Lymphocyte blastogenesis and cellular cytotoxicity in a congenital infection of bovine fetuses related to epizootic bovine abortion

The disease referred to as epizootic bovine abortion (EBA) was experimentally induced in bovine fetuses. Dark-field microscopy was used to detect congenital infection with an unclassified spirochaete-like organism. Some of the fetuses collected at abattoirs were also found to be naturally infected with a morphologically similar microorganism. Blood counts and organ weights were correlated with the presence of the microorganism. Lymphocyte blastogenesis increased, the result of in vivo stimulation among the infected fetuses. Phytomitogens (phytohaemagglutinin, concanavalin A and lipopolysaccharide) also stimulated greater responses in infected fetuses when compared to results in normal fetuses. Cellular cytotoxicity was examined by the single cell assay and results indicated that there were fewer cytotoxic lymphocytes among the diseased fetuses. The infected abattoir-collected specimens were obtained from clinically normal adult cattle, and the immunological changes in these fetuses were closely characterised with those of the EBA diseased fetuses. These naturally infected fetuses showed signs of a mild infectious disease.     

Monoclonal antibodies that distinguish bovine T and B lymphocytes

The specificities of three monoclonal antibodies (MAbs) were investigated using microcytotoxicity, fluorescence microscopy and laser flow cytometry (LFC) techniques. By microcytotoxicity, bovine thymocytes (n = 4) were estimated to be 85% B26A+, 4% TH21A+, and 1% H4+. Nylon wool enriched peripheral blood T lymphocytes (n = 3) were 90% B26A+, 10% TH21A+ and 10% H4+. Adherent B cell enriched fractions (n = 3) were 10% B26A+, 90% TH21A+ and 90% H4+. The two fluorochrome method was used to simultaneously identify lymphocytes that were sIg+ and MAb+. In these experiments, 92% of all sIg+ cells were H4+. An identical result was obtained for TH21A. 85% of all sIg- cells were B26A+. Using LFC, the mean percentages of sIg+, H4+ and TH21A+ PBL (n = 5) were not significantly different. B26A recognized a significantly greater population of cells, equivalent to the expected percentage of T lymphocytes. LFC also revealed two relatively discrete sizes of B26A+ PBL. The larger population overlapped the size range in which sIg+, H4+, TH21A+ PBL were found. The more numerous smaller B26A+ PBL were in a size range in which few sIg+, H4+ and TH21A+ PBL were found. In a study of MAb reactions with PBL of 185 cows, it was shown that in 92% of the animals H4 and TH21A were positively correlated (r = +.93), when H4 and TH21A were negatively correlated with B26A (r = -.94 and r = -.92, respectively). These correlation coefficients indicate a converse relationship between B26A and both H4 and TH21A. The remaining 8% of the animals were heterogeneous in their expression of the H4 and TH21A markers but not the B26A marker. These results provide strong evidence that: 1) B26A is a pan-T lymphocyte MAb in cattle, 2) a small but significant degree of heterogeneity exists in the expression of the epitopes recognized by H4 and TH21A. However, both MAbs recognize all B lymphocytes of most individuals, and 3) using a variety of immunological methods these three MAbs can now reliably be used to assay bovine T and B lymphocytes.     

Peanut agglutinin (PNA): binding and stimulation of bovine intestinal and peripheral blood leukocytes

The use of peanut agglutinin (PNA) as a reliable marker for bovine T lymphocytes as well as its in vitro lymphoblastogenic capacity were investigated and compared to those of concanavalin-A (ConA). The binding ability of fluorescein isothiocyanate conjugated PNA (FITC-PNA) and FITC-ConA to bovine leukocytes isolated from peripheral blood (PBL) as well as from the intraepithelium (IEL), lamina propria (LPL) and Peyer's patches (PPL) of the small intestinal mucosa of five normal adult cows (n = 5) was analyzed using laser flow cytometry (LFC) and fluorescence microscopy. Monoclonal antibodies (mAb) specific for bovine T cells (B26A), B cells (PIg45A), "null" cells (B7A1) and monocytes/granulocytes (DH59B) were employed to determine the phenotype of the cell lineage(s) expressing PNA surface receptor(s). There were no significant variations (P greater than 0.1) in the proportion of PNA-binding cells in PBL (76%), PPL (77%), IEL (79%) and LPL (81%) even though there were significant differences between the percentages of B26A+ T cells in IEL (26%) and LPL (38%) (P less than 0.001) and in PPL (44%) and PBL (57%) (P less than 0.01). These studies clearly indicate that cells other than T cells bind PNA. Although the proportions of PNA-binding cells in enriched PP-B cells (30%) and enriched PP-plastic adherent cells (44%) were significantly lower (P less than 0.001) than those in enriched PP-T cells (95%), the results indicated that a reasonable number of non-T cells can specifically bind FITC-PNA. Additional support was obtained by similar results observed with the equivalent cell subsets from PBL. Using in vitro lymphoblastogenesis, the PNA stimulating capacity significantly varied between the various cell populations (P less than 0.001 between IEL and PBL; and P less than 0.02 between PPL and PBL). In addition, marked differences were observed between the binding ability and stimulating capacity of PNA on each leukocyte population (P less than 0.01 in PBL to P less than 0.001 in IEL). Concanavalin A which bound to approximately 100% of each cell population, revealed significant variation in its mitogenic activity between IEL and PBL (P less than 0.001) but not between LPL and PPL (P greater than 0.1). The finding that PNA can bind to bovine T cells as well as to some B cells, monocytes/macrophages and possibly some granulocytes and "null" cells disputes its reliability as a specific bovine T cell marker. Furthermore, the binding abilities of PNA and ConA to bovine leukocytes are not necessarily correlated to their in vitro mitogenic capacities.     

Wheat germ agglutinin (WGA): a bovine T lymphocyte marker

Bovine peripheral blood lymphocytes (PBL) were examined for their ability to bind wheat germ agglutinin (WGA). This lectin labelled 43.8% +/- 11.95 of bovine PBL, whereas peanut agglutinin (PNA), a T cell marker, bound 59.4% +/- 8.67 cells, and surface immunoglobulin (SLG)-bearing cells constituted 24.15% +/- 8.47 of PBL. After panning fractionation of B (Slg+) and T (PNA+) lymphocytes. WGA labelled 89 to 97% of the enriched T cell population (80/87% PNA+; 2-4% Slg+) but only 6 to 8% of the enriched B cell population (85-91% Slg+; 5-7% PNA+).     

Non-immune erythrocyte rosette formation of bovine peripheral blood and thymus lymphocytes

An E-rosetting reaction is described which gave 92.1%±2.4 (mean±S.D.) E-rosettes with bovine fetal thymocytes and 48.2%±8.4 with with bovine peripheral blood leukocyte (PBL) preparations. Both culture conditions and culture medium were critical factors in obtaining maximal and reproducible E-rosette numbers. Optimum rosette formation occurred when bovine PBL and neuraminidase treated sheep erythrocytes (nSRBC) were reacted in L-15 culture medium supplemented with 10% fetal calf serum (FCS). Other media including 100% FCS, MEM with 10% FCS, and RPMI-1640 with 10% FCS were less satisfactory. Cultural conditions found to be optimal for enumeration of bovine E-rosettes are similar to those reported as optimal for detection of human T cells. The specificity of rosette formation by bovine thymus derived (T) lymphocytes was shown by demonstration of (1) rosettes and surface membrane immunoglobulins (mIg) on different cells in PBL, (2) rosette formation by the majority of fetal thymocytes, and (3) no inhibition of rosette formation by anti-immunoglobulin serum. Using the E-rosette and mIg assays for presumptive bovine T and B lymphocytes, respectively, it was possible to differentiate from 57.5 to 90% (75.2%±9.3) of cells in bovine PBL preparations, and from 90.2 to 97.5% (94.2%±2.1) of cells in bovine fetal thymocyte preparations into T and B cells.     

Bovine T lymphocyte response to bovine herpesvirus-1: cell phenotypes, viral recognition and acid-labile interferon production

The phenotype of bovine cells that proliferate to bovine herpesvirus (BHV-1) were identified by peanut agglutinin (PNA) and monoclonal antibodies with specificity for Pan T cells (B29), a T cell subset (B24), and an MHC class II (Ia-like) antigen (R-1). PNA+ cells but not PNA- cells separated by fluorescent activated cell sorting responded to BHV-1. Virally-activated T cells expressed MHC class II antigens, and possibly antigen specific receptors for virus. Binding of radiolabeled virus increased six-fold beyond the expected value for activated cells suggesting specificity of the lymphocytes for the virus. Finally, cells that responded to BHV-1 produced high levels of acid sensitive interferon. Taken together these results characterize the phenotypes of bovine lymphocytes that interact with BHV-1.     

T and B peripheral blood lymphocytes in normal and lymphocytotic sheep

Surface immunoglobulins (SIg), Peanut Agglutinin (PNA), spontaneous erythrocyte rosette (E-rosette) and Helix pomatia (HP) marker were investigated in normal and Bovine leukemia virus (BLV)-infected sheep. In normal sheep, 19.3% +/- 4.9 of peripheral blood lymphocytes (PBL) were SIg+, whereas 58% +/- 5.69 were PNA+, and 19.6 +/- 5.2 were E-rosette forming cells (E-RFC). In BLV-induced lymphocytotic sheep, SIg+ cells in PBL reached 59.4% +/- 15.06. In the same animals, PNA bound to 20.6% +/- 9.69 and E-RFC were 8.7% +/- 4.5. A panning technique was applied with an anti sheep-immunoglobulins coated plates to separate SIg+ (adherent cells = A) and SIg- cells (non-adherent cells = NA). The (A) population was 94-95% SIg+ cells and 2-3% PNA+, while the (NA) population was 0-4% SIg+ and 79-85% PNA+ cells. Thus PNA is a T cell marker in sheep species. HP, a marker for bovine T lymphocytes was also studied. Sheep PBL do not bind to HP. However, after panning separation about 50% of NA cells became HP+.     

Histopathologic changes in bovine fetuses after repeated reintroduction of a spirochete-like agent into pregnant heifers: association with epizootic bovine abortion

A spirochete-like organism was found in the plasma of bovine fetuses affected with epizootic bovine abortion (EBA). The spirochete-like organism was frequently found in abattoir-collected fetuses as an inapparent infection, and EBA was found in cattle on foothill rangeland where the vector tick Ornithodorus coriaceus could repeatedly reintroduce the infectious agent into pregnant cattle (superinfection). Epizootic bovine abortion resembled a naturally acquired superinfection in circumstances where the agent was frequently present in the environment under conditions favoring transmission. Therefore, to determine whether fetal lesions could be experimentally induced in utero, spirochete-like organisms collected from clinically normal fetuses at an abattoir were inoculated IV and subcutaneously into 2 pregnant heifers 5 times over a 4-month period to mimic repeated tick transmission in the field. Macroscopic and microscopic examinations of tissues from 2 cesarean-collected fetuses and from 3 calves born at term with the naturally acquired spirochete infection indicated that the calves had evidence of an infection that caused morphologic changes compatible with immunologic stimulation and mild reticuloendothelial hyperplasia. Compared with findings in the calves, lesions in the superinfected fetuses were more severe, and the lesion distribution in various organs was more extensive. The spirochete-like organism appeared to be a mild pathogen because of its persistence in the host. Clinical disease from the infection may only develop with repeated superinfections. Therefore, a relationship between this microorganism and EBA is probable.     

Detection of B and T cells, with lectins or antibodies, in healthy and bovine leukemia virus-infected cattle

Lectins, polyclonal antibodies and monoclonal antibodies (MAbs) were evaluated as markers for bovine lymphocytes obtained from healthy animals and from cattle infected with bovine leukemia virus (BLV). In the blood from healthy cattle the proportion of cells identified as T lymphocytes with the lectin Helix pomatia (HP) (67.8 +/- 6.2%) using the indirect immunofluorescence technique was similar to the proportion of cells identified by the MAbs P5 (66.1 +/- 3.8%) and BLT-1 (59.8 +/- 7.1%). The proportion of B cells in blood from healthy animals identified with a polyclonal antibody to bovine IgM (18.0%) was similar to that identified with a MAb to bovine IgM (16.2%). However, greater variation between individual values was detected with the MAb (SD = 8.2) than with the polyclonal antibody (SD = 4.0). In the blood from BLV-infected cattle with persistent lymphocytosis, both the polyclonal and the MAb revealed a threefold increase of B cells. A proportion of the B cells had an increased amount of immunoglobulin molecules in their plasma membrane as indicated by flow cytometry. The proportion of T lymphocytes, identified by the MAb P5, was reduced to one-third of that in non-infected cattle. The indirect HP labelling gave inconsistent results and seems not to detect solely T lymphocytes among blood lymphocytes from BLV-infected cattle.     

Erythrocyte rosettes--a marker for bovine T cells.

Many species of erythrocytes were investigated for their ability to form spontaneous rosette with bovine peripheral blood leukocytes and fetal thymocytes. Only sheep and chicken red blood cells gave rosettes. Using conditions shown optimum for the demonstration of human rosette forming cells, only low numbers of bovine rosettes were demonstrable. By changing culture conditions to include 100% fetal calf serum, neuraminidase treated erythrocytes and/or lymphocytes and optimizing the incubation times and temperature, up to 38% of peripheral blood leukocytes and 52% of thymocytes formed rosettes. A thymic origin of rosetting cells was ascribed to T cells for the following reasons: 1) thymocytes gave higher numbers than did peripheral blood leukocytes, 2) rosette forming cell numbers were increased in peripheral blood leukocyte subpopulations enriched in T cells by nylon column separation and 3) only very few rosette forming cells had surface immunoglobulin, a marker of B lymphocytes. The reasons why all T cells were not detected by the technique were discussed.     

Cattle infected with bovine leukaemia virus may not only develop persistent B-cell lymphocytosis but also persistent B-cell lymphopenia

We investigated the distribution of B and T cells in the peripheral blood of haematologically inconspicuous (non-persistent lymphocytotic, PL-) cattle infected with the bovine leukaemia virus (BLV). Flow cytometric data were obtained from six PL- cattle and compared with six age-matched animals with persistent lymphocytosis (PL+) and five non-infected healthy controls (BLV-). In the PL- group, the percentage and number of surface immunoglobulin-positive (sIg+) B cells were significantly reduced. Whereas in BLV-cattle, about 40% of the peripheral blood lymphocytes (PBL) were sIg + and 24% were sIgM + B cells. In the PL- group, less than 20% of the PBL were sIg+ and sIgM+ B cells. Only 5% of the PBL co-expressed sIgM+ and CD5+ versus 16% in BLV-. This decrease was persistent over 3 years and predominantly affected: (i) B cells that did not express sIgM; (ii) sIgM + B cells co-expressing CD5 and CD11b; and (iii) equally both lambda- and K-type light chain B-cell subpopulations. In contrast, the number of all circulating lymphocytes, CD5- and CD11b- sIgM+ B cells and CD2+ T cells did not differ. In PL+ animals, about 75% of the PBL were sIgM+ CD5+ B cells. These cells were of polyclonal origin, as light chains of the lambda- and K-type were expressed in a ratio of 4:1 (57.7% of PBL lambda+, 14% kappa+) as in BLV- animals (33.6% of PBL lambda+, 8.7% kappa+). In PL+ cattle the absolute number of B-cells and, therefore, their relative percentage is significantly increased. For this reason, even in case of absolutely increased T-cell numbers, the relative percentage of T-cells could be lower than in normal controls. The cause for the observed B cell decrease in PL- cattle is unknown, but it can be assumed that cytotoxic T cells are involved in this B-cell lymphopenia.     

Immunohistochemical studies on ontogeny of bovine lymphoid tissues.

Developing lymphoid tissues of bovine fetuses ranging from 70 to 270 days of fetal age were examined by histological and immunohistochemical procedures. In the peripheral blood, surface membrane immunoglobulin bearing cells (B-lymphocytes) and sheep red blood cell rosette forming cells (T-lymphocytes) had already appeared by 70 days of fetal age. In the lymph nodes intracytoplasmic IgM positive cells appeared at 90 days of fetal age. The cells positive for IgG appeared at 150 days of fetal age and IgA positive cells appeared at 180 days of fetal age. The spleen contained intracytoplasmic immunoglobulin positive cells at almost the same time as those in the lymph nodes. In the ileocecal region, IgM positive cells and IgG positive cells were present at 180 days of fetal age and IgA positive cells were present at 210 days of fetal age. The tonsils contained IgM positive cells and IgG positive cells at 240 days of fetal age. In the thymus, terminal deoxynucleotidyl transferase positive cells appeared at 90 days of fetal age.     

Brucella abortus-associated meningitis in aborted bovine fetuses.

Granulomatous meningitis was present in 6/33 bovine fetuses from which Brucella abortus (B. abortus) had been isolated. Meningitis was severe in three fetuses, moderate in one fetus, and mild in the remaining two fetuses. The meningitis was characterized by the infiltration of a mixed population of lymphocytes, plasma cells, and macrophages in the leptomeninges. Vasculitis characterized by the infiltration of lymphocytes and plasma cells in the vascular wall was observed in the vessels of the cerebral cortices of 4/6 fetuses. Gram negative coccobacilli were present in the cytoplasm of the leptomeningeal macrophages and extracellularly. Brucellar antigens labeled by the avidin-biotin-peroxidase complex method were present in massive amounts in leptomeningeal macrophages and in small foci of stained cells in the choroid plexus and ependyma. The findings indicate that B. abortus is one of pathogens capable of inducing meningitis in bovine fetuses.     

Separation and identification of bovine lymphocyte populations

Various methods for separation of lymphocyte populations have been modified and adapted for use in isolating and identifying bovine lymphocytes. Ficoll diatrizoate (F-D) with a specific density of 1.084 was found to be superior to those with densities of 1.080 and 1.077 which were developed originally for the mouse and human mononuclear cells, respectively. F-D with a density of 1.084 attained a lymphocyte (absolute number) recovery rate of 92% whereas those with densities of 1.080 and 1.077 yielded 81% and 71% recovery rate of lymphocytes, respectively. Subsequent separation of T lymphocytes was achieved best by nylon wool column whereas separation of B lymphocytes was attained best by complement-mediated depletion of T lymphocytes with the T lymphocyte specific monoclonal antibody (MAb), BLT-1. The former yielded 95 +/- 3% T lymphocytes with 47 +/- 9% recovery rate, and the latter gave 96 +/- 3% B lymphocytes with 71 +/- 9% recovery rate. In comparison, direct panning of F-D gradient separated mononuclear cells with goat anti-bovine IgG coated plates yielded 80% B lymphocytes with 31% recovery rate and indirect panning of MAb BLT-1 treated F-D gradient-separated mononuclear cells with goat anti-mouse IgG coated plates yielded 89% T lymphocytes with 35% recovery rate.     

Characterization and separation of bovine lymphocyte populations

Bovine lymphocyte populations were characterized by surface markers, rosette-forming ability and behaviour towards mitogens. After pre-treatment with neuraminidase 16% of the bovine blood lymphocytes and 14% of the bovine spleen cells formed spontaneous (E) rosettes with sheep erythrocytes. About 20% EAC rosette-forming cells were detected among both cell populations. Protein A receptors were detectable among 8% of the blood lymphocytes and 26% of the spleen cells. Bovine lymphocytes responded to pokeweed mitogen (PWM), phytohemagglutinin (PHA) and concanavalin A (Con A). An enrichment of bovine B and T cells was obtained by E-rosette sedimentation (81–84% B cells) and by filtration through nylon fiber columns (51–65% T cells). The T cells obtained after nylon filtration still responded to the mitogens PHA, Con A and PWM. Enriched B-cell populations responded to bacterial lipopolysaccharide (LPS). After monocyte depletion the mitogenic response of blood lymphocytes was not influenced.     

Experimental fetal infection with bovine viral diarrhea virus. I. Virological and serological studies.

The serological and virological results of an experimental infection of bovine fetuses with bovine viral diarrhea virus are presented. Four fetuses, 120-165 days gestational age, were inoculated in utero with a second passage virus strain. Two fetuses received a sham-inoculum. A humoral immune response in the virus-inoculated fetuses, was demonstrated three weeks later. In three fetuses only IgM and IgG1 were detectable. The serum from the fourth fetus also contained IgG2 and IgA. Bovine viral diarrhea virus-neutralizing antibodies were detected in two fetuses. These two fetuses inoculated at 135-150 days gestational age, represent the youngest reported bovids, giving a specific response in three weeks following an experimental infection with bovine viral diarrhea virus. The fetal sera did not contain heat-labile factors, which could mediate the neutralization. The virus was not reisolated from any of the fetuses, but viral antigen was nevertheless demonstrated by immunocytochemical methods in sections of several of the fetal organs, primarily lymphoid tissues.     

Time of appearance of esterase-positive alveolar macrophages in bovine fetal lung

Bovine fetal lung tissue was examined histochemically, using alpha-naphthyl butyrate (nonspecific method), to detect the presence of esterase-positive pulmonary alveolar macrophages (PAM). Positively stained PAM were first seen in the alveolar septa and lumina of the lungs of 4 of 8 fetuses 240 days old. Macrophages (n = 100) in 240-day-old fetuses were 6.74 +/- 0.07 microm, and in most cells, esterase-positive granules were sparsely distributed in the cytoplasm. In 10 fetuses 250 days old, the alveolar septa and lumina of lungs contained positively stained macrophages, as did those of all older fetal lungs examined. Compared with macrophages of the 240-day-old fetuses, those (n = 50) of 250-day-old fetuses were significantly larger, 7.32 +/- 0.10 micron (P less than 0.0001), and esterase-positive granules were heavily distributed throughout the cytoplasm. In all fetal lungs, T lymphocytes contained a single, small, esterase-positive granule, compared with the numerous diffuse esterase-positive granules in PAM. Type-II pneumonocytes with well-developed lamellar bodies had no evidence of positive nonspecific esterase reaction in lungs.     

Engraftment of severe combined immune deficient/beige mice with bovine foetal lymphoid tissues.

To develop a model of bovine thymus and lymph node growth in vivo, we have implanted bovine foetal tissues (16-23 weeks gestation) under the renal capsule of severe combined immune deficient (SCID)/beige (BG) mice and assayed for graft growth and characteristics 2-18 weeks after engraftment. Bovine foetal thymus and lymph node grew considerably following engraftment of SCID/BG mice. Growth was optimal if bovine foetal tissues were used before gestation Week 17. Bovine-mouse chimerism was confirmed using glucose phosphate isomerase analysis. Bovine thymus grew during the entire 18 weeks of study. Growth of bovine lymph node was initially rapid, reaching a maximum at 2 weeks after transplantation followed by a progressive decrease in size. Transplanted bovine lymph node and thymus were morphologically similar to age-matched bovine foetal tissue for a limited time period. Fibrosis, degeneration and depletion of lymphocytes were evident 6 weeks after engraftment; changes were more severe in lymph node than in thymus whereas increases in lymphocytes, lymphopoiesis and follicle formation were evident in age-matched bovine foetal tissue. Despite growth and morphological similarities of the transplanted tissue, blood counts suggested there was no peripheralization of bovine leucocytes. Bovine immunoglobulins (IgG1 and IgG2) were detected in serum of some SCID/BG chimeric mice for a limited time. The appearance of bovine immunoglobulins at 2 weeks in SCID/BG chimeric mice depended on the age of the foetal donor (> 18 weeks) and coincided with the appearance of morphologically mature lymphocytes in the donor foetus lymph nodes. The ability to produce bovine immunoglobulins decreased 8 weeks after engraftment, coinciding with the depletion of lymphocytes in the engrafted lymph node. Lymphocyte depletion and loss of function of engrafted tissues appear the result of a lack of lymphoid progenitors normally derived from hematopoietic stem cells in the bone marrow.     

Partial characterization of bovine leukemic cell populations

Peripheral blood lymphocytes (LL) from cows with chronic lymphocytic leukemia (CLL) have been studied using a number of surface markers. Cell populations were obtained by partial enrichment and depletion using Sephadex G-200-anti-F(ab')2 and Sephadex G-200-anti-LL-Ig immunoadsorbent columns. It was shown that cell populations having different markers, i.e., surface immunoglobulins (sIg) and leukemia-associated antigens (LAA), are characterized by similar parameters. Thus the adherent cells obtained by both fractionation methods formed 1.1-4.0% of rosettes with sheep red blood cells, while 90.6-94.3% were sIg positive cells. The cytotoxicity test (CTT) performed with anti-B and anti-LL sera indicated that a vast majority of adherent cells reacted to the sera. Thus the adherent cells represent a population with a high percentage of B-cells.