The CCJR® Charles A. Engh, Sr, MD. Excellence in Hip Research Award: The Evolution of Revision Hip Arthroplasty and Impact on the Trainee's Experience.

BACKGROUND: Advances in total hip arthroplasty (THA) have resulted in evolving revision indications and intraoperative techniques, which can influence the exposure of trainees to complex cases. We report 3 decades of revision experience from a tertiary referral center that trains fellows, comparing the reasons for revision and the complexity of revisions over time. METHODS: We retrospectively reviewed all revision THAs performed at our institution from 1990 to 2022. Revision diagnoses, components revised, types of revision implants used, and exposure techniques were collected. A "complex" revision was defined as a case that involved an extended trochanteric osteotomy, triflange and cup-cage construct, or acetabular augment. RESULTS: A total of 3,556 THA revisions were identified (108 revisions/year). Aseptic loosening was the most common indication in 1990 to 1999 (45 per year), but decreased to 28.3/year in 2010 to 2019. From 1990 to 1999 and 2010 to 2019, fracture increased from 3.1 to 7.3 per year, infection from 2.9/year to 16.9/year, and metallosis from 0.1 to 13.2 per year. Both component revision were common from 1990 to 1994 (42.6 per year), while polyethylene exchange was most common in 2010 to 2019 (43.3 per year). A decrease was observed in "complex" cases over time: 14.8 extended trochanteric osteotomies/year in 2000 to 2004 compared to 5.4 per year in 2018 to 2022, 4.5 triflange and cup-cage constructs/year in 2004 to 2007 compared to 0.8 per year in 2018 to 2022, and 4 acetabular augments per year in 2009 to 2012 compared to 1 per year in 2018 to 2022. CONCLUSIONS: Indications for revision have changed over the decades, while the number of "complex" revisions has gradually decreased, presumably due to advances in implants and materials. If this trend extends to other training institutions, the next generation of arthroplasty surgeons will have less exposure to complex revisions during their training.

Phenotypic Characterization of Recombinant Marek's Disease Virus in Live Birds Validates Polymorphisms Associated with Virulence.

Marek's disease (MD) is a highly infectious lymphoproliferative disease in chickens with a significant economic impact. Mardivirus gallidalpha 2, also known as Marek's disease virus (MDV), is the causative pathogen and has been categorized based on its virulence rank into four pathotypes: mild (m), virulent (v), very virulent (vv), and very virulent plus (vv+). A prior comparative genomics study suggested that several single-nucleotide polymorphisms (SNPs) and genes in the MDV genome are associated with virulence, including nonsynonymous (ns) SNPs in eight open reading frames (ORF): UL22, UL36, UL37, UL41, UL43, R-LORF8, R-LORF7, and ICP4. To validate the contribution of these nsSNPs to virulence, the vv+MDV strain 686 genome was modified by replacing nucleotides with those observed in the vMDV strains. Pathogenicity studies indicated that these substitutions reduced the MD incidence and increased the survival of challenged birds. Furthermore, using the best-fit pathotyping method to rank the virulence, the modified vv+MDV 686 viruses resulted in a pathotype similar to the vvMDV Md5 strain. Thus, these results support our hypothesis that SNPs in one or more of these ORFs are associated with virulence but, as a group, are not sufficient to result in a vMDV pathotype, suggesting that there are additional variants in the MDV genome associated with virulence, which is not surprising given this complex phenotype and our previous finding of additional variants and SNPs associated with virulence.

Newcastle disease virus (NDV) recombinant expressing Marek's disease virus (MDV) glycoprotein B significantly protects chickens against MDV and NDV challenges.

Marek's disease (MD) is a highly contagious viral neoplastic disease of chickens caused by Marek's disease virus (MDV), resulting in significant economic losses to the poultry industry worldwide. The commonly used live and/or vectored MDV vaccines are expensive to produce and difficult to handle due to the requirement of liquid nitrogen for manufacturing and delivering frozen infected cells that are viable. In this study, we aimed to develop a Newcastle disease virus (NDV) vectored MDV vaccine that can be lyophilized, stored, and transported at 4 °C. Four NDV LaSota (LS) vaccine strain-based recombinant viruses expressing MDV glycoproteins gB, gC, gE, or gI were generated using reverse genetics technology. The biological assessments showed that these recombinant viruses were slightly attenuated in vivo yet retained similar growth kinetics and virus titers in vitro compared to the parental LaSota virus. Vaccination of leghorn chickens (Lines 15I5x71 F1 cross) with these recombinant viruses via intranasal and intraocular routes conferred different levels of protection against virulent MDV challenge. The recombinant expressing the MDV gB protein, rLS/MDV-gB, protected vaccinated birds significantly against MDV-induced tumor formation when challenged at 14 days post-vaccination (DPV) but moderately at 5 DPV. Whereas the other three recombinants provided little protection against the MDV challenge. All four recombinants conferred complete protection against the velogenic NDV challenge. These results demonstrated that the rLS/MDV-gB virus is a safe and efficacious dual vaccine candidate that can be lyophilized and potentially mass-administered via aerosol or drinking water to large chicken populations at a meager cost.

Targeted Ablation of Exon 2 of the Avian Leukosis Virus-A (ALV-A) Receptor Gene in a Chicken Fibroblast Cell Line by CRISPR Abrogates ALV-A Infection.

The U.S. Department of Agriculture Avian Disease and Oncology Laboratory currently relies on live birds of specific genetic backgrounds for producing chicken-embryo fibroblasts that are used for the diagnosis and subtyping of field isolates associated with avian leukosis virus (ALV) outbreaks. As an alternative to maintaining live animals for this purpose, we are currently developing cell lines capable of achieving the same result by ablation of the entry receptors utilized by ALV strains. We used CRISPR-Cas9 on the cell fibroblast-derived cell line DF-1 to disrupt the tva gene, which encodes the receptor required for binding and entry of ALV-A into cells. We ultimately identified seven DF-1 clones that had biallelic and homozygous indels at the Cas9 target site, exon 2 of tva. When tested in vitro for their ability to host ALV-A, the five clones that had frameshift mutations that disrupted the Tva protein were unable to support ALV-A replication. This result clearly demonstrates that modified cell lines can be used as part of a battery of tests to determine ALV subtype for isolate characterization, thus eliminating the need for live birds.

Nota de investigación- La ablación dirigida del exón 2 del gene del receptor del virus de la leucosis aviar A (ALV-A) en una línea celular de fibroblastos de pollo mediante CRISPR anula la infección por ALV-A. El Laboratorio de Oncología y Enfermedades Aviares del Departamento de Agricultura de los Estados Unidos. actualmente depende de aves vivas con antecedentes genéticos específicos para producir fibroblastos de embrión de pollo que se utilizan para el diagnóstico y la subtipificación de aislamientos de campo asociados con brotes del virus de la leucosis aviar (ALV). Como alternativa al mantenimiento de animales vivos para este propósito, actualmente se están desarrollando líneas celulares capaces de lograr el mismo resultado mediante la ablación de los receptores de entrada utilizados por las cepas ALV. Se utilizó el método repeticiones palindrómicas cortas agrupadas y regularmente interespaciadas o CRISPR-Cas9 en la línea celular DF-1 derivada de fibroblastos para interrumpir el gene Tva, que codifica el receptor requerido para la unión y entrada de ALV-A en las células. Finalmente, se identificaron siete clones de DF-1 que tenían inserciones y deleciones (indeles) bialélicos y homocigóticos en el sitio blanco Cas9, exón 2 del gene tva. Cuando se probó in vitro su capacidad para albergar ALV-A, los cinco clones que tenían mutaciones que involucraban al marco de lectura y que interrumpieron la proteína Tva no pudieron admitir la replicación de ALVA. Este resultado demuestra claramente que las líneas celulares modificadas se pueden utilizar como parte de una batería de pruebas para determinar el subtipo de ALV para la caracterización de los aislamientos, eliminando así la necesidad de aves vivas.

Identification and Validation of Ikaros (IKZF1) as a Cancer Driver Gene for Marek's Disease Virus-Induced Lymphomas.

Marek's disease virus (MDV) is the causative agent for Marek's disease (MD), which is characterized by T-cell lymphomas in chickens. While the viral Meq oncogene is necessary for transformation, it is insufficient, as not every bird infected with virulent MDV goes on to develop a gross tumor. Thus, we postulated that the chicken genome contains cancer driver genes; i.e., ones with somatic mutations that promote tumors, as is the case for most human cancers. To test this hypothesis, MD tumors and matching control tissues were sequenced. Using a custom bioinformatics pipeline, 9 of the 22 tumors analyzed contained one or more somatic mutation in Ikaros (IKFZ1), a transcription factor that acts as the master regulator of lymphocyte development. The mutations found were in key Zn-finger DNA-binding domains that also commonly occur in human cancers such as B-cell acute lymphoblastic leukemia (B-ALL). To validate that IKFZ1 was a cancer driver gene, recombinant MDVs that expressed either wild-type or a mutated Ikaros allele were used to infect chickens. As predicted, birds infected with MDV expressing the mutant Ikaros allele had high tumor incidences (~90%), while there were only a few minute tumors (~12%) produced in birds infected with the virus expressing wild-type Ikaros. Thus, in addition to Meq, key somatic mutations in Ikaros or other potential cancer driver genes in the chicken genome are necessary for MDV to induce lymphomas.

Deletion of the Viral Thymidine Kinase in a Meq-Deleted Recombinant Marek's Disease Virus Reduces Lymphoid Atrophy but Is Less Protective.

Marek's disease (MD) is a ubiquitous disease of domesticated chickens and its etiologic agent is the Gallid alphaherpesvirus 2 (GaHV-2), also known as Marek's disease virus (MDV). MD is currently controlled by vaccination using live attenuated strains of MDV (e.g., CVI988/Rispens), non-pathogenic serotypes of MDV (GaHV-3), or non-pathogenic strains of the related Melagrid alphaherpesvirus 1 (MeHV-1). One attractive strategy for the production of new vaccine strains is a recombinant MDV attenuated by the deletion of the major viral oncogene meq. However, meq-deleted variants of MDV cause atrophy of the bursa and thymus in maternal antibody-negative chickens, and the resulting immunosuppression makes them unsuitable. Herein we detail our attempt to mitigate the lymphoid atrophy caused by meq-deleted MDV by further attenuation of the virus through ablation of the viral thymidine kinase (tk) gene. We demonstrate that ablation of the viral tk from the meq-deleted virus rMd5B40/Δmeq resulted in a virus attenuated for replication in vitro and which spared chickens from atrophy of the lymphoid organs in vivo. When the rMd5B40/Δmeq/Δtk/GFP was used as a vaccine it was protective against challenge with the vv+MDV strain 686, but the protection was less than that provided by the CVI988/Rispens vaccine.

Codon Deoptimization of UL54 in meq-Deleted Marek's Disease Vaccine Candidate Eliminates Lymphoid Atrophy but Reduces Vaccinal Protection.

Marek's disease (MD) is an oncogenic, lymphoproliferative, and highly contagious disease of chickens. Its etiologic agent is the alphaherpesvirus Marek's disease virus (MDV, Gallid alphaherpesvirus 2), and it is a chronic and ubiquitous problem for the poultry industry with significant economic impact in the United States and worldwide. We have previously demonstrated that MDV attenuated by dicodon deoptimization of the UL54 gene results in reduced gene product accumulation in vitro, with reduced viral genome copy number upon infection and reduced atrophy of bursa and thymus in vivo as well. In this report we detail our attempts to use the same attenuation strategy on a meq-deleted MDV mutant, rMd5B40ΔMeq. Unlike the wild-type rMd5B40 virus the rMd5B40ΔMeq is no longer oncogenic, but infected birds experience an unacceptable amount of bursa and thymus atrophy (BTA). We produced two meq-deleted MDV recombinants with a dicodon-deoptimized UL54 (rMd5B40ΔMeq/UL54deop1 and -deop2) and tested their tendency to cause BTA and to serve as a protective vaccine. We found that, although dicodon deoptimization of the UL54 gene results in a virus that spares the infected animal from atrophy of the bursa and thymus, the meq-deleted UL54-deoptimized recombinant is also less protective than the meq-deleted virus without UL54 deoptimization, the HVT + SB1 combination vaccine, or the Rispens (CVI988) vaccine.

Two Cases of Marek's Disease in Backyard Turkeys.

In two independent submissions, a 3-yr-old, dead Bourbon Red turkey tom from a zoo and a Royal Palm turkey hen from a backyard flock were submitted for necropsy. Both birds had been kept together with chickens. Findings of the necropsy of the first turkey were an enlarged and dark liver with many pale white foci and a few small white nodules, pale and enlarged spleen, prominent thymus, mottled and pale kidneys, and pale and enlarged testes. Findings of the necropsy of the second turkey were a dark and mildly enlarged liver and severely enlarged, firm, and pale kidneys. Histopathology revealed infiltration of most organs of both birds with neoplastic lymphocytes, which were uniform in the first turkey and pleomorphic in the second turkey. Immunohistochemistry with a CD3 marker identified the neoplastic lymphocytes as T cells. Marek's disease virus serotype 1 was detected with PCR in the livers of both birds, whereas PCRs for reticuloendotheliosis virus and lymphoproliferative disease virus were negative. Based on these findings, Marek's disease was diagnosed in both turkeys, which is very rare and were the first definitive cases reported in the United States. It is likely that the chickens were the source of infection.

Genomic characterization and phylogenetic analysis of Salmonella enterica serovar Javiana.

Salmonella enterica serovar Javiana is the fourth most reported serovar of laboratory-confirmed human Salmonella infections in the U.S. and in Tennessee (TN). Although Salmonella ser. Javiana is a common cause of human infection, the majority of cases are sporadic in nature rather than outbreak-associated. To better understand Salmonella ser. Javiana microbial population structure in TN, we completed a phylogenetic analysis of 111 Salmonella ser. Javiana clinical isolates from TN collected from Jan. 2017 to Oct. 2018. We identified mobile genetic elements and genes known to confer antibiotic resistance present in the isolates, and performed a pan-genome-wide association study (pan-GWAS) to compare gene content between clades identified in this study. The population structure of TN Salmonella ser. Javiana clinical isolates consisted of three genetic clades: TN clade I (n = 54), TN clade II (n = 4), and TN clade III (n = 48). Using a 5, 10, and 25 hqSNP distance threshold for cluster identification, nine, 12, and 10 potential epidemiologically-relevant clusters were identified, respectively. The majority of genes that were found to be over-represented in specific clades were located in mobile genetic element (MGE) regions, including genes encoding integrases and phage structures (91.5%). Additionally, a large portion of the over-represented genes from TN clade II (44.9%) were located on an 87.5 kb plasmid containing genes encoding a toxin/antitoxin system (ccdAB). Additionally, we completed phylogenetic analyses of global Salmonella ser. Javiana datasets to gain a broader insight into the population structure of this serovar. We found that the global phylogeny consisted of three major clades (one of which all of the TN isolates belonged to) and two cgMLST eBurstGroups (ceBGs) and that the branch length between the two Salmonella ser. Javiana ceBGs (1,423 allelic differences) was comparable to those from other serovars that have been reported as polyphyletic (929-2,850 allelic differences). This study demonstrates the population structure of TN and global Salmonella ser. Javiana isolates, a clinically important Salmonella serovar and can provide guidance for phylogenetic cluster analyses for public health surveillance and response.

Depletion of CD8αß+ T Cells in Chickens Demonstrates Their Involvement in Protective Immunity towards Marek's Disease with Respect to Tumor Incidence and Vaccinal Protection.

Marek's disease (MD) is a lymphoproliferative disease in chickens caused by Marek's disease virus (MDV), a highly oncogenic alphaherpesvirus. Since 1970, MD has been controlled through widespread vaccination of commercial flocks. However, repeated and unpredictable MD outbreaks continue to occur in vaccinated flocks, indicating the need for a better understanding of MDV pathogenesis to guide improved or alternative control measures. As MDV is an intracellular pathogen that infects and transforms CD4+ T cells, the host cell-mediated immune response is considered to be vital for controlling MDV replication and tumor formation. In this study, we addressed the role of CD8+ T cells in vaccinal protection by widely-used monovalent (SB-1 and HVT) and bivalent (SB-1+HVT) MD vaccines. We established a method to deplete CD8+ T cells in chickens and found that their depletion through injection of anti-CD8 monoclonal antibodies (mAb) increased tumor induction and MD pathology, and reduced vaccinal protection to MD, which supports the important role of CD8+ T cells for both MD and vaccinal protection.

Towards a mechanistic understanding of the synergistic response induced by bivalent Marek's disease vaccines to prevent lymphomas.

BACKGROUND: Marek's disease (MD) is a lymphoproliferative disease of chickens caused by Marek's disease virus (MDV), an oncogenic α-herpesvirus. Since 1970, MD has been controlled by widespread vaccination; however, more effective MD vaccines are needed to counter more virulent MDV strains. The bivalent vaccine combination of SB-1 and herpesvirus of turkey (HVT) strain FC126 has been widely used. Nonetheless, the mechanism(s) underlying this synergistic effect has not been investigated. METHODS: Three experiments were conducted where SB-1 or HVT were administered as monovalent or bivalent vaccines to newly hatched chickens, then challenged five days later with MDV. In Experiment 1, levels of MDV replication in PBMCs were measured over time, and tumor incidence and vaccinal protection determined. In Experiment 2, MDV and vaccine strains replication levels in lymphoid organs were measured at 1, 5, 10, and 14 days post-challenge (DPC). In Experiment 3, to verify that the bursa was necessary for HVT protection, a subset of chicks were bursectomized and these birds plus controls were similarly vaccinated and challenged, and the levels of vaccinal protection determined. RESULTS: The efficacy of bivalent SB-1 + HVT surpasses that of either SB-1 or HVT monovalent vaccines in controlling the level of pathogenic MDV in PBMCs until the end of the study, and this correlated with the ability to inhibit tumor formation. SB-1 replication in the spleen increased from 1 to 14 DPC, while HVT replicated only in the bursa at 1 DPC. The bursa was necessary for immune protection induced by HVT vaccine. CONCLUSION: Synergy of SB-1 and HVT vaccines is due to additive influences of the individual vaccines acting at different times and target organs. And the bursa is vital for HVT to replicate and induce immune protection.

Identification of Marek's disease virus genes associated with virulence of US strains.

Marek's disease virus (MDV) is the most well-cited example of vaccine-driven virulence evolution. MDV induces a lymphoproliferative disease in chickens, which is currently controlled by widespread vaccination of flocks. Unfortunately, Marek's disease (MD) vaccines, while effective in preventing tumours, do not prevent viral replication and mutation, which has been hypothesized as the major driving force for increased MDV virulence of field strains during the past 40 years in US commercial flocks. To limit future virulence increases, there is interest in characterizing MDV strain genomes collected over the years and associating genetic variations with variation in virulence. In this study, we characterized 70 MDV genomes with known virulence by complete or targeted DNA sequencing, and identified genetic variants that showed association with virulence. Our results revealed a number of MDV genes as would be expected for a complex trait. In addition, phylogenetic analysis revealed a clear separation of strains that varied by virulence. Interestingly, high virulence isolates from the same farms persisted over years despite eradication attempts, which has implications on control efforts. Given the growing ability to bioengineer the MDV genome, it should be feasible to experimentally test whether these individual variants influence virulence markers alone or combinations. Once validated, these markers may provide an alternative to live bird testing for evaluating virulence of new MDV field strains.

Educational Disabilities Among Children Born With Neonatal Abstinence Syndrome.

BACKGROUND: Neonatal abstinence syndrome (NAS) is a postnatal drug withdrawal syndrome that can occur after intrauterine opioid exposure. Adverse neurobehavioral outcomes have been documented in infants with NAS; however, educational outcomes have not been thoroughly examined. We analyzed Tennessee data to understand the need for special educational services among infants who are born with NAS. METHODS: By using Tennessee Medicaid and birth certificate data, infants who were born in Tennessee between 2008 and 2011 with a history of NAS were matched (1:3) to infants who were born during the same period without a history of NAS. Groups were matched on the basis of sex, race and/or ethnicity, age, birth region of residence, and Medicaid enrollment status. Data were linked to Tennessee Department of Education special education data during early childhood (3-8 years of age). Conditional multivariable logistic regression was used to assess associations between NAS and selected special education outcomes. RESULTS: A total of 1815 children with a history of NAS and 5441 children without NAS were assessed. Children with NAS were significantly more likely to be referred for a disability evaluation (351 of 1815 [19.3%] vs 745 of 5441 [13.7%]; P < .0001), to meet criteria for a disability (284 of 1815 [15.6%] vs 634 of 5441 [11.7%]; P < .0001), and to require classroom therapies or services (278 of 1815 [15.3%] vs 620 of 5441 [11.4%]; P < .0001). These findings were sustained in a multivariable analysis, with multiple models controlling for maternal tobacco use, maternal education status, birth weight, gestational age, and/or NICU admission. CONCLUSIONS: Results of this novel analysis linking health and education data revealed that children with a history of NAS were significantly more likely to have a subsequent educational disability.

Development of reliable techniques for the differential diagnosis of avian tumour viruses by immunohistochemistry and polymerase chain reaction from formalin-fixed paraffin-embedded tissue sections.

A variety of techniques have been developed as diagnostic tools for the differential diagnosis of tumours produced by Marek's disease virus from those induced by avian leukosis virus and reticuloendotheliosis virus. However, most current techniques are unreliable when used in formalin-fixed paraffin-embedded (FFPE) tissues, which often is the only sample type available for definitive diagnosis. A collection of tumours was generated by the inoculation of different strains of Marek's disease virus, reticuloendotheliosis virus or avian leukosis virus singularly or in combination. FFPE tissue sections from tumour and non-tumour tissues were analysed by optimized immunohistochemistry (IHC) techniques and traditional as well as quantitative polymerase chain reaction (PCR) with newly designed primers ideal for DNA fragmented by fixation. IHC and PCR results were highly sensitive and specific in tissues from single-infected birds. Virus quantity was higher in tumours compared to non-tumour spleens from Marek's disease (MD) virus-infected birds. Thus, using FFPE sections alone may be sufficient for the diagnosis of MD by demonstration of high quantities of viral antigens or genome in tumour cells, along with the absence of other tumour viruses by traditional PCR, and if standard criteria are met based on clinical history and histology. IHC furthermore allowed detection of the specific cells that were infected with different viruses in tumours from birds that had been inoculated simultaneously with multiple viruses. Following validation with field samples, these new protocols can be applied for both diagnostic and research purposes to help accurately identify avian tumour viruses in routine FFPE tissue sections.

Attenuation of Marek's disease virus by codon pair deoptimization of a core gene.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus of Gallus gallus, the domesticated chicken. Control strategies rely upon vaccination with live attenuated viruses of antigenically similar avian herpesviruses or attenuated strains of MDV. Recent studies in other viruses have shown that recoding certain viral genes to employ synonymous but rarely-used codon pairs resulted in viral attenuation. We deoptimized two MDV proteins, UL54/ICP27 and UL49/VP22, and demonstrate that the more severely deoptimized variant of UL54 accumulates significantly less gene product in vitro. Using these UL54 deoptimized mutants, we further demonstrate that animals infected with the UL54-recoded recombinant virus exhibited decreased viral genome copy number in lymphocytes, reduced lymphoid atrophy and reduced tumor incidence. This study demonstrates that codon pair deoptimization of a single viral gene can produce attenuated strains of MDV. This approach may be useful as a rational way of making novel live attenuated virus vaccines for MDV.

Evaluation and Identification of Marek's Disease Virus BAC Clones as Standardized Reagents for Research.

Marek's disease virus (MDV) is an alphaherpesvirus that causes Marek's disease (MD), a lymphoproliferative disease in chickens. Understanding of MDV gene function advanced significantly following the cloning of the MDV genome as either a series of overlapping cosmids or as a bacterial artificial chromosome (BAC), both of which could produce viable MDV. The objectives of this study were to compare multiple virulent MDV BAC clones using the Avian Disease and Oncology Laboratory's pathotyping assay, and to demonstrate the use of these clones as standardized reagents for a modified pathotyping assay by other laboratories. To date, MDV BAC clones have been produced for at least 10 MDV strains from all three serotypes including several virulent serotype 1 strains. We determined that MDV BAC clones exist for each virulent pathotype, despite the fact that these clones are not always equal in virulence to their corresponding parental strains. One clone from each pathotype was further evaluated in commercial specific-pathogen-free (SPF) chickens and found suitable for use in assays such as best-fit pathotyping, although results were variable based on the source of the SPF birds. The benefits of using BAC clones, which include easy shipping, ability to more easily manipulate, and long-term ability to use at a low passage level, are likely to result in the use of BAC clones as standard reagents for MD research. The use of the defined set of clones should allow side-by-side comparison, allowing researchers to better interpret results produced in different laboratories using different MDV field strains. Furthermore, a modified best-fit pathotyping assay has been proposed using these clones and reduced bird numbers.

Expression of Marek's Disease Virus Oncoprotein Meq During Infection in the Natural Host.

Gallid herpesvirus 2 (Marek's disease virus, MDV) causes lymphoproliferative Marek's disease (MD), and is unique among alphaherpesviruses as the viral genome encodes an oncoprotein, Meq. To elucidate the temporal relationship between Meq expression and the development of MD lymphomas in infected chickens, we generated a virulent recombinant MDV that expresses GFP simultaneously with Meq. By using this virus, we monitored the dynamics of Meq expression in vivo throughout the course of infection. In peripheral blood mononuclear cells, the percentage of Meq-expressing cells dramatically increased in the early latent phase but decreased thereafter. Furthermore, we discovered evidences that indicate some of the infected lymphocytes did not express Meq during the latent phase of MDV pathogenesis. These findings provide the first insight into the temporal relationship between Meq expression and MD progression, and new clues to refine the current MD pathogenesis model.

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.

Bilateral Uveitis and Hyphema in a Catalina Macaw (Ara ararauna × Ara macao ) With Multicentric Lymphoma.

A 20-year-old, female Catalina macaw (Ara ararauna × Ara macao ) was presented with bilateral uveitis and hyphema. The hyphema initially improved with 0.12% prednisolone acetate ophthalmic drops (1 drop OU q4h for 7 days), but the hyphema recurred after the drops were tapered. The bird subsequently developed inappetance, weight loss, regurgitation, and lethargy and was euthanatized 24 days after initial presentation. Necropsy revealed marked splenomegaly and hepatomegaly, with significant mucosal ulcerations of the proventriculus and petechiation associated with both kidneys. Histopathologic examination revealed multicentric lymphoma, with neoplastic cells observed in ocular, splenic, hepatic, renal, proventricular, intestinal, pancreatic, and choanal tissue. Neoplastic lymphocytes effaced the iris, ciliary body, and the choroid of the eyes, and neoplastic lymphocytes were attached to the corneal endothelium and infiltrated the sclera, episclera, and conjunctivae. Immunohistochemical results indicated that the neoplastic lymphocytes were CD3(+) and CD79a(-), which is consistent with T-cell lymphoma.

Fine mapping of QTL and genomic prediction using allele-specific expression SNPs demonstrates that the complex trait of genetic resistance to Marek's disease is predominantly determined by transcriptional regulation.

BACKGROUND: Marek's disease (MD) is a lymphoproliferative disease of poultry induced by Marek's disease virus (MDV), a highly oncogenic alphaherpesvirus. Identifying the underlying genes conferring MD genetic resistance is desired for more efficacious control measures including genomic selection, which requires accurately identified genetic markers throughout the chicken genome. METHODS: Hypothesizing that variants located in transcriptional regulatory regions are the main mechanism underlying this complex trait, a genome-wide association study was conducted by genotyping a ~1,000 bird MD resource population derived from experimental inbred layers with SNPs containing 1,824 previously identified allele-specific expression (ASE) SNPs in response to MDV infection as well as 3,097 random SNPs equally spaced throughout the chicken genome. Based on the calculated associations, genomic predictions were determined for 200 roosters and selected sires had their progeny tested for Marek's disease incidence. RESULTS: Our analyses indicate that these ASE SNPs account for more than 83 % of the genetic variance and exhibit nearly all the highest associations. To validate these findings, 200 roosters had their genetic merit predicted from the ASE SNPs only, and the top 30 and bottom 30 ranked roosters were reciprocally mated to random hens. The resulting progeny showed that after only one generation of bidirectional selection, there was a 22 % difference in MD incidence and this approach gave a 125 % increase in accuracy compared to current pedigree-based estimates. CONCLUSIONS: We conclude that variation in transcriptional regulation is the major driving cause for genetic resistance to MD, and ASE SNPs identify the underlying genes and are sufficiently linked to the causative polymorphisms that they can be used for accurate genomic prediction as well as help define the underlying molecular basis. Furthermore, this approach should be applicable to other complex traits.