Willie M. Reed

Marek's Disease Virus-Encoded vIL-8 Gene Is Involved in Early Cytolytic Infection but Dispensable for Establishment of Latency

ABSTRACT Mareks disease, a lymphoproliferative disease of chickens, is caused by an alphaherpesvirus, Mareks disease virus (MDV). This virus encodes a virokine, vIL-8, with general homology to cellular CXC chemokines such as interleukin-8 (IL-8) and Gro-α. To study the function of vIL-8 gene, we deleted both copies of vIL-8 residing in the terminal repeat long and internal repeat long region of the viral genome and generated a mutant virus with vIL-8 deleted, rMd5/ΔvIL-8. Growth kinetics study showed that vIL-8 gene is dispensable for virus replication in cell culture. In vivo, the vIL-8 gene is involved in early cytolytic infections in lymphoid organs, as evidenced by limited viral antigen expression of rMd5/ΔvIL-8. However, the rMd5/ΔvIL-8 virus is unimpaired in virus replication in the feather follicle epithelium. vIL-8 does not appear to be important for establishment of latency, since rMd5/ΔvIL-8 and the wild-type virus have similar viremia titers at 14 days postinfection, a period when the virus titer comes primarily from reactivated latent genomes. Nevertheless, because of the impaired cytolytic infections, the overall transformation efficiency of the virus with vIL-8 deleted is much lower, as reflected by the reduced number of transformed cells at 5 weeks postinoculation and the presence of fewer gross tumors. Importantly, the revertant virus that restored the expression of vIL-8 gene also restored the wild-type phenotype, indicating the deficient phenotypes are results of vIL-8 deletion. One of the interesting differences between the MDV vIL-8 gene and its cellular counterpart is the presence of a DKR (Asp-Lys-Arg) motif instead of ELR (Glu-Leu-Arg) preceding the invariable CXC motif. To study the significance of this variation, we generated recombinant MDV, rMd5/vIL-8-ELR, carrying the ELR motif. Both in vitro and in vivo studies revealed that the DKR motif is as competent as ELR in pathogenesis of MDV.

Four Distinct Neurologic Syndromes in Marek's Disease: Effect of Viral Strain and Pathotype

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

An outbreak of lymphomas in commercial broiler breeder chickens vaccinated with a fowlpox vaccine contaminated with reticuloendotheliosis virus.

Gross and microscopic examinations of affected tissues from chickens of two commercial broiler breeder flocks aged 27 and 31 weeks revealed lesions of visceral lymphomas with bursal involvement in some chickens. Reticuloendotheliosis virus (REV), but not avian leukosis virus (ALV), was isolated from blood of affected chickens. Furthermore, DNA extracted from tumours tested positive for REV, but not for ALV or Mareks disease virus by polymerase chain reaction (PCR) test. Attempts to determine the source of REV infection included testing a commercial fowlpox (FP) vaccine used to immunize flocks at 7 days of age. Chicken-embryo fibroblasts inoculated with the FP vaccine tested positive for REV by PCR and immunofluorescent tests. REV was also isolated from plasma of pathogen-free chickens experimentally inoculated with FP vaccine at hatch; two of eight (25%) inoculated chickens developed lymphomas by 34 weeks of age. Antigenic characterization of REV isolated from commercial broiler breeder chickens and from FP vaccine, using monoclonal antibodies, revealed that both isolates belong to subtype 3 of REV. The data represent the first report of an outbreak of REV-induced lymphomas in commercial chickens. The data also indicate that the source of REV infection is an REV-contaminated commercial FP vaccine.

Molecular Characterization of Reticuloendotheliosis Virus Insertions in the Genome of Field and Vaccine Strains of Fowl Poxvirus

Abstract 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.

An Acute Form of Transient Paralysis Induced by Highly Virulent Strains of Marek's Disease Virus

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

Cellular response of the respiratory tract of chickens to infection with Massachusetts 41 and Australian T infectious bronchitis viruses

Cellular response of chickens to infection with infectious bronchitis virus (IBV) was investigated by lavage of the respiratory tract of five 2-week-old specific-pathogen-free (SPF) chickens at 2, 8, 24, 48, 72, and 96 hours postinfection (PI) with either Massachusetts 41 (IBV-M41) or Australian T (IBV-T) IBV. Tissue response was monitored by microscopic examination of trachea and lung from five non-lavaged infected chickens collected at the same intervals. The total number of cells recovered by lavage from IBV-M41-infected chickens was dramatically higher than the total number recovered from IBV-T-infected chickens and uninfected controls. By contrast, the total number of cells recovered from IBV-T-infected chickens was no higher than that of the uninfected chickens. Heterophils constituted the majority of inflammatory cells recovered from both IBV-M41-infected and IBV-T-infected chickens. Heterophil numbers in IBV-M41-infected chickens paralleled total cell-number recovery, whereas heterophil numbers in IBV-T-infected birds were no higher than those in uninfected chickens. The number of lymphocytes recovered from IBV-M41-infected chickens increased 72 hours PI and continued to increase for the duration of the study. Lymphocyte numbers in IBV-T-infected chickens exceeded those in uninfected chickens only at 96 hours PI. The number of lavage macrophages in IBV-M41-infected chickens increased earlier than the number of lymphocytes but later reached a plateau. IBV-T macrophage numbers did not exceed those of uninfected chickens. Tissue damage occurred most consistently in the trachea and occurred when lavage heterophil numbers were rising or at their peak. Lavage cell recovery and composition reflected tracheal mucosa inflammatory cell infiltrate.

Biocharacteristics shared by highly protective vaccines against Marek's disease

Attenuated serotype 1 Mareks disease virus strains vary widely in their protection properties. This study was conducted to elucidate which biocharacteristics of serotype 1 MDV strains are related with protection. Three pairs of vaccines, each one including a higher protective (HP) vaccine and a lower protective (LP) vaccine originating from the same MDV strain, were studied. Two other highly protective vaccines (RM1 and CVI988/BP5) were also included in the study. Comparison within pairs of vaccines showed that marked differences existed between the HP and the LP vaccines. Compared with LP vaccines, HP vaccines replicated better in vivo. Also, they induced a significant expansion of total T cells and of the helper and cytotoxic T cell lineages (CD45+CD3+, CD4+CD8−, CD4−CD8+) as well as a marked increase in the expression of the antigens of MhcI and MhcII on T cells. Thus, our results show that in vivo replication and early stimulation of the T-cell lineage are two characteristics shared by HP vaccines. However, comparison among the four HP vaccines that provided protection equal to that of CVI988 (RM1, CVI988/BP5, CVI988 and 648A80) revealed variability, especially regarding in vivo replication. Strains RM1 and CVI988/BP5 showed much stronger replication in vivo than the other two vaccine strains (CVI988 and 648A80). Thus, no single set of characteristics could be used to identify the most protective Mareks disease vaccines, implying, perhaps, that multiple mechanisms may be involved.

A Marek's Disease Virus vIL-8 Deletion Mutant Has Attenuated Virulence and Confers Protection Against Challenge with a Very Virulent Plus Strain

Abstract Mareks disease virus (MDV) is an alpha-herpesvirus that causes rapid development of T-cell lymphomas in chickens. MDV-encoded vIL-8 is homologous to the cellular IL-8 gene, and its function in MDV pathogenesis has yet to be determined. Using overlapping cosmid clone–based technology, we have generated an MDV vIL-8 deletion mutant virus, rMd5/ΔvIL-8. In vivo experiments with this mutant virus demonstrated that deletion of vIL-8 results in attenuation of the virus and induction of significantly less gross tumor, both in viscera and nerves, when compared to the parental virus. Reintroduction of the vIL-8 gene in the genome of the mutant virus restored the virulence of the virus to the wild-type levels, indicating that vIL-8 plays a role in MDV-induced pathogenesis. In this study, we show that there is a significant difference in the reduction of B cells and activation of T cells in the spleen cells of chickens inoculated with parental rMd5 and vIL-8 deletion mutant virus. These results indicate that vIL-8 is involved in the early phase of pathogenesis, presumably by attracting target cells to the initial site of infection. In addition, protection studies with the vIL-8 mutant virus showed that this mildly virulent virus protects susceptible maternal antibody–positive viruses at a higher level than the commonly used serotype 1 CVI988 vaccine. These results confirm the potential of partially attenuated viruses as vaccines against very virulent plus strains and the usefulness of recombinant DNA technology to generate the next generation of MDV vaccines.

Light microscopic and ultrastructural characterization of cells recovered by respiratory-tract lavage of 2- and 6-week-old chickens.

This study characterized the cell population recovered by respiratory-tract lavage of 57 two-week-old and 59 six-week-old specific-pathogen-free chickens as a prerequisite to study the response of the avian respiratory tract to infectious agents. The respiratory tract of each bird was lavaged through the trachea with a series of three lavages of 10 ml of room-temperature, neutral phosphate-buffered saline per lavage. The three lavages per bird were pooled for analysis. Total recovery volumes were measured, lavage fluid cellularity was determined, and a 200-cell differential count of non-erythrocyte cells was performed. Lavage fluid recovery was greater from 2-week-old birds (91.3 percent) than from 6-week-old birds (86.3 percent). Total cells recovered were greater for 6-week-old chickens (6.79 x 10(5)) than for 2-week-old chickens (5.03 x 10(5)). Cells of epithelial origin included squamous cells, goblet cells, and both ciliated and non-ciliated columnar epithelial cells. Cells of non-epithelial origin consisted of heterophils, lymphocytes, macrophages, eosinophils, basophils, and erythrocytes. Cells of epithelial origin were the predominant cell type recovered from the 2-week-old chickens, followed by heterophils. In 6-week-old chickens, heterophils were the predominant cell type recovered, followed by cells of epithelial origin. In descending order of prevalence, the remainder of cell types recovered from chickens of both ages were lymphocytes, macrophages, eosinophils, and basophils.

Disseminated Mycobacterium avium infection in a dog

lSince that time Mycobacterium tuberculosis and Mycobacterium bovis have been the most frequently reported species associated with canine mycobacteriosis. Dogs are considered equally susceptible to M. tuberculosis and M. bovis but have a higher incidence of infection with M. tuberculosis. 1-7,9 Both organisms have been associated with pulmonary, cutaneous, gastrointestinal, and disseminated disease. 4 Mycobacterium avium infection has been described in many mammalian species including dogs, cats, primates, swine, cattle, sheep, and horses. 2,8 It is considered rare in