaccination protocols were evaluated in meat-type chickens: turkey herpesvirus (HVT) at manufacturer-recommended full dose; HVT diluted 1:10; HVT + SB-1 at the manufacturer-recommended full dose; and HVT + SB-1 diluted 1:10 for HVT and 1:5 for SB-1. Vaccine was administered at hatch subcutaneously. One-day-old chickens were placed in floor pens and housed together with ten 15-day-old chickens that had been previously inoculated with 500 PFU of either Md5 or 648A MDV strains. Chickens were individually identified with wing bands, and for each chicken samples of feather pulp and blood were collected at 1, 3, and 8 wk posthatch. Body weights were recorded at 8 wk for every chicken. Viral DNA load of wild-type MDV, SB-1, and HVT were evaluated by real time-PCR. Our results showed that dilution of MD vaccines can lead to reduced MD protection, reduced relative body weights, reduced vaccine DNA during the first 3 wk, and increased MDV DNA load. The detrimental effect of vaccine dilution was more evident in females than in males and was more evident when the challenge virus was 648A. However, lower relative body weights and higher MDV DNA load could be detected in chickens challenged with strain Md5, even in the absence of obvious differences in protection.     

Influence of turkey herpesvirus vaccination on the B-haplotype effect on Marek's disease resistance in 15.B-congenic chickens.

Eight recently developed 15.B congenic lines of chickens were tested for Marek's disease (MD) resistance by intra-abdominal injection of cell-associated preparations of MD virus of a virulent strain (JM), a very virulent strain (Md5), or Md5 after vaccination with turkey herpesvirus (HVT) strain FC126. Chickens of the 15.N congenic line (B15B21 or B21B21) were very resistant to JM-induced MD, in contrast to chickens homozygous for the B-haplotypes 2, 5, 12, 13, 15, or 19. After Md5 infection, more than 88% of the chickens in all of the congenic lines developed MD. However, when chickens were vaccinated with HVT before being inoculated with Md5, the B5 and B12 homozygotes were more resistant to MD than were the B2, B13, or B19 homozygotes, and B15 and B21 homozygotes had intermediate resistance. B5B5 and B2B5 F2 chicks inoculated with HVT and Md5 had a lower prevalence of MD than B2B2 sibs. These results demonstrate that a protocol involving HVT vaccination of chicks followed by infection with very virulent MD virus will allow the detection of B-haplotypes determining MD resistance, some of which are not detectable in unvaccinated chicks challenged with virulent MD.     

Resistance to Marek's disease herpesvirus-induced lymphoma is multiphasic and dependent on host genotype

Genotype-dependent differences in Marek's disease (MD) susceptibility were identified using 14-day-old line N and 6(1) (resistant) and 151 and 7(2) (susceptible) inbred chickens infected with HPRS-16 MD virus (MDV). All line 72 chickens developed progressive MD. Line 15I had fluctuating MD-specific clinical signs and individuals recovered. A novel histologic scoring system enabled indices to be calculated for lymphocyte infiltration into nonlymphoid organs. All genotypes had increased mean lesion scores (MLSs) and mean total lesion scores after MDV infection. These differed quantitatively and qualitatively between the genotypes. Lines 6(1) and 7(2) had a similar MLS distribution in the cytolytic phase, although scores were greater in line 7(2). At the time lymphomas were visible in line 7(2), histologic lesions in line 6(1) were regressing. AV37+ cells were present in similar numbers in all genotypes in the cytolytic phase, suggesting that neoplastically transformed cells were present in all genotypes regardless of MD susceptibility. After the cytolytic phase, AV37+ cell numbers increased in lines 7(2) and 15I but decreased in lines 6(1) and N. In the cytolytic and latent phases, in all genotypes, most infiltrating cells were CD4+. After this time, line 7(2) and 15I lesions increased in size and most cells were CD4+; line 6(1) and N lesions decreased in size and most cells were CD8+. In all genotypes, AV37 immunostaining was weak in lesions with many CD8+ cells, suggesting that AV37 antigen expression or AV37+ cells were controlled by CD8+ cells. The rank order, determined by clinical signs and pathology, for MD susceptibility (highest to lowest) was 7(2) > 15I > 6(1) > N.     

Selection of a recombinant Marek's disease virus in vivo through expression of the Marek's EcoRI-Q (Meq)-encoded oncoprotein: characterization of an rMd5-based mutant expressing the Meq of strain RB-1B

Marek's disease (MD) is a highly contagious viral disease of chickens (Gallus gallus domesticus) caused by MD virus (MDV), characterized by paralysis, neurologic signs, and the rapid onset of T-cell lymphomas. MDV-induced T-cell transformation requires a basic leucine zipper protein called Marek's EcoRI-Q-encoded protein (Meq). We have identified mutations in the coding sequence of Meq that correlated with virus pathotype (virulent, very virulent, and very virulent plus). The aim of this study was to determine whether recombinant viruses could be isolated based on Meq expression through in vivo selection. Chicken embryo fibroblasts (CEFs) were cotransfected with an rMd5 strain-based Meq deletion virus (rMd5deltaMeq) and meq loci from strains representing different pathotypes of MDV. Transfected CEFs were inoculated into chickens in two independent studies. We were able to isolate a single recombinant virus, rMDV-1137, in a contact-exposed chicken. rMDV-1137 had recombined two copies of the meq gene of RB-1B and was found to have pathogenicity similar to both RB-1B and rMd5 parental strains. We found the RB-1B- and rMd5-induced lymphomas showed differences in composition and that rMDV-1137-induced lymphomas were intermediate in their composition. We were able to establish cell lines from both RB-1B- (MDCC-UD35, -UD37) and rMDV-1137 (MDCC-UD36, -UD38)-induced, but not rMd5-induced, lymphomas. To date, no rMd5- or parent Md5-transformed T-cell lines have been reported. Our results suggest that 1) a recombinant MDV can be selected on the basis of oncogenicity; 2) changes in Meq sequence seem to affect tumor composition and the ability to establish cell lines; and 3) in addition to meq, other genomic loci affect MDV pathogenicity and oncogenicity.     

Mortality of one-week-old chickens during naturally occurring Marek's disease virus infection

Marek's disease (MD) is a disease of chickens that occurs worldwide and has serious economic consequences. MD can present as one of several forms, with the most commonly occurring forms being the lymphoproliferative diseases. Under experimental conditions, an early mortality syndrome has been recognized following infection by some but not all strains of MD virus (MDV). This is the first report of a confirmed case of mortality due to naturally occurring MDV infection in 1-week-old, nonvaccinated, chickens. Necrotizing lesions were observed in the bursa of Fabricius, lung, duodenum, jejunum, and proventriculus, and large intranuclear inclusion bodies were a striking feature in tissues with lesions in all birds. Immunohistochemical staining for the pp38 protein of MDV revealed abundant pp38 antigen in the affected tissues, confirming the presence of MDV within the lesions. PCR yielded an amplicon with 97% homology to the meq gene of MDV. No evidence of co-infection by either of the immunosuppressive agents chicken anemia virus and infectious bursal disease virus was detected.     

Indications of immunodepression in chickens infected with various strains of Marek's disease virus

The effect of infection by various strains of Marek's disease virus (MDV) on the immune function of 3-week-old chickens was examined. MDV strains of low (CU-2, RB-7) and high (RB-3, MD-5, and MD-11) pathogenicity were compared with prototype JM-10 strain of moderate pathogenicity. Mortality, whole body weight, relative weights of lymphoid organs, histopathology, humoral antibody responses to thymus-dependent and -independent antigens, and in vitro lymphocyte responses to mitogen stimulation were investigated at 1, 2, and 3 weeks postinfection. MDV strains of high pathogenicity significantly depressed responses at 3 weeks postinfection, seeming to indicate the ability of these viruses to induce severe immunodepression. However, the fact that the moderately pathogenic and even some of the low-pathogenicity strains induced immunodepression suggests that other viral mechanisms are also important in its determination.     

Natural killer cell activity in chickens exposed to Marek's disease virus: inhibition of activity in susceptible chickens and enhancement of activity in resistant and vaccinated chickens

Chickens of 2 genetic lines (lines P and N) were inoculated with a pathogenic strain of Marek's disease (MD) virus (MDV) and chronologically examined for disease response and natural killer (NK) cell expression. The NK cell reactivity was assayed in an in vitro cytotoxicity assay in which effector cells from the spleen of test chickens were reacted with 51Cr-labeled LSCC-RP9 target cells. Chickens of line P developed progressive debilitating disease and a high incidence of gross tumors and death. The NK cell reactivity of line-P chickens infected with MDV was significantly lower than that of uninfected control hatchmates. In contrast, NK cell levels were significantly elevated in MDV-inoculated line-N chickens that were resistant to MD and in chickens of lines P or N that had been inoculated with herpesvirus of turkeys (HVT). NK cell levels were also elevated in line P if chickens were vaccinated with HVT before infection with MDV. Inhibition of NK reactivity in susceptible chickens and elevation of reactivity in naturally resistant or vaccinated chickens may indicate a role for the NK cell system in regulating resistance to MD.     

Development of a nested polymerase chain reaction method to detect oncogenic Marek's disease virus from feather tips.

For the easy survey of Marek's disease virus (MDV), feather tip-derived DNA from MDV-infected chickens can be used because feather tips are easy to collect and feather follicle epithelium is known to be the only site of productive replication of cell-free MDV. To develop a diagnostic method to differentiate highly virulent strains of MDV from the attenuated MDV vaccine strain, CVI988, which is widely used, nested polymerase chain reaction (PCR) was performed to detect a segment of the meq gene in feather tip samples of chickens experimentally infected with MDV. In chickens infected with Md5, a strain of oncogenic MDV, the meq gene was consistently detected, whereas the L-meq gene, in which a 180-base pair (180-bp) sequence is inserted into the meq gene, was detected in CVI988-infected chickens. Moreover, the meq gene was mainly detected even in chickens co-infected with both Md5 and CVI988. These results suggest that this method is appropriate for the surveillance of the highly virulent MDV infection in the field.     

Examination of the effect of a naturally occurring mutation in glycoprotein L on Marek's disease virus pathogenesis

We recently reported a comparison of glycoprotein-encoding genes of different Marek's disease virus pathotypes (MDVs). One mutation found predominantly in very virulent (vv)+MDVs was a 12-bp (four-amino acid) deletion in the glycoprotein L (gL)-encoding gene in four of 23 MDV strains examined (three were vv+MDVs and one was a vvMDV). This mutation was noted in the gL of the TK (615K) strain, but not in the RL (615J) strain of MDV. These strains have identical mutations in the meq gene characteristic of vv+MDVs but can be distinguished by the mutation in the gL-encoding gene. The TK strain was originally isolated from vaccinated chickens and appeared to confer or enhance horizontal transmission of the vaccine virus, herpesvirus of turkeys (HVT). Because the molecular basis for increased virulence of MDV field strains is unknown, we hypothesized that one mechanism might be by coreplication of MDV-1 strains with HVT and that it could be mediated by the mutation of gL, an essential component of the glycoprotein H/L complex. In this study, we compared the pathogenicity of TK (615K) and RL (615J) strains of MDV in the presence and absence of simultaneous HVT coinfection. MDV infections were monitored at the levels of viremia (for both MDV-1 and HVT), clinical signs of MD, tumor incidence, and mortality in 1) inoculated chickens, 2) chickens exposed at 1 day of age, 3) chickens exposed at 2 wk of age, and 4) chickens exposed to both TK/HVT- and RL/HVT-infected chickens at 6 wk of age. We found high incidences of clinical MD signs in all inoculated treatment groups and all chickens exposed to TK and RL viruses, regardless of the presence of HVT. The median time to death of chickens exposed to TK1HVT-infected chickens, however, was lower than the other treatment groups for contact-exposed chickens. Although this difference was not considered to be statistically significant to a rigorously interpreted degree because of the removal of chickens for sampling from the test groups, these data suggest that replication of the TK strain and HVT, when coadministered, might incrementally affect the virulence of MDV-1 strains. The strict correlation of this enhancement of virulence with the mutation in gL, however, requires additional experiments with genetically identical MDV background strains.