Glutathione deficiency in sheep erythrocytes.

Three sheep have been found that have concentrations of erythrocyte glutathione less than 20 percent of the concentrations in normal sheep; they have no readily apparent hemolytic disorder.

Malignant lymphoma in macaques: a clinicopathologic study of 45 cases.

Malignant lymphoma was diagnosed in 42 rhesus macaques (Macaca mulatta) and 3 stumptail macaques (M. arctoides) between February 1969 and December 1977. The distribution of tumor masses in the tissues of individual animals varied widely. Solitary tumor masses were present in 14 animals and multiple masses in the remaining 31 animals. Visceral lymph nodes, gastrointestinal tract, heart, and kidneys were most commonly affected. Peripheral lymph nodes were rarely involved. Most malignant lymphomas were of an undifferentiated cell type, although tumors of histiocytic, lymphocytic, poorly differentiated, and mixed lymphocytic and histiocytic cell types were also observed. Concurrent bacterial and/or viral infections were evident in 30 of the 45 macaques with malignant lymphoma. Amyloidosis was present in 9 animals. This high incidence of malignant lymphoma suggested that their immune responses were abnormal. The development of malignant lymphoma in the macaques may have been secondary to or enhanced by immunodeficiency.

Canine malignant lymphomas: comparison of morphologic and immunologic parameters.

Twenty-three canine malignant lymphomas were studied to correlate morphologic and immunologic properties of the neoplastic cells. Morphotologic characterization included histologic classification of cell type and growth pattern, anatomic distribution of lesions, and transmission electron microscopic examination. Parameters examined to indicate B- or T-cell nature of lymphoma cells included demonstration of mitogen responsiveness, cell-surface Ig, spontaneous rosette formation with human red blood cells, and scanning electron microscope (SEM) examination of cell-surface features. Results indicated that the cells from histiocytic lymphomas were lymphocytes rather than histiocytes or macrophages. Most cells from lymphomas examined possessed cell-surface ig, indicating B-cell nature. The cell types represented by the different Ig-positive lymphomas were compatible with maturation arrest at different stages in normal lymphocyte differentiation. For the two most common histiologic cell types, nitogen responsiveness and the presence of cell-surface Ig indicated that diffuse, poorly differentiated lymphocytic cases were biologically heterogenous, whereas nodular histiocytic lymphomas were biologically homogenous. Most canine lymphomas had a multicentric anatomic distribution; however, one thymic and two alimentary forms were observed. Lymphomas with a nodular pattern in lymph nodes had multifocal splenic involvement centered on small arteries, whereas lymphomas with a diffuse pattern in lymph nodes had diffuse involvement of splenic white pulp. The cells of Ig-positive and Ig-negative neoplasms examined by SEM were predominantly of the smooth-cell type.

Regression of feline sarcoma virus-induced sarcomas in dogs. II. Immunologic investigations.

The serial development of cell-mediated immunity (CMI), cytotoxic antibody activity, serum blocking activity, and virus-neutralizing antibody levels were monitored in vitro for beagle and mongrel puppies inoculated with feline sarcoma virus (FeSV) and were compared to in vivo histologic markers of regression of induced sarcomas. CMI developed rapidly and maintained a high level of in vitro activity throughout the tumor life-span. Cytotoxic antibody levels similarly rose rapidly to peak just before clinically detectable regression and then declined during most of the regression sequence. This suggested antibody fixation at the tumor site, correlating with the histologic finding of focal necrosis and neutrophilic infiltrates. Inactivation of antibody by circulating antigen with subsequent immune complex formation was a possibility. Levels of virus-neutralizing antibody in sera paralleled those of cytotoxic antibody; their relationship in the circulation was not clear, but each related to virus-determined antigenic specificities. Serum blocking activity rose rapidly, leveled off during most of the tumor life-span, and rose slightly during the last stages of regression. This partly explained the lack of in vivo tumor lymphoid infiltrates to correlate with the striking in vitro CMI. Blocking activity was also present, however, when lymphoid infiltrates were seen histologically. Thus in vitro-in vivo correlation was best for cytotoxic antibody, which suggested that antigen-antibody reactions involving neutrophil-mediated regression sequences were important in effecting tumor-cell destruction.

Regression of feline sarcoma virus-induced sarcomas in dogs. I. Morphologic investigations.

In a study of morphologic changes in the development and regression of feline sarcoma virus (FeSV)-induced tumors in dogs, 27 weaned and newborn beagle and mongrel puppies were inoculated with FeSV in doses from 1.0 to 3.0 gEq; 2 beagle and 2 mongrel puppies were used as uninoculated contact controls. All animals were examined daily, and crude tumor volume was calculated from length, width, and depth measurements of the neoplasms. Biopsies were done at various stages of tumor development and regression. When tumors were no longer palpable, all puppies were necropsied. Two of 8 (25%) weaned beagle puppies, 10 of 12 (83%) newborn beagles, and 1 of 7 (14%) newborn mongrels developed tumors, all histologically confirmed fibrosarcomas. No metastatic tumor foci were detected. The tumor life-span was divided into approximately equal periods of growth and regression. The initial regression period was characterized by focal necrosis and accompanying neutrophil infiltration. The later stages of regression were characterized by lymphocytic or mixed mononuclear infiltrates. Thus the regression histopathology was not uniform and suggested that different immunologic mediation systems effect regression. Nonneoplastic morphologic changes consisted largely of lymphoid depletion and necrosis in lymph nodes and thymus after inoculation.

Inhibition of human lymphoma DNA-dependent RNA polymerase activity by 6-mercaptopurine ribonucleoside triphosphate.

6-Mercaptopurine was found to inhibit the growth of cultured human lymphoma P3HR-1 cells and the incorporation of [3H]-uridine into trichloroacetic acid-precipitable materials of the cells. One of the derivatives of 6-mercaptopurine, 6-mercaptopurine ribonucleoside triphosphate (6-thio-ITP), was found to inhibit in vitro RNA synthesis (both engaged and free enzyme activities) of the isolated nuclei from P3HR-1 cells. The alpha-amanitin-resistant RNA polymerase (polymerase I) and alpha-amanitin-sensitive RNA polymerase (polymerase II) of the cells were isolated and partially purified by either diethylaminoethyl cellulose or diethylaminoethyl Sephadex column chromatography, followed by DNA-cellulose affinity chromatography. It was found that these partially purified enzymes were also sensitive to 6-thio-ITP inhibition. Kinetic studies showed that the inhibition of RNA polymerase activities by 6-thio-ITP could be reversed by increasing concentrations of guanosine 5'-triphosphate in the reaction mixture, indicating that 6-thio-ITP may act as a competitive inhibitor of the enzymes by competing with guanosine 5'-triphosphate for its enzyme-binding site. These data suggest that inhibition of RNA transcription by 6-thio-ITP may be considered as one of the mechanisms of the cytotoxic action of 6-mercaptopurine in human tumor cells.

Heterogeneity of the L2 gene of field isolates of bluetongue virus serotype 17 from the San Joaquin Valley of California.

Genome segment 2 (L2) from six field isolates of bluetongue virus (BTV) serotype 17 was sequenced by cycling sequencing after the amplification of the viral cDNA by the polymerase chain reaction. The viruses were isolated from sheep, cattle and a goat in the San Joaquin Valley of California during the years 1981 and 1990. These viruses exhibit divergent patterns of neutralization with BTV 17-specific monoclonal antibodies. The six L2 genes of the BTV 17 field isolates all encode a protein of 955 amino acids. Similarity of the nucleotide sequences of the L2 genes with respect to the prototype strain ranges between 93.8% and 95.1%, whereas the similarity between the field isolates ranges from 96.8% to 99.1%. Although very closely related, the L2 gene of each virus is distinct. Furthermore, mutations in the L2 gene of field isolates of BTV do not consistently follow a linear pattern of accumulation over time. Some amino acid changes in the VP2 protein of field strains were conserved over time, whereas others were not correlated with the year of isolation and some substitutions were unique to individual viruses. The predicted VP2s constitute a group of non-identical, but closely related proteins. Phylogenetic analyses suggest that the viral variants which co-circulate in the San Joaquin Valley could evolve by different evolutionary pathways.

Genetic variation and evolutionary relationships amongst bluetongue viruses endemic in the United States.

The genetic variation and evolutionary relationships amongst the five serotypes of bluetongue virus (BTV) endemic to the United States were investigated by oligonucleotide fingerprint analysis. The viruses analyzed include prototype viruses of the five U.S. serotypes, and 32 viruses isolated from domestic and wild ruminants from the U.S. in the years 1979-1981. With the exception of serotype 2, most genes encoding the viral core and non-structural proteins were demonstrated to be highly conserved both within and between serotypes and some also appear to have reassorted in nature. Gene segments 2 and 6, which encode the outer capsid proteins VP2 and VP5 respectively, were more variable and were not consistently linked as serotype determination was dependent solely on gene segment 2. Gene segment 2 was the most variable gene between serotypes, but it was highly conserved within serotypes and stable over time. This suggests that the emergence of new BTV serotypes, which would require the stable incorporation of numerous mutations, must be a very slow process. Fingerprint comparisons further suggested that BTV serotypes 10, 11, 13 and 17 have evolved together in the U.S. over a considerable period of time, whereas serotype 2, which is genetically distinct, has evolved elsewhere and is most likely a recent introduction to North America.

Diaplacental infections with ruminant pestiviruses.

Pestiviruses are capable of causing diaplacental infections. Maternal viremias are important for localizing virus in the ruminant placentome. Placental lesions occur with cytopathic BVDV and noncytopathic BDV. The ruminant fetus is very susceptible to pestivirus infections once the virus crosses the placenta because the fetus is 1) agammaglobulinemic, 2) immunologically immature, and 3) it has many immature organ systems with undifferentiated cells. Cytopathic BVDV (NADL) in calves and noncytopathic BDV (BD-31) in lambs cause a variety of clinical syndromes including early embryonic death, abortion, stillbirth, malformed fetuses, and/or low birth weight with viral persistence and immunological tolerance. The cytopathic BVDV (NADL) reviewed herein caused pulmonary, placental and dermal lesions when infection occurred at 80-90 days gestation. In contrast, infection at 140-150 days resulted in retinal dysplasia and cerebellar hypoplasia. The lesions were attributed to direct viral cytopathology. Noncytopathic BDV (BD-31) in lambs caused weak lambs, with hairy fleece and tonic-clonic tremors. The lambs were of low birth weight, persistently viremic and immunologically tolerant. The lambs are hypothyroid and had severe hypomyelination. It is hypothesized that the central lesion leading to many of the neural, skeletal and dermal lesions was the endocrine dysfunction leading to hypothyroidism.

Border disease of sheep--aspects for diagnostic and epidemiologic consideration.

Border Disease (BD) is a condition of newborn sheep that results from congenital infection by a non-cytopathic pestivirus, occurring during the first half of gestation. The variations in expression of the virus directly relate to the age of the fetus at the time of infection. There are four distinct disease syndromes: (1) early embryonic death, (2) abortion and stillbirth, (3) birth of lambs with malformations, and (4) birth of small, weak lambs, lacking characteristic clinical signs, but bearing features of immunosuppression. In the newborn, the BD virus may be recovered from all tissues and teratogenic lesions are found in the endocrine, nervous, skeletal, integumentary and immune systems. These effects of virus infection are manifest in the clinical signs characteristic of the disease, such as tremors, ataxia, hairy birthcoat, low birth weight, facial bone malformations, short-boxy stature, and eye abnormalities. The consequences of the BD compromised immune system is an increased susceptibility to infection, a failure to produce specific antibody to BD virus, and an inability to clear the virus; features characteristic of the immuno-tolerant state. The lifelong shedding and persistence of virus is of epidemiologic importance. The persistently infected BD ewe remains a source of infection for the flock both through horizontal transmission (virus shedding) and congenital transmission (a persistently infected ewe will always bear a BD lamb). Detection of persistently infected individuals within a flock is difficult: clinical signs abate with time and most frequently no antibody to BD is produced.

A study of some pathogenetic aspects of bovine viral diarrhea virus infection.

The cytopathic (CP) strain TVM-2 of bovine virus diarrhea virus (BVDV) induced in calves a severe disease, whereas the calves inoculated with the non-cytopathic (NCP) New York-1 strain, remained clinically normal. When calves were immunosuppressed with dexamethasone (DMS) they underwent an overt, generally fatal disease. This result was obtained with either the CP and the NCP strain of BVDV. It was speculated that the immunosuppressive activity of BVDV could be a property peculiar to certain isolates of the virus.

Detecting bluetongue virus RNA in cell culture by dot hybridization with a cloned genetic probe.

A 70% copy of genome segment 7 of bluetongue virus (BTV)-17 has been cloned into the plasmid pBR-322. This cloned BTV segment when used as a radioactive probe will hybridize to BTV double-stranded RNA extracted from cell cultures and dotted onto nitrocellulose paper. This dot hybridization technique is therefore suitable for detecting and identifying BTV in cell culture. The specificity of cloned probes is discussed in relation to detecting gene sequences specific for either the bluetongue serogroup or different serotypes of BTV.

Rapid identification of bluetongue virus by nucleic acid hybridization in solution.

A liquid phase hybridization format has been adapted for the identification of bluetongue virus (BTV) with respect to serogroup and serotype. In this paper we present experiments which demonstrate the identification of bluetongue virus with respect to both serogroup and serotype. BTV serogroup-specific and BTV serotype 17-specific single-stranded RNA probes labeled with 35S-CTP were used to identify BTV viral nucleic acids in infected cell culture lysates. Cell lysates were prepared for testing by rapid solubilization in a guanidine isothiocyanate/EDTA/dithiothreitol medium. Hybridizations were done in 12.5 microliter reaction volumes for 2 h or overnight using nanogram quantities of probe. An RNAse solution was added subsequent to hybridization and incubated for 30 min. TCA-precipitable counts were collected and quantitated by scintillation counting. Specific hybridization signals were obtained in as little as 3 h in assays consisting of uninfected controls, cells infected with different BTV serotypes, and cells infected with a strain of epizootic hemorrhagic disease of deer (EHDV-1). By using asymmetric single-stranded RNA probes we were able to distinguish viral mRNA and viral double-stranded RNA components. We propose this technique as an ancillary or replacement procedure for the confirmation and classification of viral isolates using an appropriate battery of nucleic acid probes. Potential applications and methods of enhancing the technique are discussed.

Colony hybridizations using nylon membranes and RNA probes--an improved method for screening bacterial clones containing bluetongue virus genome.

Bluetongue virus (BTV) total genomic and isolated individual segment dsRNAs end-labeled with 32P were successfully used as probes in colony hybridization to detect clones of BTV genomic material. The RNA probes were highly specific for cloned BTV genomic material. DNA probes, however, gave false positive results. DNA from bacterial clones was fixed to nylon and nitrocellulose membranes. The hybridized nylon membranes could be stripped of probe and reprobed at least 6 times without loss of signal strength.

Identifying bluetongue virus ribonucleic acid sequences by the polymerase chain reaction.

A primer-directed nucleic acid amplification reaction, commonly known as the polymerase chain reaction (PCR), has been adapted to the identification of bluetongue virus (BTV) RNA. The protocol described in this article is designed for the detection of a purported globally-conserved, serogroup specific nucleic acid sequence within the BTV genome. Due to the double-stranded RNA composition of the BTV genome, the original polymerase chain reaction protocol has been modified to include chemical denaturation and reverse transcription steps that allow selective amplification from this unique template molecule. The amplification procedure yields a 210 base pair product when tested on samples of RNA from the prototypic strains of U.S. BTV serotypes 2, 10, 11, 13, 17. The amplification product is tentatively identified by agarose gel electrophoresis of the PCR samples. Final positive identification of the amplified BTV-specific product is determined by Southern blot hybridization of the PCR samples. RNA samples from uninfected cell culture controls and from cell cultures infected with two serotypes of epizootic hemorrhagic disease of deer (EHDV) yielded negative results. Preliminary experiments to quantify the threshold of sensitivity of this protocol, as described here, indicate positive detection of the BTV target RNA sequence at a level of less than 2 fg, or the amount of target sequence derived from less than 7500 viral particles. While this falls short of the theoretical limit of sensitivity of the PCR, the enhanced threshold of sensitivity of the PCR reaction compared to standard nucleic acid hybridization methodology suggests that rapid detection of BTV RNA directly within clinical specimens may be feasible with further refinements. Potential methods of enhancing this reaction are discussed.

Comparison of dot-blot and Northern blot hybridizations in the determination of genetic relatedness of United States bluetongue virus serotypes.

Dot-blot and Northern blot hybridization methods to determine the genetic relatedness of United States bluetongue virus serotypes 2, 10, 11, 13, and 17 were compared. Both plasmid and insert DNA probes from cloned BTV-17 dsRNA segments 2, 5, 6, and 8 were hybridized to dsRNA from the BTV serotypes and epizootic hemorrhagic disease virus (EHDV). Stringencies of hybridization were kept identical, and experiments differed only in the method in which dsRNA was applied to the membranes (dot-blot or Northern blot). The Northern blot hybridization method yielded more consistent results, and it was visually and unequivocally shown to which dsRNA segment a cDNA probe bound, since the dsRNA segments were separated by PAGE prior to blotting and hybridization. In contrast, the dot-blot hybridization method gave less consistent results. Identical results for plasmid and insert probes were obtained for Northern blot hybridizations but not for dot-blot hybridizations. At least two probes could be used simultaneously in Northern blot hybridizations.

Virological, clinical and serological responses of sheep infected with tissue culture adapted bluetongue virus serotypes 10, 11, 13 and 17.

Sheep were experimentally infected with cloned strains of tissue culture adapted bluetongue virus (BTV) serotypes 10, 11, 13 and 17. All the infected animals developed viremia by Day 2 or 3 post-inoculation (P.I.) and reached maximum viremia on Day 7 P.I. The viremia lasted for 2 to 3 weeks. Animals infected with the different serotypes showed mild clinical bluetongue (BT) responses, characterized by pyrexia and leukopenia, which coincided with the peak of viremia. Antibodies appeared by Day 10 P.I. and reached maximum by Day 28 P.I. There was a temporal relationship between the increase in neutralizing antibody titer, the drop in titer and clearance of virus from the peripheral circulation. Recovery from primary infection protected the animals against secondary challenge with homologous virus.

Recovery of dual serotypes of bluetongue virus from infected sheep and cattle.

Dual serotypes of bluetongue virus (BTV) were recovered from field-collected samples of sheep and cattle blood. Two sheep, each infected with both BTV serotypes 10 and 17, were found in a flock with bluetongue disease associated with these two serotypes. One sheep infected with BTV serotypes 11 and 17 was found in a second flock; it was the only viremic sheep detected and was clinically ill. Dual serotype infections of one beef and two dairy cattle were found in three geographically separate herds: mixtures recovered were of BTV serotypes 10 and 17 and serotypes 11 and 17. Clinical signs of illness were absent in the cattle in two herds, but severe conjunctivitis was seen in several cows in a third herd, including the cow with a dual serotype infection (BTV 11 and 17). Two of the cattle with dual infections had no serological evidence of BTV as determined by the agar gel precipitin test; serum was not available from the other cow with a dual serotype infection. The significance of dual infections and immune tolerance are discussed.

Temporal development of bluetongue virus protein-specific antibody in sheep following natural infection.

Humoral immune responses of sheep to natural bluetongue virus (BTV) infection were studied on a temporal basis. The temporal development of viral protein-specific IgG was determined by western immunoblotting; virus neutralization and agar gel immunodiffusion (AGID) were conducted for comparative purposes. Prior to the emergence of the arthropod vector and the associated transmission of BTV, virus-neutralizing antibody was absent from all sentinel sheep; 3 sheep had pre-existing AGID antibody and all sheep had IgG, specific for 4 viral proteins, as determined by immunoblotting. Following emergence of the BTV vector, 9 of 11 sheep became infected, as determined by virus isolation, with BTV. All sheep developed virus-neutralizing and AGID antibody. However, only those sheep with a demonstrable viremia experienced an increase in viral protein-specific antibody. Development of viral protein-specific IgG varied with the individual animal and no obvious correlation between a specific response and protective immunity or viral clearance was noted. From a diagnostic viewpoint, the immunoblotting procedure was superior in identifying past exposure to BTV, as compared with neutralization and AGID. In addition, the application of immunoblotting to paired serum samples appeared to be a sensitive indicator of viremia.

A nested PCR for detection of North American isolates of bluetongue virus based on NS1 genome sequence analysis of BTV-17.

A nested polymerase chain reaction (PCR)-based assay, for detection of bluetongue virus (BTV) ribonucleic acid in cell culture and tissue samples, was developed. Two pairs of oligonucleotide primers (BTV1 and BTV4 and BTV2 and BTV3), selected from non-structural protein 1 (NS1) gene of BTV-17, were used for the nested PCR in two amplification steps. First a 826-bp product was amplified using an outer primer pair BTV1 and BTV4. The second amplification, using nested or internal primer pair BTV2 and BTV3, produced a 517-bp PCR product. RNA from North American prototype serotypes 2, 10, 11, 13 and 17, propagated in cell cultures, were detected by this nested PCR-based assay. The nested primers BTV2 and BTV3 increased the sensitivity of the BTV PCR assay, and as little as 0.1 fg of BTV RNA (equivalent to 5 viral particles) could be detected. Amplification products were not detected when the PCR-based assay was applied to RNA from a closely related orbivirus, epizootic hemorrhagic disease virus (EHDV) prototype serotypes 1 and 2; total nucleic acid extracts from uninfected BHK-21 cells; or whole blood from calves and deer that were BTV-seronegative and virus isolation negative. Application of this nested BTV PCR-based assay to clinical samples resulted in detection of BTV RNA from a variety of tissues collected from calves and deer with natural and experimental BTV infections. The described BTV PCR-based assay provides a valuable tool to study the epidemiology of BTV infection in susceptible wild ruminants and domestic livestock.