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N J MacLachlan

Publications and source records attributed to N J MacLachlan.

At least 73 records · Page 4Linked to original sources
PubMed1992

Neutralizing epitopes of the serotypes of bluetongue virus present in the United States.

Neutralizing epitopes present on the five serotypes of bluetongue virus (BTV) which have been isolated in the United States were investigated with a panel of monoclonal antibodies (MAbs). Neutralizing MAbs were raised against the U.S. prototype viruses of BTV serotypes 2, 10, 11, 13 and 17, and were reacted with each virus in both neutralization and immune precipitation assays. All MAbs neutralized and precipitated VP2 of the virus against which they were raised. Five MAbs raised against BTV-10 also precipitated VP2 of the prototype strain of BTV-17, and four of these MAbs neutralized BTV-17. To characterize further the neutralizing epitopes of BTV, the MAbs raised against BTV-10 and BTV-17 were reacted by immune precipitation and neutralization assays with four field strains each of BTV-17 and BTV-10 isolated from ruminants in the U.S. All MAbs raised against BTV-10 both precipitated VP2 and neutralized the four field isolates of BTV-10, whereas none of the MAbs raised against BTV-17 reacted with these viruses. By contrast, all seven MAbs raised against BTV-17 and four of the seven MAbs raised against BTV-10 precipitated VP2 of the four BTV-17 field isolates. Another MAb raised against BTV-10 precipitated VP2 of three of the four field isolates of BTV-17. Whereas neutralization of the BTV-17 field isolates by several MAbs was inconsistent, all 10 isolates of BTV-10 and BTV-17 were neutralized by three MAbs raised against BTV-10. Results of this and other studies indicate that multiple neutralizing epitopes exist on each serotype of BTV. Some of these epitopes are conserved whereas others apparently vary in their significance to the neutralization of individual field isolates of BTV-17 and perhaps other BTV serotypes. These findings have implications for the future development of efficacious subunit vaccines to prevent BTV infection of ruminants.

Animals↗
PubMed1992

Flow cytometric analysis of in vitro bluetongue virus infection of bovine blood mononuclear cells.

Cultures of adherent and non-adherent bovine peripheral blood mononuclear (PBM) cells were inoculated with bluetongue virus (BTV) serotype 10. Some cultures of non-adherent cells were stimulated with interleukin 2 (IL-2) and concanavalin A for 24 h prior to virus inoculation. Cells were harvested at various intervals up to 72 h after inoculation. A panel of leukocyte differentiation antigen-specific monoclonal antibodies (MAbs), specific for bovine CD2, CD4 or CD8, monocytes and granulocytes, B cells, gamma delta T cells or the IL-2 receptor (IL-2r), was directly conjugated to fluorescein isothiocyanate, and a MAb specific for the BTV major core protein VP7 was directly conjugated to phycoerythrin. Cells were labelled with conjugated MAbs in single- and double-label immunofluorescence studies to identify specifically the BTV-infected cells in inoculated cultures. The viability of cells was determined by propidium iodide exclusion, and all analyses were done using flow cytometry. Productive infection of cultures of PBM cells was confirmed by virus titration. The data revealed a clear difference between subsets of bovine PBM cells in susceptibility to infection with BTV in vitro. Monocytes were readily infected with BTV, as were stimulated CD4+ cells, and infection was cytopathic to monocytes and stimulated lymphocytes. The proportion of infected cells decreased after 24 h and virus titres dropped markedly by 72 h in all cultures. CD4+ cells in cultures of unstimulated non-adherent cells inoculated with BTV showed increased expression of IL-2r. The possible relevance of these findings to the pathogenesis of BTV infection of cattle is discussed.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

A competitive ELISA for detection of antibodies to the group antigen of bluetongue virus.

A competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect antibodies to the group antigen of bluetongue virus (BTV). The epitope recognized by the BTV-specific monoclonal antibody was confirmed, by immunofluorescence staining of monolayers of virus-infected Vero cells, to be present on BTV serotypes 2, 10, 11, 13, and 17 but not on epizootic hemorrhagic disease virus (EHDV) serotypes 1 and 2. Sera from BTV-inoculated ruminants and rabbits were used to evaluate the cELISA and to compare its specificity and sensitivity with that of the conventional BTV-specific agar gel immunodiffusion (AGID) and serum neutralization (SN) tests. Rabbit antisera to the 5 serotypes of BTV present in the United States had cELISA titers (inverse of the final dilution of serum that gave greater than 20% inhibition) that ranged from 32 to greater than 1.024. Seroconversion of the 8 calves and lambs inoculated with BTV was detected by all 3 serologic tests (SN, AGID, cELISA) by 6 weeks after inoculation. Specificity of the cELISA test was confirmed with bovine sera that contained neutralizing antibodies to EHDV but not to the 5 serotypes of BTV present in the United States; these sera gave positive results by AGID test but were negative by cELISA. The sensitivity and specificity of the cELISA test was further confirmed by analysis of a panel of bovine test sera supplied by the National Veterinary Services Laboratories, indicating that the cELISA is a superior test for detection of BTV group-specific antibodies in sera from ruminants in the United States.

Animals↗
PubMed1990

Identification of four distinct neutralizing epitopes on bluetongue virus serotype 10 using neutralizing monoclonal antibodies and neutralization-escape variants.

Neutralizing sites on bluetongue virus serotype 10 (BTV-10) were investigated with a panel of seven neutralizing monoclonal antibodies (MAbs). Each MAb was coupled to agarose beads and tested against the other MAbs and a nonneutralizing control MAb in a competitive immueprecipitation assay. In addition, neutralization-escape viral variants of BTV-10 were identified and cloned by selecting individual plaques that formed in the presence of neutralizing MAbs. Four antigenic variants were isolated, each under the selective pressure of a different MAb. Parental BTV-10 and the four antigenic variants were compared by microneutralization assay using the MAb panel. The panel of neutralizing MAbs was subdivided into four groups on the basis of these assays, indicating that at least four distinct neutralizing epitopes exist on the BTV-10 virion. These epitopes are individually defined by representative MAbs 034, 039, 041, and 045, and the results indicate that the four epitopes are interacting sites within a single antigenic domain on BTV-10 outer capsid protein VP2. This conclusion is further supported by the fact that a double-site neutralization-escape variant designated DE34/39 (sequentially produced against MAbs 034 and 039) was not neutralized by any MAb of the panel.

Animals↗
PubMed1990

The pathogenesis of experimental bluetongue virus infection of calves.

Eleven seronegative calves were intravenously inoculated with bluetongue virus (BTV) serotype 10, and two calves were inoculated with a placebo. Cellular association of BTV during viremia was investigated in three of the calves by titrating virus present in plasma and different blood cell fractions at weekly intervals after infection. Viremia persisted 35 to 49 days in individual calves. Virus was transiently isolated from blood mononuclear cells and plasma collected from two of the calves but was consistently isolated from erythrocytes throughout infection of all three animals. Titers of BTV present in the erythrocyte fraction were comparable to those of the unseparated blood cell fraction. Tissue tropism of BTV was determined by viral isolation from tissues collected from calves euthanatized at 1, 2, 3, 4, 5, 7, 14, 28, 42, and 56 (2 calves) days after inoculation with BTV. Tropism was also determined by immunohistochemical staining of selected tissues with an avidin-biotin complex immunoperoxidase staining procedure using three BTV-specific monoclonal antibodies. The BTV infected calves remained healthy throughout the study. Virus was isolated from at least one tissue collected from calves euthanatized at 1 through 28 days after inoculation, but not thereafter. High titers of BTV were present in the lungs, prescapular and mesenteric lymph nodes, thymus, and spleen of calves euthanatized at 1 to 4 days after inoculation, whereas BTV was either not isolated or isolated in low titer from bone marrow collected from these animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Evaluation of age-related effects on the antiviral activity of interferon and induction of 2-5A synthetase in testicular cell cultures derived from swine of various ages.

The antiviral activity of recombinant DNA-derived bovine alpha 1(-1) interferon on an established swine testicular cell line and primary testicular cell cultures derived from swine of various ages (2 days, 3 weeks, and 5 weeks) was determined. Bovine interferon induced a dose-dependent increase in 2-5A synthetase in testicular cells, regardless of the source of the cells. Furthermore, interferon inhibited replication of vesicular stomatitis virus to an equivalent extent in all testicular cell cultures. The results indicate that 2-5A synthetase is a reliable marker of interferon activity in swine testicular cell cultures and that the induction of 2-5A synthetase and antiviral effects of recombinant bovine interferon in primary testicular cell cultures are not dependent on the age of the donor animal.

2',5'-Oligoadenylate Synthetase↗
PubMed1990

Temporal appearance of structural and nonstructural bluetongue viral proteins in infected cells, as determined by immunofluorescence staining and flow cytometry.

The temporal appearance of 4 viral proteins was detected in bluetongue virus (BTV)-infected Vero cells by indirect immunofluorescence staining with monoclonal antibodies (MAb) to BTV structural proteins VP2 and VP7 and nonstructural proteins NS1 and NS2. Bluetongue viral proteins were detected at distinct intervals after inoculation of Vero cells; VP7 was first detected 3 hours after inoculation, NS1 and NS2 at 5 hours after inoculation, whereas VP2 was not detected until 8 hours after inoculation. Patterns of fluorescence varied with the fixative used, but each MAb induced a distinct pattern of fluorescence in infected cells. Flow cytometry, which was used with each of the 4 MAb, proved to be an accurate and sensitive method of detecting BTV-infected P3 mouse myeloma cells. The temporal appearance of each viral protein in BTV-infected P3 cells was similar to that detected in BTV-infected Vero cells. Advantages of flow cytometry over conventional immunofluorescence staining to detect BTV-infected cells included: (1) enumeration of the proportion of infected cells in a population; (2) further characterization of infected cells, including estimates of their viability; and (3) computer-assisted storage and analysis of data obtained.

Animals↗
PubMed1990

Modulation of arachidonic acid metabolism by bovine alveolar macrophages exposed to interferons and lipopolysaccharide.

Stimulation of bovine alveolar macrophages with calcium ionophore A23187 resulted in marked production of leukotriene (LT)B4 and a lesser increase in thromboxane (TX)B2, whereas opsonized zymosan (OPZ) resulted in production of TXB2 and relatively small increases in LTB4 and prostaglandin (PG)F2 alpha. Alveolar macrophages incubated with recombinant bovine interferon-gamma or lipopolysaccharide, and subsequently stimulated with A23187 or OPZ, had altered arachidonic acid metabolism, producing markedly increased amounts of TXB2 and PGF2 alpha, and slightly increased LTB4. Incubation of alveolar macrophages with lipopolysaccharide had a more profound effect on the increased amounts of TXB2 and PGF2 alpha, observed in response to stimulation with A23187 or OPZ, than did incubation with interferon-gamma. Alveolar macrophages incubated with recombinant bovine interferon-alpha 1-1 also produced slightly increased amounts of LTB4 when stimulated with A23187 or OPZ. Altered arachidonic acid metabolism by alveolar macrophages exposed to interferons and lipopolysaccharide may contribute to the development of pulmonary inflammation, such as in the early stages of bacterial pneumonia following viral infections that induce interferon production.

Animals↗
PubMed1989

Isolation of Eubacterium suis from sows with cystitis.

Eubacterium suis was isolated from the bladders of 3 of 64 sows (4.7%) examined at slaughter. Two of the sows had cystitis similar to other descriptions of E suis infections in sows. There was no histologic evidence of cystitis in the third sow from which E suis was recovered. In all 3 instances, a mixed infection was present. Other bacteria present were predominantly gram-positive organisms. Eubacterium suis cystitis may be more prevalent than previously suspected. The special culture conditions required to grow this organism may account for the paucity of reports in the United States.

Abattoirs↗
PubMed1989

A simplified procedure for oligonucleotide fingerprint analysis of multiple bluetongue virus genome segments utilizing small gels.

The technique of oligonucleotide fingerprinting is a useful tool for analyzing sequence homology among RNA molecules. A rapid method for the simultaneous production of multiple fingerprints has been developed using a commercially available electrophoresis apparatus. The system makes use of relatively small gels, yielding fingerprints that when compared to conventional systems are of reduced size but of comparable resolution. The system described should have particular application to the analysis of RNA viruses with multiple genome segments, and in epidemiologic studies concerned with the analysis of multiple virus isolates.

Animals↗
PubMed1989

Induction of the 2-5A system by interferon and transmissible gastroenteritis virus.

Interferon (IFN) treatment increased the level of 2',5'-oligoadenylate (2-5A) synthetase and inhibited replication of transmissible gastroenteritis virus (TGEV) in cultured swine testicular cells. Despite a minimal increase in TGEV titer in IFN-treated swine testicular cells, there was rapid cellular destruction. IFN-treated swine testicular cells had detectable levels of 2-5A (3.6 nM 6 h post-infection) after infection with 30 pfu TGEV/cell. Infection of neonatal pigs with a virulent strain of TGEV caused a significant increase in serum IFN and enterocyte 2-5A synthetase activity, as compared to control pigs. The level of enterocyte 2-5A synthetase in TGEV-infected pigs was increased 25-fold before viral-induced damage of the intestine consistently was present. 2-5A was not detected in enterocytes of either TGEV-infected or control pigs (less than 0.5 nM). Preparations of enterocytes contained two subpopulations of cells, one of which does not support replication of the virus. The considerable dilution of TGEV-infected cells (villous enterocytes) with uninfected cells (crypt cells) may be responsible for our inability to detect 2-5A in enterocytes from TGEV-infected pigs. These results indicate that the 2-5A system in porcine enterocytes and cultured testicular cells is modulated by TGEV infection and/or interaction with IFN.

2',5'-Oligoadenylate Synthetase↗
PubMed1988

Elevated serum IgA associated with immunoproliferative enteropathy of Basenji dogs: lack of evidence for alpha heavy-chain disease or enhanced intestinal IgA secretion.

Immunoglobulin isotypes in serum and intestinal secretions of Basenji dogs with chronic diarrhea, asymptomatic Basenji dogs, and healthy control dogs were quantitated and their molecular sizes characterized to detect alpha-chain, gamma-chain, or mu-chain fragments. Quantitation of immunoglobulin isotypes in serum showed that affected Basenjis have significantly elevated serum IgA values as compared to asymptomatic Basenjis and normal control dogs. However, IgA concentrations in intestinal wash fluids were not significantly different for the three groups. Immunoelectrophoresis (IEP) and polyacrylamide gel electrophoresis (PAGE) demonstrated that virtually all IgA was in the dimeric form. Using IEP and immunoselection, we were unable to detect evidence for the presence of alpha-chain or other heavy-(H)-chain fragments. Hyperimmune serum obtained from rabbits immunized with serum or a globulin fraction of affected Basenjis also failed to detect H-chain fragments. The results of this study indicate that immunoproliferative enteropathy of Basenjis resembles closely the nonsecretory form of human immunoproliferative small intestinal disease (IPSID).

Animals↗
PubMed1988

Comparison of virologic and serologic responses of lambs and calves infected with bluetongue virus serotype 10.

Four lambs and 3 calves, seronegative to bluetongue virus (BTV), were inoculated intravenously with a highly plaque-purified strain of BTV Serotype 10. A single calf and lamb served as controls and were inoculated with uninfected cell culture lysate. All BTV-inoculated lambs exhibited mild clinical manifestations of bluetongue, whereas infected calves were asymptomatic. Viremia persisted in BTV-infected lambs for 35-42 days, and for 42-56 days in BTV-infected calves. Neutralizing antibodies were first detected in sera collected at Day 14 post-inoculation (PI) from 2 BTV-infected calves and all 4 infected lambs, and at Day 28 PI in the remaining calf. The appearance of neutralizing antibody in serum did not coincide with clearance of virus from blood; BTV and specific neutralizing antibody coexisted in peripheral blood of infected lambs and calves for as long as 28 days. The sequential development, specificity and intensity of virus protein-specific humoral immune responses of lambs and calves were evaluated by immunoprecipitation of [35S]-labelled proteins in BTV-infected cell lysates by sera collected from inoculated animals at bi-weekly intervals PI. Sera from infected lambs and calves reacted most consistently with BTV structural proteins VP2 and VP7, and nonstructural protein NS2, and less consistently with structural protein VP5, and nonstructural protein NS1. Lambs developed humoral immune responses to individual BTV proteins more rapidly than calves, and one calf had especially weak virus protein-specific humoral immune responses; viremia persisted longer in this calf than any other animal in the study. The clearance of virus from the peripheral blood of BTV-infected lambs and calves is not caused simply by the production of virus-specific neutralizing antibody, however the intensity of humoral immune responses to individual BTV proteins might influence the duration of viremia in different animals.

Animals↗
PubMed1988

Bluetongue virus genome remains stable throughout prolonged infection of cattle.

Infection of three calves with a highly plaque-purified strain of bluetongue virus (BTV) resulted in prolonged infections, during which virus and neutralizing antibodies co-circulated in peripheral blood. Oligonucleotide fingerprint analyses of the original challenge virus and of the final virus isolate obtained from each calf demonstrated the BTV genome to remain stable throughout prolonged infection as no differences in fingerprint patterns were detected. Six neutralizing monoclonal antibodies (MAbs), and a polyclonal rabbit antiserum, were produced against the challenge virus. This panel of MAbs recognized at least two distinct neutralizing epitopes as demonstrated by immune precipitation. Neutralizing epitopes remained stable through the prolonged infections, as all MAbs and the polyclonal rabbit antiserum neutralized the challenge virus and the final calf isolates to equivalent titres. These results suggest that antigenic drift is not the mechanism by which BTV is able to persist in cattle in spite of a strong humoral immune response.

Animals↗
PubMed1988

Gastroenteritis of basenji dogs.

Intestinal digestive and absorptive function and the gross and histologic appearance of the gastrointestinal tract were evaluated in Basenji dogs with chronic diarrhea, asymptomatic Basenji dogs, and healthy control dogs. Gastric rugal hypertrophy, lymphocytic gastritis, and gastric mucosal atrophy occurred in asymptomatic and affected Basenji dogs. All affected dogs had moderate or severe intestinal lesions characterized by villous clubbing and fusion, increased tortuosity of intestinal crypts, and diffuse infiltration of mononuclear inflammatory cells. Intestinal lesions in asymptomatic Basenji dogs invariably were less severe than those in affected dogs, but the small intestinal lamina propria of asymptomatic Basenji dogs consistently contained greater numbers of mononuclear inflammatory cells than did that of control dogs. The proportion of cells containing each immunoglobulin isotype (IgG, IgM, IgA) was similar among affected Basenji dogs, asymptomatic Basenji dogs, and control dogs. As compared to healthy beagle controls, intestinal function was abnormal in both affected and asymptomatic Basenji dogs evaluated by combined N-benzoyl-L-tyrosyl-p-aminobenzoic acid and d-xylose test, but malabsorption and maldigestion were most pronounced in affected Basenji dogs.

Animals↗