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The effect of serotype 2 and 3 Marek's disease virus (MDV) vaccines on the pathogenesis of reticuloendotheliosis virus (REV)-induced bursal and non-bursal lymphomas was examined in chickens of RPRL lines 15I(5) X 7(1) and 6(3) X 0, respectively. At hatch, chickens were vaccinated with strain 301B/1 of serotype 2 MDV or strain FC126 of turkey herpesvirus (HVT), a serotype 3 MDV and inoculated with spleen necrosis virus (SNV), a non-defective strain of REV. In another experiment, bursas from 14-week-old 15I(1) X 7(1) chickens coinfected with strain 301B of serotype 2 MDV and SNV strain of REV at hatch were examined microscopically for REV-induced transformed follicles by methyl green pyronin stain and for the presence of MDV by in situ hybridization. The incidence of REV-induced bursal lymphomas was significantly higher in 15I(5) X 7(1) chickens vaccinated with serotype 2 MDV than in unvaccinated chickens or chickens vaccinated with HVT. On the other hand, the incidence of REV induced nonbursal lymphoma in chickens of line 63 X 0 vaccinated with serotype 2 MDV was comparable to that in unvaccinated chickens or chickens vaccinated with HVT. The average number of hyperplastic follicles in bursas from REV-infected 15I(5) X 7(1) chickens was significantly higher in chickens vaccinated with serotype 2 MDV than that in unvaccinated chickens or chickens vaccinated with HVT, and the MDV was more frequently detected in REV-transformed than in untransformed bursal follicles. Data from this study suggest that serotype 2, but not serotype 3, of MDV may enhance the development of REV-induced bursal lymphomas, and that neither serotype 2 nor serotype 3 MDV influence the development of REV-induced nonbursal lymphomas. The data also suggest that the enhancement effects of serotype 2 MDV on REV bursal lymphomas may be at the stage of formation of hyperplastic or transformed bursal follicles.

Integration of two different avian retroviruses, reticuloendotheliosis virus (REV) and avian leukosis virus (ALV), into the genome of two different avian herpesviruses, the herpesvirus of turkeys (HVT) and Marek's disease virus (MDV), was investigated. Integration events occurred by the fourth and sixth in vitro passage of cells coinfected with REV/HVT and ALV/MDV, respectively. In order to further characterize the integration events, integrated proviruses and surrounding herpesviral genetic material were cloned and analyzed. In the REV/HVT coinfection experiment, one of the three unique integrated proviruses was found to have integrated into the HVT gD gene, resulting in disruption of the coding region of this gene. The two additional unique integrations were localized to the UL and IRL border regions of HVT, two previously described common sites of REV integration into MDV. Interestingly, one of the integrated proviruses in the HVT genome appeared to be full length, was infectious when transfected into CEF cells, and therefore could potentially function to produce infectious REV from an HVT infectious platform. In the ALV/MDV coinfection experiment, one of two unique integrated proviruses was found to have integrated into the gD gene, resulting in disruption of the coding region of this gene. The second unique integration site was in the polyadenylation site of the SORF2 gene at the boundary of the IRs and Us, once again a common site of REV integration into MDV. These results demonstrate that the U/IR-TR border regions of herpesviruses are common sites of retroviral integration. In addition gD, in the Us region of the herpesvirus, is a common site of retroviral integration in multiple herpesvirus, indicating a possible selective advantage for disruption of this gene in the in vitro growth of a herpesvirus. Finally, this is the first instance of a full-length provirus found integrated into a herpesvirus genome, indicating that a retrovirus could alter its route of infection by being carried in a herpesvirus genome.

Current poultry vaccines are based either on live attenuated organisms or on killed organisms. Future vaccines also may be based on deletion mutants, live viral or bacterial vectors that express foreign genes, and naked DNA. Vaccines have different purposes, depending on the disease, which govern their intrinsic characteristics. Improvement of vaccine efficacy can be addressed by modifications of the vaccine and its administration, modifications in the capacity of the host to mount an immune response, and modifications of environmental factors. The concept of "designer vaccines" for matching vaccines that deliver specific antigenic peptides to chickens with the MHC haplotype that best presents those peptides to T cells is discussed.

A radioactive PCR test was developed that amplified the very virulent Marek's disease virus-1 (vvMDV-1) DNA sequence containing the 132 bp repeats. In apathogenic MDV-1 (CVI 988, Rispens), amplified DNA bands containing multiple copies of 132 bp repeats were identified. In the present study this PCR technique was used to monitor the passage level of vvMDV-1 in chicken embryo fibroblasts (CEF) in which the number of tandem 132 bp repeats was increased. It was found that at passage level 32 of vvMDV-1-B isolate, the 132 bp tandem repeat was already markedly amplified and its pattern resembled that of the MDV-1 (CVI 988, Rispens) vaccine virus DNA. In the vvMDV-1Z strain, amplification of the 132 bp repeat was not detectable at a similar passage level. The PCR test demonstrated that the apathogenic MDV-1 Md11/75c virus developed by extensive in vitro passaging has amplified 132 bp DNA repeats similar to those of the commercial vaccine virus (CVI 988, Rispense). It was also found that the pattern of viral RNA from infected cells detectable by Northern blot hybridization was markedly changed from a 2.4 kb RNA species in cells infected with vvMDV-1 viruses, to four RNA species (ranging from 2.2 to 4.4 kb) in cells infected with passage 32 of MDV-1-B strain, to a very large number of undefined RNA species synthesized in cells infected with attenuated MDV-1 viruses (CVI 988, Rispens and Md 11/75c).

The enhanced protective effect (synergism) when certain Marek's disease (MD) vaccine viruses are combined has been widely used in the development of improved vaccines, but the mechanism is poorly understood. To better characterize the basis for synergism among MD vaccine viruses, three vaccine viruses from each of the three MD viral serotypes were evaluated alone and in various combinations for protection against early challenge with very virulent MD viruses in four replicate trials. Synergism seemed to be influenced by viral serotype because significant enhancement occurred frequently between viruses of serotypes 2 and 3 (five of nine bivalent vaccines positive), but rarely between viruses of serotypes 1 and 3 (one of nine bivalent vaccines positive) and 1 and 2 (one of nine bivalent vaccines positive), and was not detectable between viruses of the same serotype (none of nine bivalent vaccines positive). With some exceptions, the degree of synergism tended to vary inversely with the mean protective efficacy of the most protective component virus. Little effect of virus dose, virus dose ratio or type and route of viral challenge was noted. The combination of strains 281MI/1 (serotype 2) and WTHV-1/1 (serotype 3), both poorly protective as monovalent vaccines, consistently demonstrated high levels of synergism (over 300%) in antibody-positive chickens challenged 5 days post-vaccination with Md5 virus. This protocol may be a useful model system for further studies on mechanisms of synergism. However, mixtures that optimize synergism are not necessarily as protective as commercial vaccines.

Outbreaks of Marek's disease (MD) were diagnosed in two flocks from the same company. Clinical signs, mainly blindness (90%), but also depression, mild paralysis, and 11 to 12% mortality by 20 weeks of age were observed. MD virus, serotype 1 was isolated. The isolates were designated NC-1 (flock 1) and NC-2 (flock 2). Challenge experiments were conducted with these isolates and with two reference MD virus strains (JM/102W and Md5) in unvaccinated, turkey herpesvirus- (HVT) vaccinated and bivalent- (HVT and SB-1) vaccinated chickens. Blindness, gross ocular lesions and tumour formation were observed in a high proportion of all groups challenged with NC-1 and NC-2 when compared with chickens challenged with JM/102W and Md5. In chickens challenged with isolates NC-1 and NC-2, corneal changes included oedema, midstromal cellular infiltration consisting of macrophages, lymphocytes, plasma cells and lesser numbers of heterophils, collagen degeneration and keratic precipitates consisting primarily of macrophages covering the central endothelium. Eosinophilic intranuclear inclusion bodies were present in mononuclear cells infiltrating the cornea. Changes in the uveal tract consisted of inflammatory cell infiltrates similar to those present in the cornea. Retinal lesions included disruption of the retinal pigmented epithelium, inflammatory cell infiltration in the subretinal space, photoreceptor degeneration and in severely affected eyes, necrosis of retinal cellular elements. Pecten changes consisted of necrosis and mononuclear cell infiltration. Intranuclear inclusion bodies were abundantly present in cells of the retina's ganglion and inner nuclear cell layers. The unusual clinical manifestation of MD, the unusual tropism and virulence of NC-1 and NC-2 for ocular tissues and the incomplete protection afforded by conventional vaccination suggest that these isolates may be new pathotypes.

We have developed a quantitative-competitive (QC) polymerase chain reaction (PCR) for the detection of Marek's disease virus (MDV) DNA. The assay utilizes a competitor DNA that differs from the viral DNA of interest by having a small insertion. The competitor DNA acts as an internal standard for the estimation of viral DNA in an unknown sample. The amount of viral DNA in a sample is quantitated by coamplification in the presence of a known amount of competitor DNA. The same PCR primers that amplify the viral DNA also amplify the competitor DNA. When the amount of competitor is equal to the amount of viral DNA in a sample, there is equal amplification of the competitor and the virus. Thus, we are able to quantitate the viral DNA in an unknown sample. To establish the utility of this assay, in vivo correlations between virulence and virus replication were studied. Our data demonstrated that a more virulent strain of MDV (648A) replicated better in thymus during cytolytic infection than did a less virulent strain (GA). However, no differences in virus titer were observed when these two viruses were propagated in tissue culture. Our data are consistent with the generally held idea that "hot" strains of MDV replicate earlier and better in birds. Thus, QC-PCR is extremely specific and sensitive to measure MDV DNA over a wide range and can be applied to in vivo studies of viral pathogenesis.

Profiles of infection with avian leukosis virus subgroup J (ALV-J) and factors that predict virus transmission to progeny were studied. Eggs from an infected broiler breeder flock were hatched at the laboratory. The flock was reared in a floor pen, transferred to laying cages at 22 wk, and inseminated to produce fertile eggs. A cohort of 139 chickens was tested at frequent intervals over a 62-wk period for virus, viral antigens, or antibodies in plasma, cloacal swabs, egg albumen, and embryos. Virus was detected in 7% of chicks at hatch but spread rapidly so that virtually all chicks became infected between 2 and 8 wk of age. Mortality due to myeloid leukosis and related tumors was 22%. Over 40% of the chicks developed persistent infections, whereas the remainder experienced transient infections. Five types of infection profiles were recognized. Novel responses included hens that were positive for virus intermittently or started late in life to shed viral antigens into the cloaca. ALV-J was isolated from 6% of 1036 embryos evaluated between 26 and 62 wk. However, over 90% of the virus-positive embryos were produced between 29 and 34 wk of age. Of 80 hens that produced embryos, 21 produced at least one infected embryo and were identified as transmitters. All but one transmitter hen would have been detected by a combination of viremia, cloacal swab, and albumen tests conducted between 18 and 26 wk. However, virus was transmitted to embryos from hens that were not persistently viremic or that rarely shed viral group-specific antigen into the albumen of their eggs. Intermittent patterns of both antigen shedding and virus transmission to embryos were observed in some hens. These results validate current screening procedures to identify potential transmitter hens and provide some suggestions for improvement but also show that identification of all transmitter hens by such procedures is unlikely. Thus, eradication programs based solely on dam testing may be less effective than those where dam testing is combined with procedures to mitigate early horizontal transmission in progeny chicks.

A novel syndrome was observed after inoculation of 3-wk-old chickens with highly virulent Marek's disease virus (MDV) strains. This syndrome was characterized by the acute onset of neurologic signs including flaccid paralysis of neck and limbs 9-10 days postinoculation, typically resulting in death 1-3 days after the onset of clinical signs. Most affected birds died, and spontaneous recovery was rare. Few if any gross tissue changes were found. Histologic brain lesions included acute vasculitis with vasogenic edema and perivascular cuffing. The syndrome was influenced by the virus strain and dose and by chicken strain and B haplotype and was prevented by vaccination with turkey herpesvirus. Chickens up to 18 wk of age were susceptible. On the basis of clinical signs and histopathology, the syndrome was determined to be an acute form of transient paralysis (TP); its more acute nature and virtual lack of spontaneous recovery differentiated this syndrome from classical TP. Affected birds were viremic, and brains were positive for viral DNA by polymerase chain reaction assays, but these tests were also positive in inoculated chickens without clinical signs and may have limited value for diagnosis. Although acute TP should occur only rarely in Marek's disease-vaccinated commercial flocks, this syndrome may be important in laboratory studies, where it could interfere with pathogenesis trials. Finally, acute TP appears to be one component in the pathogenesis of the early mortality syndrome, a previously described immunodepressive disease induced by inoculation of 1-day-old chicks with highly virulent MDV.

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.

Hemorrhagic enteritis (HE) is an economically important disease of turkeys caused by a type II aviadenovirus, hemorrhagic enteritis virus (HEV). The vaccines currently available to the commercial poultry producer are highly effective in preventing disease outbreaks; however, they are immunosuppressive. A recombinant fowl poxvirus (rFPV) expressing the native hexon of HEV has been shown to induce an anti-HEV humoral immune response in turkeys. In this study, the rFPV expressing the native hexon of HEV was compared with a commercial HEV vaccine (vxHEV) for its ability to protect turkeys from virulent HEV challenge. Complete protection from the enteritis of HE was achieved in experimental groups vaccinated with either the rFPV or the vxHEV. Lymphocyte stimulation was measured in turkeys inoculated with rFPV, vxHEV, or a sublethal dose of HEV or not inoculated. No statistically significant immunodepression was observed in turkeys receiving the rFPV.

Indirect immunofluorescence (IFA), polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) were used for detection of reticuloendotheliosis virus (REV) as a contaminant in a live virus fowl pox (FP) vaccine of poultry. A FP vaccine known to be contaminated with REV was tested by in vitro and in vivo assays in chicken embryo fibroblasts (CEFs) and day-old specific-pathogen-free (SPF) chicks, respectively. Using in vitro assays, IFA and PCR were more sensitive than ELISA in detection of REV in CEFs inoculated with REV-contaminated FP vaccine. However, when the vaccine was tested by in vivo assays using SPF chickens, the sensitivity of ELISA was comparable with that of IFA and PCR. Antibody to REV was not detected in SPF chickens within 4 wk postinoculation with REV-contaminated FP vaccine at hatch. Filtration of vaccine to eliminate vaccine virus from the inoculum before testing in CEFs resulted in a significant reduction in the frequency of REV detection by PCR or IFA. The data suggest that the sensitivity of IFA, PCR, and ELISA depends on the concentration of REV in the vaccine and that in vivo assays of vaccines for contamination with REV should include a test for virus because a negative antibody test may be misleading.

Our earlier studies have shown that retrovirus insertion into herpesvirus is an efficient process that engenders recombinant herpesviruses with altered biological properties. The RM1 clone is derived from the JM strain of Marek's disease virus (MDV) through retrovirus insertional mutagenesis and contains sequences of reticuloendotheliosis virus inserted at the junction of the internal repeat and unique short regions of the genome. In previous studies, the RM1 clone appeared attenuated for oncogenicity but caused marked atrophy of the thymic lobes. The present studies represent a detailed analysis of the biological characteristics of the RM1 clone in order to better understand mechanisms of oncogenicity and gene function of MDV. RM1 was almost fully attenuated for oncogenicity but retained other in vivo properties of virulent viruses such as thymic and bursal atrophy, early immunosuppression, early cytolytic infection followed by efficient replication, and contact spread--all normally absent in attenuated strains. This suggests that, for serotype 1 MDV, oncogenicity is not tightly linked with immunodepression or viral replication and that these properties may be controlled by different genes or mechanisms. The mutation was stable through serial passage of the virus in chickens as determined by molecular analysis. None of the mutant viruses demonstrated expansion of the 132-bp repeat region of the genome, indicating that such expansion is not required for attenuation. Chickens vaccinated with RM1 clones were protected against challenge with virulent MDV, and levels of protection exceeded those of other attenuated serotype 1 vaccine viruses. Thus, attenuation by selective mutation may be an advantageous strategy for development of serotype 1 Marek's disease vaccines.

The continuation of an apparent evolutionary trend of Marek's disease virus (MDV) towards greater virulence may explain recent increased losses from Marek's disease (MD) in vaccinated flocks. To address this question, the virulence of 31 isolates of serotype 1 MDV obtained from layer or broiler flocks between 1987 and 1995 were characterized. Each isolate was cultured in duck embryo fibroblasts for four to six passages, and ascertained to be free from contamination with avian retroviruses, chicken anemia virus, and MDVs of other serotypes. The viruses, along with prototype viruses JM/102W and Md5, were tested for virulence by inoculation at 6 days of age into laboratory strain 15I5 x 7(1) chickens of three types: nonvaccinated, vaccinated with turkey herpesvirus (HVT) and bivalent (HVT + SB-1)-vaccinated. The results showed that three isolates did not differ from JM/102W and were classified in the virulent (vMDV) pathotype. Twenty-one isolates produced significantly higher levels of MD in HVT-vaccinated chickens than did the JM/102W control and were classified in the very virulent (vvMDV) pathotype. Seven isolates, five of which were isolated in 1994 or 1995, produced significantly higher levels of MD in bivalent-vaccinated chickens than did the Md5 (vvMDV) control. These isolates, provisionally designated as the vv+MDV pathotype, appeared to be at the high end of a virulence continuum. Several MD response parameters, including lymphoma mortality, early mortality with bursal/thymic atrophy, and frequency of visceral lymphomas or ocular lesions in nonvaccinated chickens were positively correlated with virulence. These findings support the continued evolution of MDV towards greater virulence.

Studies were focused on two attenuated serotype 1 Marek's disease (MD) vaccine viruses, CVI988/Rispens (passage 42) and R2/23 (passage 105). Both serotype 1 vaccine viruses provided much higher levels of protection than the prototype MD vaccine, turkey herpesvirus (HVT); the best protection was generally provided by CVI988/Rispens when compared with other vaccines. The efficacy of neither serotype 1 vaccine was improved by mixture with viruses of other serotypes (synergism). No differences between the two serotype 1 vaccines were revealed by cross-neutralization tests, thus excluding preferential in vivo neutralization by maternal antibodies as an explanation for differences in protective efficacy. Neither vaccine strain induced MD lesions or reduced growth rates in 8- or 18-week trials. Neither virus depressed humoral or cellular immune responses to antigenic challenge at 3 or 15 days after vaccination. Both virus strains exhibited altered characteristics during serial backpassage; R2/23 acquired increased oncogenic potential, and CVI988/Rispens acquired the potential for increased viremia titers, accompanied by an increased frequency of both histologic nerve lesions and gross thymic atrophy. During backpassage trials, contact spread was not observed for R2/23 and, surprisingly, seemed relatively limited for CVI988/Rispens. Studies on these two serotype 1 strains generally support the safety and efficacy of the serotype 1 class of MD vaccines.

SUMMARY. The increased use of serotype 2 Marek's disease virus (MDV) and serotype 3 turkey herpesvirus (HVT) as components of effective bivalent vaccines against Marek's disease (MD) prompted studies on the possible interactions of these two viruses in vitro and in vivo. The replication of the SB-1 strain of MDV was compared with replication of the FC126/2 strain of HVT in chickens and cell cultures infected with one or both viruses. Replication of MDV was reduced in the presence of HVT in both in vitro and in vivo systems. MDV plaque counts in dually infected chicken embryo fibroblast cultures inoculated with tissue-culturepropagated viruses were reduced by up to 91%; however, no inhibition was noted when inocula consisted of virus-infected buffy-coat cells. Plaque formation by MDV in chicken embryo fibroblast cultures was inhibited by virus-free conditioned medium from HVT-infected cultures. This conditioned medium also inhibited growth of vesicular stomatitis virus in a standard interferon assay. In chickens inoculated with both MDV and HVT, MDV viremia titers were lower and the dose required to infect 50% of susceptible chickens was increased 13-fold compared with chickens inoculated with MDV alone. In spite of these findings, there was no evidence that high concentrations of HVT interfered with either the ability of MDV to induce protective synergism in vivo or the protective efficacy of bivalent vaccines. No reciprocal inhibitory effects of MDV on the replication of HVT in vivo or in vitro were noted.

SUMMARY. B-haplotype genes in the chicken were previously shown to differentially influence vaccine efficacy against challenge with very virulent Marek's disease virus according to the type of Marek's disease (MD) vaccine used. To determine whether MD vaccines of the same serotype gave comparable levels of protection against MD in chickens of the same haplotype challenged with MD virus strain Md5, two serotype 1 and two serotype 2 vaccines were compared with one serotype 3 vaccine using chickens of 15.B-congenic lines. There was a strong correlation in development of MD lesions among chickens of the different lines receiving the two serotype 2 vaccines (r = 0.94) as well as among chickens receiving the two serotype 1 vaccines (r = 0.76). The serotype 1 vaccines were preferable for B2, B'3, B'5, and B21, but serotype 2 vaccines were more protective for B5 chickens. The two serotype 2 vaccines gave equivalent protection; however, of the serotype 1 vaccines, CVI988/Rispens provided more protection than Mdl 1/75c/R2/23. We conclude that the B-haplotype influence on MD vaccine efficacy is dependent on the serotype of the vaccine.

SUMMARY. To determine if B-haplotype differentially influences vaccinal immunity to very virulent Marek's disease (MD) virus challenge, chickens of five 15.B-congenic lines were vaccinated with vaccines representing serotypes 1, 2, and 3. B-haplotype differentially influenced vaccinal immunity to very virulent MD virus challenge using vaccines of all three serotypes, and different MD vaccines were optimal for some B-haplotypes. Regarding specific haplotypes, the 15.B-congenic chickens with B2 and B13 developed less protection against MD than chickens with B'5 following vaccination with all three serotypes of MD vaccine, whereas the chickens with B5 and B2' developed variable protection with different MD vaccines. Thus, for induction of maximum MD resistance, it may be necessary to select a vaccine appropriate for the predominant B-haplotypes of the chicken flock.

SUMMARY. 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 B2'B21) 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, B%3, or B19 homozygotes, and B'5 and B2' homozygotes had intermediate resistance. B5B5 and B2B5 F2 chicks inoculated with HVT and MdS 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.

In earlier studies, a revertant serotype 1 Marek's disease virus (MDV), clone Md11/75C/R2, was found to be a highly protective vaccine virus but was mildly pathogenic for susceptible chickens. The term "revertant" indicates that the virus, after attenuation, gained virulence following backpassage in chickens. The present study is an attempt to develop a more attenuated but still protective vaccine virus from Md11/75C/R2. Forty-two derivative viruses or clones from Md11/75C/R2 were evaluated. Two of these, designated clones R2/23 and R2/29, induced viremia but little or no pathology in preliminary trials and were selected for further study. In a series of nine trials, both clones provided protection against challenge with very virulent MDV strains that was superior to that induced by turkey herpesvirus (HVT) and was not significantly different (P greater than 0.05) from that induced by a bivalent (HVT + SB-1) vaccine. Both clones appeared fully attenuated based on pathogenicity tests in susceptible antibody-negative chickens. Both clones gained virulence on backpassage in chickens, but this seemed of little concern because neither virus spread by contact to other chickens. Although the two clones were very similar, clone R2/23 appeared to have a slightly lower pathogenic potential following backpassage and thus best meets the combined criteria of safety and efficacy.

When inactivated Md11/75C vaccine was inoculated into 1-day-old chickens, it stimulated antibodies detectable by enzyme-linked immunosorbent assay (at a titer of 6400) and indirect fluorescent antibody test (at a titer of 640), but lacking virus-neutralizing activity. Chickens passively inoculated with these antibodies were protected against bursal atrophy, weight loss, and early mortality when challenged with the virulent Md5 strain of Marek's disease virus (MDV). That led to the conclusion that virus-neutralizing activity is not a prerequisite for protection. In another experiment, antibody titers of adult chickens previously primed by exposure to live turkey herpesvirus and MDV did not increase after immunization with inactivated oil-emulsion MDV vaccines. This result provides little hope that Marek's disease can be controlled in progeny chickens by maternal immunity derived from hyperimmunized parents.

SUMMARY. Selected biological characteristics were determined for 14 low-passage serotype 2 Marek's disease virus (MDV) isolates. Four of these isolates were also tested after extensive serial passage in chicken embryo fibroblast cultures. Observations were made on replication in vitro and in vivo, pathogenicity by in ovo inoculation, antigenicity, and protection against virulent MDV challenge. Among the low-passage isolates, there were some differences in pathogenicity after in ovo inoculation but relatively little difference in other characteristics, with the exception of the HN-1 strain, which replicated more rapidly in cell culture but produced generally lower in vivo responses than other isolates. After extended in vitro passage, isolates replicated much more readily in cell culture and produced lower pathologic responses in vivo than low-passage isolates, as has been reported for serotype 1 isolates. No antigenic differences among isolates were detected, but high-passage isolates induced lower levels of precipitating antibodies than low-passage isolates, indicating a possible reduction in A antigen production. The observed diversity associated with strain and passage level may be of value in the selection of optimum vaccine strains.

The kinetics of phytohemagglutinin (PHA) response of peripheral blood lymphocytes from chickens infected with oncogenic Marek's disease (MD) virus (MDV) or nononcogenic herpesvirus of turkeys (HVT) was studied with a whole blood microassay. At about 7 days after inoculation, a depression in PHA response was observed in MDV-inoculated resistant line N or susceptible line 7(2) chickens and in HVT-inoculated line 7(2) chickens. All chickens initially regained their PHA responsiveness. Susceptible chickens that died of MD or developed MD lymphoma in later stages of virus infection showed a second severe depression in PHA response. No depression was observed in HVT-vaccinated chickens when challenged with MDV. The PHA response of MDV-inoculated chickens that survived MD, HVT-inoculated chickens, and HVT-vaccinated MDV-challenged chickens showed evidence of enhancement. The depression of PHA response was studied and was attributed to the suppressive effect of macrophages on T-cell response, a finding consistent with our previous studies on MDV suppression of PHA response.

Cells of the JMV Marek's disease (MD) tumor, originally produced by rapid serial passage of MD lymphoma cells in chickens, were characterized to determine whether they were of host or donor origin and to ascertain certain virus-host cell interrelationships. Differences noted in blood group B surface alloantigens between tumor cells and host lymphocytes indicated a probable nonhost origin (i.e., transplantability) of the tumor. JMV spleen tumors contained predominantly large lymphoblasts bearing MD tumor-associated surface antigen. DNA from JMV tumor cell suspensions hybridized significantly with MD virus cRNA, which indicated that JMV cells contained at least a portion of the MD virus genome. No MD virus was rescued from JMV tumors by techniques suitable for rescue of virus from MD lymphomas. The JMV tumor cells were also devoid of MD virus-specific antigens. These properties differed markedly from those of MD lymphoma cells and make the JMV tumor cell a unique, potentially valuable, tool for further study of oncogenic herpesvirus infection and tumor immunity in the chicken.

We injected chick syncytial strain of reticuloendotheliosis virus (CS-REV) and wild type and recombinant avian leukosis virus (ALV) near the blastoderm of unincubated fertilized embryos and CS-REV intra-abdominally at day of hatch, and we progeny tested the surviving ALV viremic males and REV viremic males and females for transmitted viral genetic material. A number of positive progeny were identified and their deoxyribonucleic acid (DNA) analyzed for restriction enzyme fragments that hybridized with viral genetic material. Most of the progeny had simple restriction enzyme patterns unlike the viremic parents or congenitally infected progeny. This is suggestive evidence that retroviral genetic information has been inserted into the germ line of chickens.

Response of turkeys and 151(5)x7(1) chickens to prenatal or neonatal inoculation with the avian leukosis virus RAV-1 was compared. Virus-inoculated turkeys and chickens developed viraemia and antibody to. RAV-1. Many of the chickens remained persistently viraemic through the duration of the experiment, whereas in turkeys viraemia was transient. Circulating antibodies were detected earlier in turkeys than in chickens. Inoculation of turkeys with RAV-1 resulted in a high incidence of inflammatory and lymphoproliferative, but non-neoplastic, lesions in various visceral organs, including spleen, pancreas, heart, bursa and thymus, 3 to 5 weeks after virus inoculation. The lesions in chickens were those typical after RAV-1 infection, i.e. neoplastic, and appeared after a latent period of 9 weeks.

Of chickens either spontaneously producing or exogenously infected in ovo with Rous-associated virus, type O (RAV-O), an endogenous virus of the chicken, only 1 died with lymphoid leukosis (LL), the most common neoplasm associated with the leukosis-sarcoma virus group. Because the chickens were not kept in strict isolation, it could not be assumed that the one LL was induced by RAV-O. In contrast, RAV-1-infected chickens from the same lines had a high incidence of LL and other neoplasms. Over 800 chickens of several inbred lines were maintained in plastic isolators free of exogenous avian leukosis-sarcoma virus infection for from 500 to nearly 1,000 days of age. No LL was observed, even though some lines are known to produce RAV-O spontaneously or to express inherited gs antigen. Three neoplasms of unknown etiology were observed, but none generally associated with leukosis virus infection. We concluded that avian endogenous virus expression had little, if any, oncogenic potential, and that exogenous avian leukosis viruses were responsible for most naturally occurring neoplasms.

Coinfection of chicken embryo fibroblasts with reticuloendotheliosis virus (REV) and avian sarcoma virus leads to the formation of avian sarcoma viral pseudotypes which carry envelope determinants of REV. These pseudotypes can be neutralized by REV antiserum, have a host range which is different from that of any known avian sarcoma virus, and are unable to form foci in cells preinfected with REV. The REV stocks used in these experiments were plaque-purified. They were free of avian leukosis virus detectable in the COFAL tests, and their ability to form pseudotypes with avian sarcoma virus was neutralized with specific REV antiserum.

Chickens free of exogenous avian leukosis virus (ALV) infection, replicating endogenous ALV (Rous-associated virus-O), gs antigen, and chick helper factor were fully susceptible to induction of Marek's disease (MD) by ALV-free MD viruses. Dual infection with Rous-associated virus-2 and MD virus did not significantly alter the character of the MD lesions. Thus exogenous ALV infection was not requisite for MD virus-induced oncogenesis. Although participation of endogenous RNA tumor virus genes in MD lesion induction could not be excluded, expression of such genes in MD tumors as gs antigen was not established.

Summary Chick syncytial virus (CSV), a strain of avian reticuloendotheliosis virus (REV) causes lymphoid tumours in chickens after a prolonged incubation period. A number of CSV-induced tumours were examined for cell surface antigen and were found to be of the B cell type and to produce immunoglobulin. Attempts were made to grow in vitro cell lines from CSV-induced tumours and a lymphoblastoid cell line was established from a liver tumour of a chicken that was inoculated with CSV via the yolk sac in embryo. The donor chicken was viraemic at the time the tumour was removed. The cell line is designated RECC-RP13, it produces non-defective REV, is a B cell type and it produces IgM. It is free from infection with endogenous and exogenous avian leukosis virus (ALV) and has an increased number of chromosomes. Sequences specific to REV were detected in at least four sites in cellular DNA from RECC-RP13. Sequences specific to ALV DNA, beyond that normally found in 15I5 × 71 cells, were not found in DNA from this cell line.

A phosphonoacetate (PA)-resistant mutant of the herpesvirus of turkeys (HVT) was isolated and characterized. The mutant of HVT resistant to PA (HVTpa) replicated in duck embryo fibroblast (DEF) culture in media containing 300 microgram PA/ml, whereas the replication of the wild type of HVT (HVTwt) was completely inhibited in DEF culture in media containing 100 microgram PA/ml. The HVTpa was distinct from the HVTwt in plaque morphology, but was indistinguishable antigenically and showed in vitro temperature sensitivity at 41 degrees C (3741 degrees C efficiency of replication was about 5). It replicated poorly in chickens and failed to provide complete protection against challenge with Marek's disease virus (MDV). The HVTpa-induced DNA polymerase had an apparent inhibition constant for PA, an apparent inhibition constant for pyrophosphate, and an apparent Michaells constant for dCTP about 10, 2, and 2.5 times, respectively, greater than the constants for the HVTwt-induced enzyme and was also more thermolabile.

Three Marek's disease (MD) vaccines were evaluated for safety and protective efficacy in chickens with maternal antibodies against serotype 1, 2 and 3 MD viruses and in chickens with no maternal antibodies. The vaccines were: (1) Md11/75C, an attenuated serotype 1 MD virus, (2) a trivalent mixture of MD virus strains Md11/75C and SB-1, and turkey herpesvirus (HVT) strain FC 126, and (3) bivalent mixtures of these three viruses. These vaccines were compared with HVT or SB-1 vaccines in some trials. None of the vaccines was pathogenic or immunodepressive in susceptible chickens with or without maternal antibodies. No interference with in vivo HVT replication by additional viral components was noted, although some interference was demonstrated in vitro. Md11/75C replicated to limited titres in vivo and did not spread by contact, however, it acquired mild pathogenicity upon serial back-passage. Although Md11/75C provided good protection against highly virulent MD viruses in chickens without maternal antibodies, it was poorly protective in chickens with homologous maternal antibodies, and appeared more susceptible to in vivo neutralisation than did SB-1 or HVT. The trivalent vaccine, in contrast, was highly efficacious against very virulent MD virus challenge, even in chickens with maternal antibodies of all three serotypes, and was significantly more effective than monovalent vaccines. A bivalent vaccine composed of SB-1 and HVT was superior to the other two bivalent combinations of the three viruses. Protective synergism among all three vaccine viruses was confirmed. The efficacy of HVT was enhanced by as little as 80 PFU of SB-1, and even fractional doses of HVT and SB-1 together were superior to full doses of HVT alone.

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

Summary Tests confirmed that turkey herpesvirus (HVT) vaccine protected chickens poorly against challenge with the highly virulent Md5 strain of Marek's disease (MD) virus, especially in chickens with homologous HVT antibodies. The naturally avirulent SB‐1 vaccine virus was likewise poorly protective against challenge with the Md5 strain. Homologous antibodies reduced the protective efficacy of both vaccines, but SB‐1 was not affected by HVT antibodies. In order to provide better protection against strains of MD virus poorly protected against by HVT, such as Md5, the Md11 strain of MD virus was attenuated by 75 cell culture passages and evaluated for protective efficacy. This vaccine virus, designated Mdl 1/75C, provided good protection against challenge with Md5 and most other highly virulent MD viruses tested, but was less efficacious against challenge with the JM/102W strain, a prototype MD virus protected against well by HVT and SB‐1 vaccines. Furthermore, its efficacy was consistently lower in chicks...

A moribund wild turkey (Meleagris gallopavo) died shortly after it was discovered in Martin County, North Carolina (USA). The 4.3-kg female turkey appeared in good condition with no visible external lesions or evidence of injury. There were 2- to 5-mm yellow-white plaques on the mucosal surfaces of the oral cavity and mid-esophagus. The liver had large, multifocal, irregular pale areas on cut and uncut surfaces. The spleen contained multifocal, pale, hard, nodules. Microscopic changes in the liver consisted of large multifocal coalescing areas of necrosis. Occasional spherical 10 to 15 microns in diameter organisms consistent with Histomonas meleagridis were present in the necrotic areas. Viable hepatic parenchyma contained multifocal infiltrations of numerous mononuclear cells interpreted as neoplastic cells resembling lymphoblasts and plasma cells. Similar neoplastic cell infiltrates, consistent with the lymphoproliferative disease reticuloendotheliosis, were present in spleen, lung, and esophageal and oral mucosa. Reticuloendotheliosis virus, subtype 2, was isolated from samples of liver and spleen.

Spleen cells from chickens 7 days after inoculation with Marek's disease virus (MDV) responded poorly to stimulation by phytohemagglutinin (PHA). Addition of these cells to syngeneic normal spleen cells caused a marked suppression of the PHA response of the normal cells. The MDV spleen cells also inhibited the DNA synthesis of MSB-1 lymphoblastoid cells in vitro. The suppressive activity is attributed to the presence in MDV spleen cells of a population of suppressor cells with characteristics typical of macrophages. The suppressor cell activity was not removable by treatment with anti-T or anti-B serum with C, but it was reversible by treatment with carrageenan or carbonyl iron/magnet, by passage through glass wool column, and by adherence to plastic Petri dishes. The adherent MDV spleen cells also showed strong suppressor cell activity against syngeneic normal spleen cells.

A chicken lymphoblastoid cell line (TLT)-6855 originally established from an avian oncornavirus-induced lymphoma (Siegfried and Olson, 1972) was studied for the presence and expression of Marek's disease virus (MDV) genome. By nucleic acid hybridization a significant amount of MDV DNA was found in this cell line. This virus DNA, however, was not expressed in either virus-specific intracellular or membrane antigens or the MD-associated tumour-specific surface antigen (MATSA). Moreover, MDV-specific antigens could not be activated in this cell line by treatment with 5-IdUrd. In several experiments, when chickens were inoculated with the cell line a herpesvirus was repeatedly isolated from the kidneys. This herpesvirus was antigenically similar to MDV but was low in oncogenicity for chickens.

Lymphoid leukosis viruses and viral group-specific antigen were found in albumen of unincubated chicken eggs stored at 8 degrees C. Infectious virus was detected for up to 22 days and antigen was stable for 63 days. Tests for virus were conducted on albumen withdrawn from eggs prior to incubation and on extracts of embryos from the same eggs. When albumen was from eggs stored no longer than 6 days, virus was isolated from 20% more albumen samples than embryo extracts. The techniques described should be useful in programmes to eradicate lymphoid leukosis viruses from commercial poultry.

Chickens inoculated with herpesvirus of turkeys or with apathogenic or attenuated vaccine strains of Marek's disease virus (MDV) developed a T-cell-mediated immune response to Marek's disease (MD) tumor cells. This immune response was detected in a 4-hour 51Cr-release assay in which effector cells obtained from spleens of vaccinated chickens were reacted with 51Cr-labeled target cells of an MD lymphoblastoid cell line (MSB-1). The cytotoxic effector cells generated by the vaccine viruses had characteristics similar to those noted previously for anti-MSB-1 effector cells generated by MDV. The immune response was specific to MSB-1 cells, because another target cell line (TLT) antigenically unrelated to MSB-1 cells was not lysed by the effector cells nor did the unrelated target cells inhibit the cytotoxicity of effector cells against MSB-1 target in a cold-target inhibition assay. Because MSB-1 cells contain MD tumor-associated surface antigen, we postulated that the immune response detected in the vaccinated chickens may be directed against this antigen and that the antitumor antigen immunity may play a role in the mechanism of vaccine protection against lymphoma development by pathogenic MDV.

The emergence of viral strains of increased virulence has been associated with increased Marek's disease losses in certain vaccinated flocks. Methods are described for the isolation and typing of these strains, and for their control by means of improved vaccines.

Chickens inoculated as embyros or at hatching with the chick syncytial strain of reticuloendotheliosis virus developed a high incidence of lymphoid neoplasms between the 17th and 43rd weeks of age, involving principally the liver and bursa of Fabricius. On the basis of organ distribution, latent period, pathology and surface IgM production, the lymphomas closely resembled those of lymphoid leukosis. One inoculated chicken developed a myxosarcoma. No tumors were observed in uninoculated controls. The tumor-bearing chickens were free of infection with Marek's disease virus and exogenous avian leukosis virus (ALV) of subgroups A, B, C or D. However, the chickens were known to express endogenous ALV genes to varying degrees.

SummaryA focal cytopathic effect (CPE) was observed in duck embryo fibroblast (DEF) cultures 11-25 days postinoculation with blood from the JM strain of Marek's disease (MD). All cell suspensions from such cultures reproduced MD when inoculated into chicks, while all of the morphologically normal DEF cultures were noninfectious. The CPE and infectivity were maintained in DEF cultures for 182 days. Both the induction of CPE in cell cultures and MD in chickens required intact cells in the inoculum. These data indicate that the JM strain of MD was successfully propagated in DEF cultures and produced a characteristic CPE.

Chickens of 2 strains, 20 to 22 weeks of age and free of prior infection, were substantially more resistant than l-day-old chicks to mortality and tumor induction caused by exposure to Marek's disease virus. These older chickens were refractory to inoculation with up to 10(4) chick tumor-inducing doses of Marek's disease virus and, following contact infection, lesion frequencies were 8 to 30% of corresponding responses in chick controls. Resistance in contact exposed chickens was observed as early as the 8th week and, although variable, did not increase appreciably through the 20th week. Median latent periods to death were similar regardless of age at exposure. Older birds were fully susceptible to infection with Marek's disease virus and were only slightly resistant to development of microscopic lesions, thus suggesting that this resistance might be 'mediated through enhancement of lesion regression. Age-related resistance was confirmed to be unrelated to previously described tumor resistance caused by prior infection or genetic constitution

Morphological changes induced by a herpesvirus of turkeys (HVT) in duck embryo fibroblast, chicken embryo fibroblast, and chicken kidney cell cultures were studied by light and electron microscopy and compared with those of high cell culture passaged Marek's disease virus (MDV) in cell culture. Both viruses produced somewhat similar microplaques with different degrees of polykaryocytosis depending on the type of cell culture used. A high concentration of 35 nm nuclear particles, occasionally in crystalline form, were seen in cells infected with HVT. These cells also demonstrated crystalline arrays composed of particles about the same size and electron density of ribosomes. Morphologically, virions of HVT were similar to those of MDV and went through the same stages of maturation. However, a high percentage of naked virions of HVT were morphologically distinct; i.e., they lacked a single dense core and had an inner structure resembling an electron lucent cross.

Chickens of various age levels, free from prior infection, were simultaneously exposed to Marek's disease virus, and the response of each age group was recorded. Four- and 20-week-old chickens of lines 15×7 and CM (commercial source) had substantial resistance to mortality and gross lesions. In contrast, in line 7, which was tested at 1-day, 2-, 4-, 8-, 12- and 16-week age levels, 4-week-old chickens were fully susceptible to clinical Marek's disease (MD), although resistance was demonstrated at 8-week and older age levels. Genetically resistant chickens of line 6 maintained their resistance at all age levels tested. Pathogenesis of MD was compared in 12-week-old and 1-day-old chickens of line 15×7. Within the 1-day-old group, 23% of the chickens died because of MD, whereas there were no deaths in the 12-week-old group. Both groups developed viremia although duration, incidence, and levels of virus in the 1-day-old group were higher than in the 12-week-old group. Although initially the 12-week-old group responded by producing higher levels of antibody, the long term incidence of agar gel precipitin, immunofluorescent, and virus neutralization antibody in the two groups was similar. Gross and microscopic lesions of MD developed in both groups, but lesions regressed in the 12-week-old group and persisted in the 1-day-old group. It was concluded that age resistance to MD was expressed through lesion regression.

SummaryDuck embryo fibroblast (DEF) monolayer cultures seeded with whole blood from birds inoculated with the JM strain of Marek's disease (MD) and which showed a cytopathic effect (CPE), also contained intranuclear herpes-like virus particles. Complete virus particles with envelope essential for infectivity of herpesviruses were not found in either the infected cells or the extracellular materials of cultures containing such cells. All cultures showing CPE contained virus particles, and when inoculated into susceptible chicks caused MD. Cytopathic effect and viral synthesis could not be induced in fresh DEF cultures with spent media from infected cultures passed through 450 mμ Millipore filters. The perfect correlation obtained between CPE, presence of virus particles, and reproduction of MD by these cells suggests a cause and effect relationship and circumstantially implicates this virus in the etiology of MD.

MAREK'S disease virus (MDV) of chickens induces lymphomas and other lymphoproliferative changes, which, depending on the virulence of the strain used, may terminate in paralysis and death. Chickens genetically selected for resistance, however, are refractory to clinical MD and most chickens that are fully susceptible when young develop resistance as they become older. Resistance acquired with age is expressed through lesion regression1,2. Our studies with genetic and age resistance established that humoral immunity did not play a major role in either type of resistance3,4 and now we present evidence that the thymus-dependent immune system is of principal importance in the natural resistance of old chickens.

(1984). A new strategy for Marek's disease immunisation ‐Bivalent vaccine. Avian Pathology: Vol. 13, No. 2, pp. 133-135.