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Biomedical subjects

R L Witter

Publications and source records attributed to R L Witter.

At least 19 recordsLinked to original sources

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

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

Aging↗

Classification of Marek's disease viruses according to pathotype: philosophy and methodology.

The concept of pathotype in Marek's disease (MD) probably dates from the recognition of a more virulent form of the disease in the late 1950s (Benton & Cover, 1957). Distinctions between MD virus strains were further expanded with the description of the vv pathotype in the early 1980s and of the vv+ pathotype in the 1990s. Pathotype designations reflect important biological properties that correlate with the break-through of vaccinal immunity in the field. However, pathotyping methods applied by various laboratories have not been uniform, preventing critical comparison of results. Better uniformity of pathotyping procedures is desirable.The Avian Disease and Oncology Laboratory (ADOL) method is based on induction of lymphoproliferative lesions in vaccinated chickens. This method has been used to pathotype more than 45 isolates and is the basis for the current pathotype classification of MD virus strains. Its limitations include requirements for a specific type of chickens (15x7 ab+), large numbers of animals, and a statistical method to compare lesion responses to those of JM/102W and Md5 control strains. Because of these limitations, it has not been and is not likely to be used in other laboratories. Comparability in pathotyping can be improved by the comparison of field isolates with standard prototype strains such as JM/102W, Md5 and 648A (American Type Culture Collection) or their equivalents. Data may be generated by different in vivo procedures that measure tumour induction, neurological disease (both neoplastic and non-neoplastic lesions), or solely non-neoplastic criteria (such as lymphoid organ weights or virus replication). Methods based on neoplastic criteria, especially when generated in MD-immunized chickens, will probably correlate most closely with that of the ADOL method and be most relevant to evolution of MD virus in the field. Based on data from several trials, a modification of the ADOL method that utilizes fewer chickens and can be conducted with commercial specific pathogen free strains is proposed. The modified method is based on "best fit" comparisons with prototype strains, and is expected to provide results generally comparable with the original method. A variety of other alternative criteria (see earlier) are also evaluated both for primary pathotyping and as adjuncts to other pathotyping methods. Advantages and disadvantages of alternative methods are presented.

Animals↗

Novel criteria for the diagnosis of Marek's disease virus-induced lymphomas.

Several novel criteria have been tested to assist in the differential diagnosis of tumours induced by Marek's disease virus (MDV) from those induced by avian leukosis virus (ALV) and reticuloendotheliosis virus (REV). A collection of tumours induced by inoculation of specific strains of MDV, ALV and REV, alone or in combination, were tested for quantification of MDV DNA by real-time polymerase chain reaction, expression of the MDV oncogene Meq, expression of several cell markers associated with transformation (CD30, Marek's disease-associated surface antigen, and p53), and level of DNA methylation in the tumour cells. In addition, tissues latently infected with MDV and non-infected tissues were tested as controls. Tumours induced by MDV had about 10(2)-fold more copies of MDV DNA than either tissues latently infected by MDV or tumours induced by retrovirus in MDV-vaccinated chickens. Moreover, the MDV antigen Meq was consistently expressed in all MDV tumours but it could not be detected in tissues latently infected with MDV or in tumours induced by retrovirus in MDV-vaccinated chickens. Other markers studied were not specific for MDV and therefore had limited value for diagnosis. Nonetheless, some of these markers might have potential value in research as they will help to identify transformed cells.

Animals↗

The pp38 gene of Marek's disease virus (MDV) is necessary for cytolytic infection of B cells and maintenance of the transformed state but not for cytolytic infection of the feather follicle epithelium and horizontal spread of MDV.

Marek's disease virus has a unique phosphoprotein, pp38, which is suspected to play an important role in Marek's disease pathogenesis. The objective of the present study was to utilize a mutant virus lacking the pp38 gene (rMd5Deltapp38) to better characterize the biological function of pp38. This work shows that the pp38 gene is necessary to establish cytolytic infection in B cells but not in feather follicle epithelium, to produce an adequate level of latently infected T cells, and to maintain the transformed status in vivo.

Animals↗

Serotype 1 viruses modified by backpassage or insertional mutagenesis: approaching the threshold of vaccine efficacy in Marek's disease.

Improved vaccines to control Marek's disease (MD) in chickens are desired by the poultry industry but have been difficult to develop. Studies were conducted to evaluate strategies for deriving MD vaccines of high protective efficacy, irrespective of virulence. Candidate viruses from parent strains representing v and vv+ pathotypes were modified by cell culture passage, backpassage in chickens, or insertional mutagenesis following cocultivation with retroviruses. Ten strains considered most likely to exhibit high protective efficacy were selected for further study. The ability of these modified viruses to protect commercial or maternal antibody-positive (ab+) chickens against virulent MD virus (MDV) challenge was compared with that of strain CVI988, the standard commercial MD vaccine. Modified strains were also evaluated for the ability to induce lymphomas or other pathologic changes in ab+ and antibody-negative (ab-) chickens. Two of the 10 modified viruses, strains RM1 and CVI988/BP5, provided high levels of protection against highly virulent MDV challenge. The magnitude of protection was greater than that of one laboratory and two commercial preparations of CV1988, but was approximately equal to that of two other commercial preparations of CVI988 in laboratory and field tests. Three of the strains, including RMI and CVI988/BP5, induced lymphoid organ atrophy in ab-chicks but not in ab+ commercial chicks, a property designated here as L phenotype. Seven strains, including two L+ strains, were mildly oncogenic for ab- chicks, a property designated here as O phenotype. Five of these strains caused no tumors in ab+ chickens. The two fully attenuated strains induced neither lymphomas nor lymphoid organ atrophy. The L and O phenotypes appeared not to be linked, and both (especially the L phenotype) appeared associated with high levels of protection. These studies also illustrated differences in the protective efficacy of different preparations of CVI988 vaccine, indicating the need to choose carefully the most protective strains as controls for efficacy studies. A new vv+ strain, designated as 686, is described and appears useful as a challenge virus; it is the most virulent of the 48 field isolates of MDV thus far pathotyped in this laboratory. These findings support the conclusion that new virus strains with high levels of protective immunity comparable to that of CVI988 can be developed. However, the question of whether strains can be developed that exceed the efficacy of current CVI988-based vaccines remains unanswered. After more than 30 years of unsuccessful endeavor by many laboratories toward this goal, it now may be useful to consider whether the efficacy of MD vaccines is limited by some type of biologic threshold.

Animals↗

Molecular indications for in vivo integration of the avian leukosis virus, subgroup J-long terminal repeat into the Marek's disease virus in experimentally dually-infected chickens.

Marek's disease virus, a herpesvirus, and avian leukosis virus, subgroup J, a retrovirus, are oncogenic viruses of poultry. Both viruses may infect the same flock, the same bird and the same cell. In a double-infected cell, the retroviral DNA can integrate into the cellular or the Marek's disease virus (MDV) genome. The retroviral-long terminal repeat (LTR) integration into MDV was first described by Isfort et al., (Proc Natl Acad Sci 89, 991-995, 1992) following tissue culture co-infection. The recombinant virus isolated, RM1, had altered biological properties compared to the parental MDV (Witter R.L., Li D., Jones D., and Kung H.-J., Avian Dis 41, 407-421, 1997) . The issue of retroviral sequence integration into herpesviruses in vivo, in cases of double-virus infection is of wide significance in general virology and veterinary medicine; it also represents a special case of gene transposition. Using the avian system, we aimed to determine occurrence of such integrations in vivo. Chickens were experimentally co-infected with both avian leukosis virus (ALV) subgroup J and with MDV. To demonstrate the presence of the retroviral LTR in the MDV genome we applied the Hot Spot-combined PCR assay (Borenshtain R. and Davidson I., J Virol Meth 82, 119-127, 1999) that consisted of two consecutive steps of amplification. By that HS-cPCR assay, certain MDV genomic sites, defined as HS for integration were specifically amplified, the HS step, and then subjected to screening in an attempt to detect LTR inserts. The screening was achieved by amplification using heterologous primer sets, one for the MDV hot spot and the other for the retroviral LTR, the cPCR step. The products were Southern blotted and hybridized with MDV and ALV-LTR probes. Chimeric molecules were detected and evidenced by an intense signal in 3/10 chickens and weakly in other 3/10 birds. Detection was by LTR amplification, sequencing and multiple alignment to the ALV-J-LTR sequence. The present study indicated that chimeric molecules were produced in vivo.

Animals↗

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

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

Animals↗

The complete unique long sequence and the overall genomic organization of the GA strain of Marek's disease virus.

We have determined the DNA sequence of the unique long (UL) region and the repeat long (RL) region in the genome of serotype 1 GA strain of Marek's disease virus (MDV), a member of the alpha-herpesvirus family. With this information, the complete nucleotide sequence of GA-MDV is now known. The entire GA-MDV genome is predicted to be about 174 kbp in size, with an organization of TRL-UL-IRL-IRS-US-TRS, typical of a alpha-herpesvirus. The UL sequence contains 113,508 bp and has a base composition of 41.7% G + C. A total of 67 ORFs were identified completely within the UL region, among which 55 are homologous to genes encoded by herpes simplex virus-1. Twelve of them are unique with presently unknown functions. The sequence of RL reported here together with those published earlier reveal the major structural features of the RL. Virtually all of the ORFs encoded by RL are specific to serotype I of MDV. These ORFs are likely to contribute to some of the unique biological properties of MDV. Among the proteins encoded by MDV-specific ORFs are Meq, a jun/fos family of transcriptional factor implicated in transformation and latency, virus-encoded interleukin-8, a CXC chemokine, and pp38 and pp24, two phosphoproteins with undefined functions. There is also a putative lipase gene (LORF2) that has homologies in HPRS-24 (serotype II) strain of MDV and in various avian adenoviruses. An additional unique feature of MDV is the presence of long terminal repeat remnant sequences of avian retrovirus reticuloendotheliosis virus. These remnant sequences are derived from the U3-enhancer region through ancestral insertions by reticuloendotheliosis virus proviruses.

DNA Replication↗

High resolution mapping and identification of new quantitative trait loci (QTL) affecting susceptibility to Marek's disease.

Marek's disease (MD) is a lymphoproliferative disease of chickens that costs the poultry industry approximately $1 billion annually. Genetic resistance to MD is gaining increased attention to augment vaccinal control as disease outbreaks occur more frequently. Previously, analysis of a 272 F2 White Leghorn resource population measured for many MD traits and genotyped for 78 microsatellite markers revealed two and four quantitative trait loci (QTL) with significant and suggestive association, respectively, to one or more MD associated traits. Additional genetic markers have since been scored on the MD resource population to increase QTL resolution and genome coverage. Saturation of four of the QTL regions with 17 markers revealed five new QTL while 32 markers extended the genome coverage by 400 + CM and uncovered three more QTL. QTL analysis by single-point and interval mapping algorithms agreed well when marker saturation was approximately 20 CM or less. Currently 127 genetic markers cover approximately 68% of the genome that contain up to 14 MD QTL associated to one or more MD trait; seven at the significant level and seven at the suggestive level. Individually each QTL accounts for 2-10% of the variation and, in general, resistance was dominant although the resistant allele may come from either parental line. This study suggests that a limited number of genomic regions play a major role in the genetic control of MD resistance. Markers linked to these loci may be useful for selection of MD resistant stock by the poultry industry following verification of the association within their breeding populations.

Animals↗

Genetic mapping of quantitative trait loci affecting susceptibility to Marek's disease virus induced tumors in F2 intercross chickens.

Marek's disease (MD) is a lymphoproliferative disease caused by the MD virus (MDV), which costs the poultry industry nearly $1 billion annually. To identify quantitative trait loci (QTL) affecting MD susceptibility, the inbred lines 6(3) (MD resistant) and 7(2) (MD susceptible) were mated to create more than 300 F2 chickens. The F2 chickens were challenged with MDV JM strain, moderately virulent) at 1 wk of age and assessed for MD susceptibility. The QTL analysis was divided into three stages. In stage 1, 65 DNA markers selected from the chicken genetic maps were typed on the 40 most MD-susceptible and the 40 most MD-resistant F2 chickens, and 21 markers residing near suggestive QTL were revealed by analysis of variance (ANOVA). In stage 2, the suggestive markers plus available flanking markers were typed on 272 F2 chickens, and three suggestive QTL were identified by ANOVA. In stage 3, using the interval mapping program Map Manager and permutation tests, two significant and two suggestive MD QTL were identified on four chromosomal subregions. Three to five loci collected explained between 11 and 23% of the phenotypic MD variation, or 32-68% of the genetic variance. This study constitutes the first report in the domestic chicken on the mapping of non-major histocompatibility complex QTL affecting MD susceptibility.

Analysis of Variance↗

Control strategies for Marek's disease: a perspective for the future.

Marek's disease virus is an evolving pathogen, acquiring virulence in response to increasingly effective vaccines. Although vaccine efficacy is generally good, industry has placed a high priority on more effective products. The search for better vaccines has been conducted mainly in the arena of molecular biology, and has been thus far disappointing. Various conditions prevail that currently limit the potential to develop suitable long-term solutions. A new paradigm based on reduction of early exposure, multiple levels of host resistance, and improved cooperation among stakeholders is proposed for consideration.

Animals↗

Reticuloendotheliosis in captive greater and Attwater's prairie chickens.

Reticuloendotheliosis in captive greater (Tympanuchus cupido pinnatus) and Attwater's (T. cupido attwateri) prairie chickens is reported for the first time. Between September 1993 and August 1994, two adult female wild-caught greater prairie chickens housed at Texas A&M University (College Station, Texas, USA) were observed with multiple subcutaneous nodules. Both birds were euthanatized. Complete necropsy examinations revealed lesions limited to the skin of each bird. Histopathologic examination of lesions revealed pleomorphic lymphoreticular cells suggestive of reticuloendotheliosis and reticuloendotheliosis virus (REV) was demonstrated in tumor tissue by polymerase chain reaction and virus isolation. Between September 1994 and June 1995, five additional greater prairie chickens and two Attwater's prairie chickens were euthanatized or found dead with evidence of lymphoreticular neoplasia in multiple organ systems. Initial testing of the captive flock in December 1994 for evidence of viremia and antibody to reticuloendotheliosis virus revealed over 50% of the tested birds were viremic, but none developed antibodies. Subsequent testing between January 1995 and January 1996 indicated that once infected with reticuloendotheliosis virus, Attwater's prairie chickens tended to remain outwardly healthy despite persistent viremia compared to infected greater prairie chickens which had higher morbidity and mortality rates within 60 to 90 days after initial detection of viremia and did not usually develop persistent viremia. Antibodies to REV were detected in only three captive greater prairie chickens and only in 1995. Six of the nine birds that were euthanatized or found dead due to reticuloendotheliosis developed viremia prior to death; three birds were not tested prior to death. Testing of free-ranging greater and Attwater's prairie chickens for reticuloendotheliosis is recommended prior to translocation or release.

Animals↗

Non-association between Rfp-Y major histocompatibility complex-like genes and susceptibility to Marek's disease virus-induced tumours in 6(3) x 7(2) F2 intercross chickens.

Marek's disease (MD) is a lymphoproliferative disease caused by a member of the herpesvirus family, and the best understood genetic resistance to MD involves the chicken major histocompatibility complex (MHC) B-complex. Preliminary observations have suggested that MHC-like Rfp-Y genes might also influence the incidence of MD. This study describes the differentiation and definition of unique Rfp-Y genes in inbred lines 6(3) and 7(2), lines that possess identical B-complex genes, but that are resistant or susceptible to MD, respectively. To assess if Rfp-Y genes affect susceptibility to MD, 265 6(3) x 7(2) F2 chickens were challenged with the JM strain of MD virus at 1 week of age and were evaluated for MD lesions at up to 10 weeks of age. Genotyping of the F2 chickens for Rfp-Y haplotypes was performed by restriction fragment length polymorphism analysis of genomic DNA using TaqI and a B-FIV probe. Analysis of variance and interval mapping procedures were used to determine association between the Rfp-Y haplotypes and the phenotypic MD values of the F2 chickens. The cosegregation analysis of 265 F2 chickens indicated that there was no association between Rfp-Y haplotypes and MD susceptibility. Furthermore, the fact that the Rfp-Y haplotypes fit the 1:2:1 segregation ratio and the Rfp-Y allele frequencies did not differ significantly from 0.5 in the full population or in selected subpopulations (of either 40 MD-resistant or 39 MD-susceptible chickens) also indicated that Rfp-Y haplotypes do not significantly influence MD susceptibility. We conclude that Rfp-Y haplotypes do not play a major role in determining the genetic susceptibility to MD in 6(3) x 7(2) F2 White Leghorn chickens.

Animals↗

Avian tumor viruses: persistent and evolving pathogens.

Most neoplasias of lymphoid and other hematopoietic cells in commercial poultry are caused by viruses which belong to one of four distinct groups. Marek's disease virus (MDV) is an oncogenic herpesvirus. Avian leukosis virus (ALV), reticuloendotheliosis virus (REV) and lymphoproliferative disease virus (LPDV) are oncogenic retroviruses. Each group is distinguished by nucleic acid type, molecular structure, antigenicity, epidemiology, host range and other characteristics. However, most of these viruses have in common a unique ability to persist, both in the host and in the ecosystem. In addition, both the viruses and the virus-host relationships for several members of the group have demonstrated a propensity to evolve with time, creating new dilemmas for diagnosis and control. A focus on the persistence and evolution of avian tumor viruses will be used to address a number of current issues with individual viruses of economic importance. Issues of primary concern include (1) the evolution of MDV towards greater virulence with concomitant reduction of vaccine efficacy and expansion of host range, (2) the emergence of subgroup J ALV as a major pathogen in meat-type breeder stocks, and (3) the increasing prevalence of REV and its evolving role as a pathogen in chickens and turkeys.

Animals↗

The immunodominant proteins of reticuloendotheliosis virus.

The antigenic profiles of three REV prototype strains, CSV, SNV and REV-T and eight Israeli isolates were analysed by SDS-PAGE and immunoblotting with convalescent chicken serum, three mAbs, 11A25, 11C237 and 11C100, a rabbit antiserum to REV-T whole virus (Cui et al., 1986) and a rabbit antiserum to REV-A p30 gag protein (Tsai et al., 1985). Under both reducing (+DTT) and non-reducing conditions of SDS-PAGE, a major immunodominant 75-100 kDa band was shared by all strains examined. In contrast to the chicken serum that recognized both continuous and discontinuous epitopes on the 75-100 kDa band of all the isolates, the mAbs and the two rabbit sera behaved otherwise. Only the DTT-resistant epitopes on the 75-100 kDa band of REV-T were recognized by the rabbit antisera and the mAb 11C237, and only the DTT-labile epitopes of REV-T 75-100 kDa antigen were detected by mAb 11C100. The two mAbs 11A25 and 11C237 detected discontinuous epitopes of all the strains except SNV, while the rabbit antisera recognized the discontinuous epitopes on the 75-100 kDa band of all the 11 strains. The rabbit antisera and mAb 11C237 detected additional lower molecular weight proteins and the mAb 11C237 also detected three proteins of high molecular weight under non-reducing conditions only. The p30 antiserum detected the low molecular weight proteins demonstrating their gag gene-encoded identity. From these results we conclude that the major immunogen of REV is the 75-100 kDa protein that contains both continuous and discontinuous epitopes. With this panel of antibodies the eight new isolates appeared to belong antigenically to REV subtype 3 (Chen et al., 1987).

Animals↗

B haplotype influence on the relative efficacy of Marek's disease vaccines in commercial chickens.

The objectives were to investigate whether or not B haplotypes influence vaccinal immunity against Marek's disease (MD) in commercial chickens and to evaluate whether retrospective analysis would detect the influence. This method involved evaluating the B haplotypes of turkey herpesvirus (HVT)-vaccinated sick vs normal chickens from a flock afflicted with MD symptoms. An analysis of the retrospective data disclosed that MD symptoms were present in a higher proportion of B2B19 than B2B21 chickens. A prospective study was then conducted with blood-typed chickens of the strain vaccinated with HVT or HVT + 301B bivalent MD vaccines prior to inoculation of the very virulent Md5 virus. The bivalent vaccine provided better protection than HVT alone, but with either vaccine fewer B2B21 chickens developed MD lesions. We conclude that the B haplotype influence on vaccinal immunity against MD previously demonstrated in B-congenic strains of chickens is also significant in commercial chickens and that the influence can be detected through analysis of B haplotypes in sick vs normal chickens of an affected flock.

Animals↗