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.

Natural variation of canine parvovirus.

Canine parvovirus was first recognized during 1978. Analysis of isolates collected since its emergence revealed that viruses circulating after 1980 were antigenically different from earlier isolates. Monoclonal antibodies clearly distinguished the two strains, some being specific for either the old or the new viruses. Restriction enzyme analysis of viral DNA's showed that the post-1980 viruses were similar to earlier isolates, but some restriction site differences were present in the new strain. These results suggest that the canine parvoviruses infecting dogs in the seven areas of the United States that were sampled derive from a variant virus that replaced the original strain during 1980.

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.

Influence of genetic resistance of the chicken and virulence of Marek's disease virus (MDV) on nitric oxide responses after MDV infection.

Nitric oxide (NO), a free radical produced by the enzyme NO synthase (NOS), is a potent antiviral agent in addition to having immune regulating functions. Recently, it was reported that chickens resistant (N2a, MHC: B21B21) to the development of Marek's disease (MD) had a greater potential to produce NO than MD-susceptible chickens (P2a, MHC: B19B19). This difference was shown by measuring NO levels in chick embryo fibroblast cultures obtained from these chickens after treatment with lipopolysaccharide and recombinant chicken interferon-gamma (IFN-gamma). To extend these results, the levels of NO in blood plasma from N2a and P2a chickens inoculated with the nonattenuated JM-16 strain of MD virus (MDV) were examined. In four out of five experiments, N2a chickens had increased NO levels at 7 days postinoculation (DPI). In contrast, P2a chickens challenged with JM-16 had a significant increase in NO in only one of four experiments, and in that experiment the increase was delayed (10 DPI) compared with N2a chickens. Attenuation abrogated MDV-induced NO in chickens. Inoculation with MDV strains ranging from mild to very virulent plus showed that the more virulent strains induced the highest level of NO in blood plasma, suggesting a role of NO in the pathogenesis of MD with more virulent strains. On the basis of quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assays for analysis of mRNA expression, IFN-gamma does not appear to be the primary inducer of inducible (i)NOS gene expression during MDV infection. iNOS gene expression and NO production are mediated during the cytolytic phase of MDV infection on the basis of real-time RT-PCR assays with primers specific for glycoprotein B, a late gene expressed only during the cytolytic phase of MDV infection. These findings implicate NO as a factor potentially involved in increasing virulence of MDV, possibly through immune suppression.

Isolation of a reovirus from poult enteritis and mortality syndrome and its pathogenicity in turkey poults.

Poult enteritis and mortality syndrome (PEMS) is an acute, infectious intestinal disease of turkey poults, characterized by high mortality and 100% morbidity, that decimated the turkey industry in the mid-1990s. The etiology of PEMS is not completely understood. This report describes the testing of various filtrates of fecal material from control and PEMS-affected poults by oral inoculation into poults under experimental conditions, the subsequent isolation of a reovirus, ARV-CU98, from one of the PEMS fecal filtrates, and in vivo and in vitro studies conducted to determine the pathogenicity of ARV-CU98 in turkey poults. In order to identify a filtrate fraction of fecal material containing a putative etiologic agent, poults were challenged in two independent experiments with 220- and 100-nm filtrates of fecal material from PEMS-negative and PEMS-positive poults. The 100-nm filtrate was chosen for further evaluation because poults inoculated with this filtrate exhibited mortality and significantly lower (P < or = 0.05) body weight and relative bursa weight, three clinical signs associated with PEMS. These results were confirmed in a third experiment with 100-nm fecal filtrates from a separate batch of PEMS fecal material. In Experiment 3, body weight and relative bursa and thymus weights were significantly lower (P < or = 0.05) in poults inoculated with 100-nm filtrate of PEMS fecal material as compared with poults inoculated with 100-nm filtrate of control fecal material. Subsequently, a virus was isolated from the 100-nm PEMS fecal filtrate and propagated in liver cells. This virus was identified as a reovirus on the basis of cross-reaction with antisera against avian reovirus (FDO strain) as well as by electrophoretic analysis and was designated ARV-CU98. When inoculated orally into poults reared under controlled environmental conditions in isolators, ARV-CU98 was associated with a higher incidence of thymic hemorrhaging and gaseous intestines. In addition, relative bursa and liver weights were significantly lower (P < or = 0.05) in virus-inoculated poults as compared with controls. Virus was successfully reisolated from virus-challenged poults but not from control birds. Furthermore, viral antigen was detected by immunofluorescence in liver sections from virus-challenged poults at 3 and 6 days postinfection and virus was isolated from liver at 6 days postinfection, suggesting that ARV-CU98 replicates in the liver. In addition to a decrease in liver weight, there was a functional degeneration as indicated by altered plasma alanine aminotransferase and aspartate aminotransferase activities in virus poults as compared with controls. Although this reovirus does not induce fulminating PEMS, our results demonstrated that ARV-CU98 does cause some of the clinical signs in PEMS, including intestinal alterations and significantly lower relative bursa and liver weights. ARV-CU98 may contribute directly to PEMS by affecting the intestine, bursa, and liver and may contribute indirectly by increasing susceptibility to opportunistic pathogens that facilitate development of clinical PEMS.

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.

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.

A hypervariable region in VP1 of chicken infectious anemia virus mediates rate of spread and cell tropism in tissue culture.

Chicken infectious anemia virus (CIAV) is a unique infectious agent with an amino acid composition that has been found to be remarkably conserved even in isolates from different parts of the world. We have characterized field isolates of CIAV which vary significantly in terms of their abilities to replicate in culture, demonstrating a biological difference between isolates. Two sublines of MDCC-MSB1 cells that differ in their abilities to support CIAV were identified. In the MSB1(S) subline the CIA-1 isolate of CIAV was found to be less cytopathogenic than the prototype Cux-1(C) isolate; the MSB1(L) subline, which supports Cux-1(C) replication, was found to be nonpermissive for CIA-1. Alignments of the VP1 sequences of previously examined isolates with those of the field isolates CIA-1 and L-028 and the culture-adapted ConnB isolate revealed a previously unreported hypervariable region spanning amino acid positions 139 to 151. Chimeras of Cux-1(C) and CIA-1 were constructed to examine the potential for this region to affect cytopathogenicity. Transfer of a 316-bp region of Cux-1(C) open reading frame 1 into CIA-1 produced a virus with a cytopathogenic profile typical of Cux-1(C), indicating that one or both of the amino acid differences at positions 139 and 144 affect the rate of replication or the spread of infection. Transfection experiments with additional chimeras indicated that the inability of CIA-1 to replicate in MSB1(L) cells is mediated by a larger region of the genome which contains the hypervariable region in addition to upstream amino acid differences. Analysis of chimeras excluding the entire region of open reading frame 1 suggested the presence of a secondary mediator in the progression of infection in culture that was localized to a region containing a single nucleotide difference which results in amino acid differences in both VP2 (V-153) and the nuclear localization signal of VP3 (C-118). Immunofluorescence assays indicated an increased cytoplasmic distribution of VP3 and a general lack of VP3-associated apoptotic bodies in infections of CIA-1 and chimeras containing V-153 or C-118, as opposed to a primarily nuclear distribution and association with well-formed apoptotic bodies in Cux-1(C)-infected cells.