Endogenous Avian Leukosis Virus in Combination with Serotype 2 Marek's Disease Virus Significantly Boosted the Incidence of Lymphoid Leukosis-Like Bursal Lymphomas in Susceptible Chickens.

In 2010, sporadic cases of avian leukosis virus (ALV)-like bursal lymphoma, also known as spontaneous lymphoid leukosis (LL)-like tumors, were identified in two commercial broiler breeder flocks in the absence of exogenous ALV infection. Two individual ALV subgroup E (ALV-E) field strains, designated AF227 and AF229, were isolated from two different breeder farms. The role of these ALV-E field isolates in development of and the potential joint impact in conjunction with a Marek's disease virus (MDV) vaccine (SB-1) were further characterized in chickens of an experimental line and commercial broiler breeders. The experimental line 0.TVB*S1, commonly known as the rapid feathering-susceptible (RFS) line, of chickens lacks all endogenous ALV and is fully susceptible to all subgroups of ALV, including ALV-E. Spontaneous LL-like tumors occurred following infection with AF227, AF229, and a reference ALV-E strain, RAV60, in RFS chickens. Vaccination with serotype 2 MDV, SB-1, in addition to AF227 or AF229 inoculation, significantly enhanced the spontaneous LL-like tumor incidence in the RFS chickens. The spontaneous LL-like tumor incidence jumped from 14% by AF227 alone to 42 to 43% by AF227 in combination with SB-1 in the RFS chickens under controlled conditions. RNA-sequencing analysis of the LL-like lymphomas and nonmalignant bursa tissues of the RFS line of birds identified hundreds of differentially expressed genes that are reportedly involved in key biological processes and pathways, including signaling and signal transduction pathways. The data from this study suggested that both ALV-E and MDV-2 play an important role in enhancement of the spontaneous LL-like tumors in susceptible chickens. The underlying mechanism may be complex and involved in many chicken genes and pathways, including signal transduction pathways and immune system processes, in addition to reported viral genes.IMPORTANCE Lymphoid leukosis (LL)-like lymphoma is a low-incidence yet costly and poorly understood disease of domestic chickens. The observed unique characteristics of LL-like lymphomas are that the incidence of the disease is chicken line dependent; pathologically, it appeared to mimic avian leukosis but is free of exogenous ALV infection; inoculation of the nonpathogenic ALV-E or MDV-2 (SB-1) boosts the incidence of the disease; and inoculation of both the nonpathogenic ALV-E and SB-1 escalates it to much higher levels. This study was designed to test the impact of two new ALV-E isolates, recently derived from commercial broiler breeder flocks, in combination with the nonpathogenic SB-1 on LL-like lymphoma incidences in both an experimental egg layer line of chickens and a commercial broiler breeder line of chickens under a controlled condition. Data from this study provided an additional piece of experimental evidence on the potency of nonpathogenic ALV-E, MDV-2, and ALV-E plus MDV-2 in boosting the incidence of LL-like lymphomas in susceptible chickens. This study also generated the first piece of genomic evidence that suggests host transcriptomic variation plays an important role in modulating LL-like lymphoma formation.

Protective efficacy of a recombinant bacterial artificial chromosome clone of a very virulent Marek's disease virus containing a reticuloendotheliosis virus long terminal repeat.

Marek's disease virus (MDV), an alphaherpesvirus, causes Marek's disease (MD), a lymphoproliferative disease in poultry characterized by T-cell lymphomas, nerve lesions, and mortality. Vaccination is used worldwide to control MD, but increasingly virulent field strains can overcome this protection, driving a need to create new vaccines. Previous studies revealed that insertion of reticuloendotheliosis virus (REV) long terminal repeat (LTR) into a bacterial artificial chromosome (BAC) clone of a very virulent strain of MDV, Md5, rendered the resultant recombinant virus, rMd5 REV-LTR BAC, fully attenuated in maternal antibody positive (Mab+) chickens at passage 40. In the current study, the protective efficacy of rMd5 REV-LTR BAC was evaluated. First, passage 70 was identified as being fully attenuated in maternal antibody negative chickens and chosen as the optimal passage level for use in protective efficacy studies. Second, three protective efficacy trials were conducted comparing the rMd5 REV-LTR p70 BAC to the CVI988/Rispens vaccine. Groups of Mab+ and Mab- 15I5 × 71 chickens were vaccinated in ovo at 18 days of embryonation or intra-abdominally at day of hatch, and challenged at 5 days post-hatch with the vv+MDV strain 686. Vaccination at day of hatch and in ovo with rMd5 REV-LTR p70 BAC protected chickens against MDV-induced bursa and thymic atrophy, but did not provide the same level of protection against MD tumours as that afforded by the commercial vaccine, CVI988/Rispens.

Development, characterization and optimization of a new suspension chicken-induced pluripotent cell line for the production of Newcastle disease vaccine.

Traditionally, substrates for production of viral poultry vaccines have been embryonated eggs or adherent primary cell cultures. The difficulties and cost involved in scaling up these substrates in cases of increased demand have been a limitation for vaccine production. Here, we assess the ability of a newly developed chicken-induced pluripotent cell line, BA3, to support replication and growth of Newcastle disease virus (NDV) LaSota vaccine strain. The characteristics and growth profile of the cells were also investigated. BA3 cells could grow in suspension in different media to a high density of up to 7.0 × 10(6) cells/mL and showed rapid proliferation with doubling time of 21 h. Upon infection, a high virus titer of 1.02 × 10(8) EID50/mL was obtained at 24 h post infection using a multiplicity of infection (MOI) of 5. In addition, the cell line was shown to be free of endogenous and exogenous Avian Leukosis viruses, Reticuloendotheliosis virus, Fowl Adenovirus, Marek's disease virus, and several Mycoplasma species. In conclusion, BA3 cell line is potentially an excellent candidate for vaccine production due to its highly desirable industrially friendly characteristics of growing to high cell density and capability of growth in serum free medium.

Avian oncogenesis induced by lymphoproliferative disease virus: a neglected or emerging retroviral pathogen?

Lymphoproliferative disease virus (LPDV) is an exogenous oncogenic retrovirus that induces lymphoid tumors in some galliform species of birds. Historically, outbreaks of LPDV have been reported from Europe and Israel. Although the virus has previously never been detected in North America, herein we describe the widespread distribution, genetic diversity, pathogenesis, and evolution of LPDV in the United States. Characterization of the provirus genome of the index LPDV case from North America demonstrated an 88% nucleotide identity to the Israeli prototype strain. Although phylogenetic analysis indicated that the majority of viruses fell into a single North American lineage, a small subset of viruses from South Carolina were most closely related to the Israeli prototype. These results suggest that LPDV was transferred between continents to initiate outbreaks of disease. However, the direction (New World to Old World or vice versa), mechanism, and time frame of the transcontinental spread currently remain unknown.

Characterization of reticuloendotheliosis virus isolates obtained from broiler breeders, turkeys, and prairie chickens located in various geographical regions in the United States.

Nine reticuloendotheliosis virus (REV) isolates obtained from broiler breeders, turkeys, and prairie chickens located in three different geographical regions in the USA, and three isolates obtained from known contaminated live-virus vaccines were characterized using polymerase chain reaction (PCR) and indirect immunofluorescence (IFA) assays. All isolates were propagated in chicken embryo fibroblasts obtained from a specific pathogen free breeder flock. PCR analysis of all 12 isolates resulted in the amplification of the 291-bp REV long-terminal repeat region (LTR); none of the isolates exhibited a different pattern or shift from the expected PCR product of REV LTR. The subtype of the REV isolates was determined by IFA using REV-specific monoclonal antibodies, 11B118.22, 11C237.8, and 11D182. Results from sub-typing indicated that all nine isolates from broiler breeders, turkeys, and prairie chickens belonged to subtype 3, and are antigenically related to the chick syncytial virus (CSV) strain of REV, the prototype of subtype 3 REV. In contrast, the three isolates from contaminated vaccines were classified as subtype 2, and were antigenically related to spleen necrosis virus (SNV) strain of REV, the prototype of subtype 2 REV. Three isolates representing REV isolated from broiler breeders, turkeys, and prairie chickens were cloned and further evaluated by DNA sequence analysis of the envelope gene. Results from DNA sequence analysis confirmed those from sub-typing and indicated that the three REV isolates representing those from broiler breeders, turkeys, and prairie chickens are closely related to CSV of REV, with an amino acid homology of 98% or greater as compared with SNV with an amino acid homology of 95% or less. Data from this study clearly indicate that subtype 3 is the most common subtype of REV circulating in three different avian species, namely broiler breeders, turkeys and prairie chickens, located in three different geographical regions in the United States.

Development of an endogenous virus-free line of chickens susceptible to all subgroups of avian leukosis virus.

Primary chicken embryo fibroblasts (CEF) from special specific pathogen-free chicken lines are used for detection of contamination of adult or embryonic tissues, meconium, or tissue culture fluids with avian leukosis viruses (ALV). The suitability and efficiency of such tests depend on the susceptibility of CEF to the various subgroups of exogenous as well as endogenous ALV. The ideal CEF for such tests should be not only susceptible to all retroviruses, but also free of endogenous viruses so that such tests are immune to any interference that may occur between the endogenous and the tested (exogenous) viruses. CEF and/or chickens free of endogenous viruses are also desirable for gene transfer studies using retroviral vectors, such as RNA interference (RNAi) experiments and transgenic work. The absence of ev genes in CEF or chickens can empower clean detection of successful RNAi construct delivery or gene transfer. CEF free of ev genes are also essential reagents routinely used in growing and detecting unknown retroviruses in varied viral assays. This report documents the development of a new line of chickens, 0.TVB*S1, that is free of endogenous viruses and susceptible to all subgroups of ALV identified in chickens.

Development of a polymerase chain reaction to differentiate avian leukosis virus (ALV) subgroups: detection of an ALV contaminant in commercial Marek's disease vaccines.

Avian leukosis viruses (ALVs) are common in many poultry flocks and can be detected using an enzyme-linked immunosorbent assay or any other test designed to identify p27, the group-specific antigen located in gag. However, endogenous retroviruses expressing p27 are often present and can be confused with exogenous ALVs. A more specific and informative assay involves targeting the variable envelope glycoprotein gene (gp85) that is the basis for dividing ALVs into their different subgroups. We designed polymerase chain reaction (PCR) primers that would specifically detect and amplify viruses from each of the six ALV subgroups: A, B, C, D, E, and J. Subgroup B and D envelopes are related, and our B-specific primers also amplified subgroup D viruses. We also designed a set of common primers to amplify any ALV subgroup virus. To demonstrate the usefulness of these primers, we obtained from the Center for Veterinary Biologics in Iowa culture supernatant from chicken embryo fibroblasts infected with an ALV that was found to be a contaminant in two commercial Marek's disease vaccines. Using our PCR primers, we demonstrate that the contaminant was a subgroup A ALV. We cloned and sequenced a portion of the envelope gene and confirmed that the ALV was a subgroup A virus. Unlike typical subgroup A viruses, the contaminant ALV grew very slowly in cell culture. We also cloned and sequenced a portion of the long terminal repeat (LTR) from the contaminant virus. The LTR was found to be similar to those LTRs found in endogenous ALVs (subgroup E) and very dissimilar to LTRs normally found in subgroup A viruses. The E-like LTR probably explains why the contaminant grew so poorly in cell culture.

Influence of strain, dose of virus, and age at inoculation on subgroup J avian leukosis virus persistence, antibody response, and oncogenicity in commercial meat-type chickens.

The effects of viral strain, viral dose, and age of bird at inoculation on subgroup J avian leukosis virus (ALV J) persistence, neutralizing antibody (VNAb) response, and tumors were studied in commercial meat-type chickens. Chickens were inoculated on the fifth day of embryonation (5 ED) or on day of hatch (DOH) with either 100 or 10,000 50% tissue-culture infective dose (TCID50) of one of three ALV J strains, namely ADOL Hcl, ADOL 6803, or ADOL 4817. At 1, 3, 7, 11, 15, 19, 23, 27, and 32 wk posthatch, chickens were examined for ALV J viremia and VNAb against the inoculated strain of ALV J. A high incidence (83%-100%) of ALV J persistence was observed in all treatment groups. Development of VNAb did not always lead to viremia-free status; even though 18% of the chickens developed VNAb, only 4% were able to clear viremia. The viral strain, dose, and age of bird at inoculation seemed to have an effect on the incidence of VNAb; however, the differences were statistically significant in only some treatment groups. Chickens infected with ADOL 6803 had higher incidence of VNAb than chickens infected with ADOL Hc1 and ADOL 4817 (P < 0.05 in groups 5 ED at 100 TCID50 and DOH at 10,000 TCID50). There was a trend in all groups inoculated with 100 TCID50 to have higher incidence of VNAb than that of groups inoculated with 10,000 TCID50 (ADOL 6803 at 5 ED and ADOL 4817 at DOH [P < 0.05]; ADOL Hc1 at DOH [P < 0.08]). In most treatment groups (ADOL Hc1 at 100 and 10,000 TCID50, ADOL 6803 at 10,000 TCID50, and ADOL 4817 at 100 TCID50), chickens inoculated at DOH had higher incidence of VNAb than that of chickens inoculated at 5 ED (ADOL 6803 at 10,000 TCID50 [P < 0.05], ADOL Hc1 at 100 TCID50 [P < 0.08]). Incidence of ALV J-induced tumors and tumor spectrum were influenced by viral strain, age at inoculation, and VNAb response.

Susceptibility of various parental lines of commercial white leghorn layers to infection with a naturally occurring recombinant avian leukosis virus containing subgroup B envelope and subgroup J long terminal repeat.

Chickens from seven different parental lines of commercial White Leghorn layer flocks from three independent breeders were inoculated with a naturally occurring avian leukosis virus (ALV) containing an ALV-B envelope and an ALV-J long terminal repeat (LTR) termed ALV-B/J. Additional groups of chickens from the same seven parental lines were inoculated with ALV-B. Chickens were tested for ALV viremia and antibody at 0, 4, 8, 16, and 32 wk postinfection. Chickens from all parental lines studied were susceptible to infection with ALV-B with 40%-100% of inoculated chickens positive for ALV at hatch following embryo infection. Similarly, infection of egg layer flocks with the ALV-B/J recombinant virus at 8 days of embryonation induced tolerance to ALV with 86%-100% of the chickens viremic, 40%-75% of the chickens shedding virus, and only 2/125 (2%) of the chickens producing serum-neutralizing antibodies against homologous ALV-B/J recombinant virus at 32 wk postinfection. In contrast, when infected with the ALV-B/J recombinant virus at hatch, 33%-82% of the chickens were viremic, 28%-47% shed virus, and 0%-56% produced serum-neutralizing antibodies against homologous ALV-B/J recombinant virus at 32 wk postinfection. Infection with the ALV-B/J recombinant virus at embryonation and at hatch induced predominately lymphoid leukosis (LL), along with other common ALV neoplasms, including erythroblastosis, osteopetrosis, nephroblastomas, and rhabdosarcomas. No incidence of myeloid leukosis (ML) was observed in any of the commercial White Leghorn egg layer flocks infected with ALV-B/J in the present study. Data suggest that the parental line of commercial layers may influence development of ALV-B/J-induced viremia and antibody, but not tumor type. Differences in type of tumors noted in the present study and those noted in the field case where the ALV-B/J was first isolated may be attributed to differences in the genetics of the commercial layer flock in which ML was first diagnosed and the present commercial layer flocks tested in the present study.

Molecular and biological characterization of a naturally occurring recombinant subgroup B avian leukosis virus with a subgroup J-like long terminal repeat.

Infection of broiler chickens with subgroup J avian leukosis virus (ALV) results in the induction of myeloid tumors. However, although egg-type chickens are susceptible to infection with ALV-J, the tumor incidence is very low, and on rare occasions the tumors observed are of the myeloid lineage. We recently described the isolation of an ALV (AF115-4) from commercial egg-type chickens suffering from myeloid leukosis. AF115-4 was initially identified as an ALV-J isolate based on PCR analysis of the long terminal repeat (LTR). However, further characterization of the viral envelope indicated that the virus is recombinant with subgroups B envelope and J LTR. Here we further characterize this recombinant virus at both the molecular and biological levels. We show that the AF115-4 isolate expresses a recombinant envelope glycoprotein encoded by a subgroup B gp85 region and a subgroup E gp37 region. The host range ofAF115-4 was analyzed using cells resistant to infection by subgroups A/B, J, or E; this shows that no ALV-J was present in the isolates obtained from the affected chickens. Additional antigenic characterization of AF115-4 using chicken sera specific for subgroups B or J indicated that no ALV-J was present in the samples examined. Inoculation of AF 115-4 into ALV-susceptible 1515 X 71 chickens resulted in the induction of lymphoid leukosis but not the expected myeloid leukosis affecting the commercial chickens. These results suggest that differences in the genetic makeup of the chickens from which AF115-4 was isolated and the line 1515 X 71 used in the present experiments may be responsible for the observed differences in pathogenicity. In addition, the results suggest that ALV-J continues to evolve by recombination, generating new viruses with different pathological properties.

Response of white leghorn chickens of various B haplotypes to infection at hatch with subgroup J avian leukosis virus.

White leghorn chickens from seven 15.B congenic lines (genetically similar except for genes linked to the major histocompatibility complex [MHC] B haplotype) and two Line 0.B semicongenic lines were infected at hatch with strain ADOL Hc-1 of subgroup J avian leukosis virus (ALV-J). At 5, 8, 16, and 36 wk of age, chickens were tested for viremia, serum-neutralizing antibody, and cloacal shedding. Chickens were also monitored for development of neoplasia. In the 15.B congenic lines (B*2, B*5, B*12, B*13, B*15, B*19, and B*21) there were no significant differences in the incidence of viremia between B haplotypes. In fact, infection at hatch in all of the 15.B congenic lines induced tolerance to ALV-J because 100% of these chickens were viremic and transient circulating serum-neutralizing antibody was detected in only a few chickens throughout the 36 wk experiment. However, at 16 wk of age more B*15 chickens had antibody and fewer B*15 chickens shed virus than did the 16-wk-old B*2, B*5, or B*13 chickens. Moreover, compared with B*15 chickens, a higher percentage of B*13 chickens consistently shed virus from 8 wk postinfection to termination at 36 wk postinfection. The B haplotype had a transient effect on viral clearance in Line 0.B semicongenics, as more B*13 than B*21 chickens remained viremic through 5 wk of age. Very few (0%-18%) of the Line 0.B semicongenic chickens shed virus. By 36 wk of age, all Line 0 B*13 and B*21 chickens produced serum-neutralizing antibodies and cleared the virus. These results show that following ALV-J infection at hatch the immune response is influenced transiently by the B haplotype and strongly by the line of chicken. Although this study was not designed to study the effect of endogenous virus on ALV-J infection, the data suggest that endogenous virus expression reduced immunity to ALV-J in Line 15I5, compared with Line 0, a line known to lack endogenous virus genes.

Comparison of serological and virological findings from subgroup J avian leukosis virus-infected neoplastic and non-neoplastic flocks in Israel.

Blood samples from nine broiler breeder flocks comprising five flocks clinically affected with myeloid leukosis tumours (ML+) and four tumour-free flocks from the same commercial background (ML-) were compared for avian leukosis virus subgroup J (ALV-J) serum antibodies by enzyme-linked immunosorbent assay (ELISA), for antigenemia (group-specific antigen) by antigen-trapping ELISA and for viremia. Group-specific antigen was detected in the sera of 58.1% of ML+ birds and 46.4% of the ML- birds (P=not significant), while 45.5% of ML+ birds and 24.1% of the ML- birds had ALV-J antibodies (P=0.065). In inoculated cell culture, 64.1% of the ML+ sera were viremic compared with 16.7% of the ML- sera (P=0.001). Similar significant differences were found between the two groups of flocks when ALV-J viremia was detected by immunofluorescence using a monoclonal env antibody (P=0.004), and for proviral DNA by polymerase chain reaction using two different sets of env-gene primers, H5-H7 (P=0.001) and R5-F5 (P=0.001). Using the primer pair R5-F5 the product size was approximately 1 kbp, while some heterogeneity in size among isolates was discernable. Our results indicate that a combination of diagnostic tests should be adopted in routine examination of tumour material in order to rule out false-negative findings.

Response of white leghorn chickens of various genetic lines to infection with avian leukosis virus subgroup J.

In Experiment 1, chickens from various white leghorn experimental lines were inoculated with strain ADOL-Hcl of subgroup J avian leukosis virus (ALV-J) either as embryos or at 1 day of age. At various ages, chickens were tested for ALV-J induced viremia, antibody, and packed cell volume (PCV). Also, at 4 and 10 wk of age, bursal tissues were examined for avian leukosis virus (ALV)-induced preneoplastic lesions with the methyl green-pyronine (MGP) stain. In Experiment 2, chickens harboring or lacking endogenous virus 21 (EV21) were inoculated with strain ADOL-Hcl of ALV-J at hatch. All embryo-inoculated chickens in Experiment 1 tested positive for ALV-J and lacked antibody throughout the experimental period of 30 wk and were considered viremic tolerant, regardless of line of chickens. By 10 wk of age, the incidence of ALV-J viremia in chickens inoculated with virus at hatch varied from 0 (line 0 chickens) to 97% (line 1515); no influence of ALV-J infection was noted on PCV. Results from microscopic examination of MGP-stained bursal tissues indicate that ALV-J can induce typical ALV-induced transformation in bursal follicles of white leghorn chickens. Lymphoid leukosis and hemangiomas were the most common ALV-J-induced tumors noted in chickens in Experiment 1. At termination of Experiment 2 (31 wk of age), 54% of chickens harboring EV21 were viremic tolerant compared with 5% of chickens lacking EV21 after inoculation with ALV-J at hatch. The data indicate that genetic differences among lines of white leghorn chickens, including the presence or absence of EV21, can influence response of chickens to infection with ALV-J.

Tissue tropism and bursal transformation ability of subgroup J avian leukosis virus in White Leghorn chickens.

In Experiment 1, a monoclonal antibody against the envelope glycoprotein (gp85) of subgroup J avian leukosis virus (ALV-J) was used to study the distribution of ALV-J in various tissues of White Leghorn chickens inoculated as embryos with the strain ADOL-Hcl of ALV-J. At 2 and 6 wk of age, various tissues from infected and control uninfected chickens were tested for the presence of ALV-J gp85 by immunohistochemistry. In Experiment 2, using the methyl green-pyronine (MGP) stain, sections of bursa of Fabricius (BF) from chickens of line 15I5 x 7(1), inoculated with ALV-J or Rous-associated virus-1 (RAV-1), a subgroup A ALV, at hatch were examined for transformation of bursal follicles at 4 and 10 wk of age. In Experiment 1, specific staining indicative of the presence of ALV-J gp85 was noted at both 2 and 6 wk of age in the adrenal gland, bursa, gonads, heart, kidney, liver, bone marrow, nerve, pancreas, proventriculus, spleen, and thymus. In Experiment 2, by 10 wk of age, transformed bursal follicles were detected in MGP-stained sections of BF in only one of five (20%) chickens inoculated with ALV-J at hatch, compared with five of five (100%) chickens inoculated with RAV-1. The data demonstrate distribution of ALV-J gp85 in various tissues of White Leghorn chickens experimentally inoculated as embryos with the virus. The data also confirm our previous observation that ALV-J is capable of inducing transformation of bursal follicles, albeit the incidence is less frequent than that induced by subgroup A ALV.

Pathogenicity of two recombinant avian leukosis viruses.

We have recently described the isolation and molecular characteristics of two recombinant avian leukosis subgroup J viruses (ALV J) with an avian leukosis virus subgroup A envelope (r5701A and r6803A). In the present study, we examined the role of the subgroup A envelope in the pathogenesis of these recombinant viruses. Chickens of line 151(5) x 7(1) were inoculated at 1 day of age with r5701A, r6803A, Rous-associated virus type 1 (RAV-1), or strain ADOL-Hcl of ALV-J. At 2, 4, 10, 18, and 32 wk postinoculation (PI), chickens were tested for avian leukosis virus (ALV)-induced viremia, shedding, and neutralizing antibodies. All except one chicken inoculated with the recombinant viruses (98%) developed neutralizing antibodies by 10 wk PI compared with only 16% and 46% of the ADOL-Hcl and RAV-1-inoculated birds, respectively. ALV-induced tumors and mortality in the two groups inoculated with recombinant viruses were different. The incidence of tumors in groups inoculated with r5701A or RAV-1 was 100% compared with only 9% in the groups inoculated with r6803A or ADOL-Hcl. The data suggest that differences in pathogenicity between the two recombinant viruses might be due to differences in the sequence of the 3' untranslated region (presence or absence of the E element), and, therefore, not only the envelope but also other elements of the viral genome play an important role in the pathogenesis of ALV.

Molecular characterization of reticuloendotheliosis virus insertions in the genome of field and vaccine strains of fowl poxvirus.

Evidence of the widespread occurrence of reticuloendotheliosis virus (REV) sequence insertions in fowl poxvirus (FPV) genome of field isolates and vaccine strains has increased in recent years. However, only those strains carrying a near intact REV provirus are more likely to cause problems in the field. Detection of the intact provirus or REV protein expression from FPV stocks has proven to be technically difficult. The objective of the present study was to evaluate current and newly developed REV and FPV polymerase chain reaction (PCR) assays to detect the presence of REV provirus in FPV samples. The second objective was to characterize REV insertions among recent "variant" FPV field isolates and vaccine strains. With REV, FPV, and heterologous REV-FPV primers, five FPV field isolates and four commercial vaccines were analyzed by PCR and nucleotide sequence analysis. Intact and truncated REV 5' long terminal repeat (LTR) sequences were detected in all FPV field isolates and vaccine strains, indicating heterogeneous REV genome populations. However only truncated 3' LTR and envelope sequences were detected among field isolates and in one vaccine strain. Amplifications of the REV envelope and 3' LTR provided strong evidence to indicate that these isolates carry a near intact REV genome. Three of the four FPV vaccine strains analyzed carried a solo complete or truncated 5' LTR sequence, indicating that intact REV provirus was not present. Comparison of PCR assays indicated that assays amplifying REV envelope and REV 3' LTR sequences provided a more accurate assessment of REV provirus than PCR assays that amplify the REV 5' LTR region. Therefore, to differentiate FPV strains that carry intact REV provirus from those that carry solo 5' LTR sequences, positive PCR results with primers that amplify the 5' LTR should be confirmed with more specific PCR assays, such as the envelope, or the REV 3' LTR PCR.

Characterization of T-cell lymphomas associated with an outbreak of reticuloendotheliosis in turkeys.

Increased mortality and decreased egg production associated with disseminated lymphoma were observed in a turkey breeding flock for more than 20 weeks. A few unrelated meat turkey flocks, from the same integrator, experienced increased condemnation due to neoplasia in a few organs. Lymphoma was characterized by a uniform population of large lymphocytes with large vesicular nuclei containing one or two nucleoli and with little, faintly staining, basophilic cytoplasm. Neoplastic cells replaced normal tissue and were consistent with lesions seen with reticuloendotheliosis virus (REV) infection. Immunoperoxidase and fluorescent antibody staining characterized the neoplastic cells as CD3+, CD4+ and CD8- lymphocytes. Infection with REV was confirmed by virus isolation, polymerase chain reaction, serology and indirect fluorescent antibody. Poults hatched from these breeders tested positive for REV antibodies at hatch, but the performance of these flocks was normal and lymphoma was not observed. The origin of REV infection in this outbreak could not be determined. This is the first documented report of T-cell lymphomas associated with REV in commercial flocks. Furthermore, this is the first time that lymphomas have been characterized as T helper cells (CD3+ CD4+ and CD8-) in an outbreak of REV in turkeys.

Diagnosis of myeloid leukosis induced by a recombinant avian leukosis virus in commercial white leghorn egg laying flocks.

Commercial white leghorn egg layer flocks being used to produce fertile eggs for human vaccine production exhibited dramatically low peaks in egg production, two to four times higher than normal weekly mortality, and high numbers of cull, nonlaying birds after the onset of sexual maturity. These lower production characteristics could not be associated with management-related problems. Gross lesions of cull and fresh dead birds necropsied showed approximately 60% lacked ovarian activity and had lesions of a bacterial bursitis or synovitis, whereas the other 40% had tumors of the viscera but not of the bursa of Fabricius. Histologic examination of tumor-containing tissues showed lesions typical of myelocytomatosis. The diagnosis of myeloid leukosis was confirmed by the isolation of a recombinant avian leukosis virus (ALV) containing the LTR of subgroup J and the envelope of subgroup B ALV. A positive polymerase chain reaction with primers specific for the 3' untranslated region LTR confirmed the presence of LTR of ALV-J. The source of infection with this recombinant ALV was not determined; however, it is likely that commingling of the day-old egg-type chicks with ALV-J-infected meat-type chicks in a common hatchery had contributed to this outbreak.