Characterization of the host response in systemic isosporosis (atoxoplasmosis) in a colony of captive American goldfinches (Spinus tristis) and house sparrows (Passer domesticus).

Systemic isosporosis, also known as atoxoplasmosis, is a common parasitic disease of passerines. Infection is thought to be endemic in wild birds with fulminant, fatal disease occurring under the influence of stress, concurrent infections, or immunosuppression. Here, we describe the histologic and immunohistochemical characteristics of the cellular infiltrate occurring in captive colonies of American goldfinches and house sparrows. Necropsies were performed on 9 birds, and histologic examination was performed on the intestines of 7 additional birds. Lesions were most severe in the proximal small intestines. Histologically, the changes ranged from variably intense infiltrates of lymphocytes that filled the lamina propria to sheets of large, atypical cells that expanded and obliterated normal mucosal epithelium and invaded through the wall of the intestine and into the ceolomic cavity. Both the smaller lymphocytes and large atypical cells were immunoreactive for CD3. Intracellular parasites consistent with Isospora were detected in the large atypical cells, but they were more easily detectable in the more differentiated lymphocytes. Polymerase chain reaction and virus isolation performed on tissues from 7 birds were negative for retroviruses and herpesvirus. The immunohistochemical results of this study and the destructive nature of the cellular infiltrate suggest that the lesion represents T-cell lymphoma. In birds, lymphomas are most often associated with herpes and retroviruses; the absence of these viruses suggests that the parasite initiated neoplastic transformation. Though much work needs to be done to prove the transformative nature of the lesions, these preliminary results suggest that passerine birds may be susceptible to parasite-associated lymphomas.

Open reading frame L1 of Marek's disease herpesvirus is not essential for in vitro and in vivo virus replication and establishment of latency.

Two mutant CV1988 Marek's disease virus (MDV) strains were developed in which a part of ORF L1 was replaced by lacZ with the SV40 early promoter. These mutant strains, CVIL1LacZ-A and -B, were inoculated into chickens to test the hypothesis that ORF L1 is involved in the induction and/or maintenance of latency. Mutant virus could be reisolated from lymphocytes obtained from chickens during both the lytic and latent phase of infection, indicating that ORF L1 is not essential for the induction and/or maintenance of latency or the reactivation from latency. Beta-galactosidase-positive lymphocytes were detected during the latent infection demonstrating that the SV40 early promoter can be active in recombinant MDV strains during latent infection. Although the insertion of lacZ was stable in cell culture, recombination within lacZ and the BamHI-L fragment was observed during in vivo infection.

Syngeneic lysis of reticuloendotheliosis virus-transformed cell lines transfected with Marek's disease virus genes by virus-specific cytotoxic T cells.

Cell-mediated immune responses against Marek's disease virus (MDV) antigens were examined using reticuloendotheliosis virus (REV)-transformed cell lines of two haplotypes (B19B19 and B13B13). These cell lines were stably transfected with cloned fragments of MDV DNA resulting in the expression of the MDV-specific phosphoprotein pp38. Effector cells were obtained from P2a (B19B19) and S13 (B13B13) chickens at 7 days post inoculation with REV, oncogenic or attenuated serotype 1 MDV (JM-16/O and JM-16/A, respectively), serotype 2 MDV (SB-1), or herpesvirus of turkeys (HVT). Transfection of MDV genes did not influence the expression of Class I major histocompatibility complex antigens. The optimal effector to target cell ratio was determined to be 100:1. REV-sensitized effector cells lysed REV cell lines and REV cell lines transfected with MDV DNA in a syngeneic fashion. Effector cells from chickens inoculated with JM-16/O, JM-16/A, SB-1 or HVT lysed only the syngeneic, transfected cell lines, but not the parent REV cell lines. The percentage specific release caused by the MDV-sensitized effector cells was low, but statistically significant.

Characterization of Marek's disease virus BamHI-A-specific cDNA clones obtained from a Marek's disease lymphoblastoid cell line.

A cDNA library was constructed from poly(A)+ RNA fractions obtained from a Marek's disease (MD) lymphoblastoid cell line, MDCC-CU41, in which viral gene expression is very limited. Three independent groups (1, 2, and 3) of MD virus (MDV)-specific clones were obtained, which were mapped in the inverted repeat region of the BamHI-A fragment of the MDV genome. Northern blot analysis showed that probes prepared from these cDNA clones hybridized with several transcripts of different sizes in poly(A)+ RNA of MDCC-CU41, although the amounts of these transcripts were relatively small compared to those in MDV lytically infected cells. Moreover, a small open reading frame, which can encode a 94-amino-acid protein, was identified in the A41 cDNA clone (Group 3). By RNase protection assays, the 1.2-kb Group 3 transcriptional unit has been defined. In indirect immunofluorescent antibody assays, antiserum against the bacterially expressed fusion protein, glutathione S-transferase-A41, reacted specifically with the cytoplasmic regions of MDV (strain RB1B)-infected chick kidney cells. However, MDCC-CU41 did not contain a detectable level of the protein determined by these methods.

Characterization of a Marek's disease virus BamHI-L-specific cDNA clone obtained from a Marek's disease lymphoblastoid cell line.

Two Marek's disease (MD) virus BamHI-L-specific cDNA clones were isolated from a cDNA library constructed from poly(A)+ RNA fractions of an MD lymphoblastoid cell line, MDCC-CU41 (CU41). These clones were mapped to the region corresponding to the BamHI-Q2 and L-regions. These clones hybridized with 2.5-, 0.8-, and 0.6-kb transcripts prepared from CU41. The transcriptional unit of the 0.6-kb transcript was determined by RNase protection assays. An open reading frame encoding a 107-amino-acid polypeptide was identified in the 0.6-kb transcript. Reverse transcriptase-PCR demonstrated the presence of this transcript in both CU41 and a reticuloendotheliosis virus-transformed cell line latently infected with MD virus.

Detection of retrovirus sequences in budgerigars with tumours.

Renal tumours are a common neoplastic disease of budgerigars. Although a retro-virus has been implicated as the aetiological agent, there is no definitive proof for this hypothesis. Sixteen birds suspected to have renal tumours were examined in an attempt to elucidate the possible role of retroviruses. Thirteen birds had renal tumours and the majority of these birds showed abdominal enlargement and paresis. Renal masses were detected by radiography in nine birds. Post-mortem examination confirmed the presence of abdominal tumours which were mostly confined to the kidneys. All of the renal tumours were carcinomas. ELISA tests to detect the presence of p27 of avian leukosis virus and virus isolation attempts were negative. DNA from eight tumours was examined by dot-blot hybridization for the presence of sequences hybridizing with a full length clone of the RAV-2 strain of the avian leukosis virus. A positive reaction was detected with DNA from 6/8 tumours. Southern blot hybridization demonstrated the presence of a 7.2 kb fragment following restriction with BamHI and a 4.6 kb fragment in an additional tumour following digestion with EcoRI that were recognized by the RAV-2 probe. These results suggest the presence of a retrovirus in tumours of budgerigars.