Bovine pulmonary artery cells in cell culture were exposed to lipopolysaccharide (LPS) purified from Pasteurella haemolytica serotype A1. This resulted in severe membrane damage, which caused a time- and dose-dependent release of lactate dehydrogenase that was first detected 4 hours after exposure and reached a maximal mean release of 67% after 24 hours of exposure to 1 microgram of LPS/ml. Mean release of 51chromium followed by a similar pattern and reached a maximum of 61% following 24 hours of exposure to 10 micrograms of LPS/ml. Morphologically, endothelial cells responded to LPS by marked cell membrane retraction, the formation of numerous cytoplasmic blebs, and ruffling of the cell membrane. Subsequently, the cells became round and detached. Cell detachment reached a mean of 95% following 8 hours of exposure to 1 microgram of LPS/ml. These studies demonstrated that P haemolytica LPS is capable of causing direct damage to bovine pulmonary arterial endothelial cells, which may be important in the pathogenesis of bovine pneumonic pasteurellosis.

The amphotropic murine leukemia virus (MLV) has been shown to infect mammalian species other than mice. If this virus infects and expresses genes in cells of livestock species (cattle, sheep, and pigs) it has potential for use as a vector to produce transgenic livestock. Because the gene-injection technique for producing transgenic animals is inherently inefficient, our laboratory was interested in identifying or constructing retroviral vectors capable of infecting livestock embryos. The infectivity of an amphotropic MLV-based vector for ovine, bovine, and porcine cells was tested. Experiments were also conducted to test the ability of the amphotropic MLV promoter, compared with known strong promoters, to express genes in cells from these species. Results indicated that amphotropic MLV infects and expresses genes efficiently in porcine cells and is, therefore, a potential vector for producing transgenic pigs. Infection was not detected in cells from adult bovine and ovine species; however, low levels of infection, with subsequent gene expression, were detected in cells derived from bovine embryos.

The effects of Moraxella bovis on the morphologic features of purified bovine neutrophils and bovine corneal epithelial cells were examined, using transmission and scanning electron microscopy and light microscopy. Within 2 minutes after incubation of bovine neutrophils with living M bovis, electron microscopic cellular changes included vacuolation, swelling, and loss of microplicae. Most of the neutrophils were lysed by 10 minutes of incubation. Human neutrophils phagocytosed the M bovis and remained intact, even after 30 minutes of incubation with the bacteria. Living M bovis killed bovine corneal epithelial cells in vitro. Sterile filtrates prepared from 6-hour shaker cultures of M bovis also killed bovine corneal epithelial cells, but the cytotoxic activity was less than that produced by the living bacteria. Cellular changes were first observed in specimens collected 1 hour after corneal cell monolayers were inoculated with sterile culture filtrates. The changes in these cells included pit-like lesions on the cellular surface, cellular separation, and vacuolation.

Bovine pulmonary artery cells in cell culture were exposed to lipopolysaccharide (LPS) purified from Pasteurella haemolytica serotype A1. This resulted in severe membrane damage, which caused a time- and dose-dependent release of lactate dehydrogenase that was first detected 4 hours after exposure and reached a maximal mean release of 67% after 24 hours of exposure to 1 microgram of LPS/ml. Mean release of 51chromium followed by a similar pattern and reached a maximum of 61% following 24 hours of exposure to 10 micrograms of LPS/ml. Morphologically, endothelial cells responded to LPS by marked cell membrane retraction, the formation of numerous cytoplasmic blebs, and ruffling of the cell membrane. Subsequently, the cells became round and detached. Cell detachment reached a mean of 95% following 8 hours of exposure to 1 microgram of LPS/ml. These studies demonstrated that P haemolytica LPS is capable of causing direct damage to bovine pulmonary arterial endothelial cells, which may be important in the pathogenesis of bovine pneumonic pasteurellosis.

Turkey enteric coronavirus (TCV) from intestinal contents of diarrheal poults was isolated and serially propagated in HRT-18 cells, an established cell line derived from a human rectal adenocarcinoma. In these cells, TCV induced cytopathic changes, including polykaryocytosis, which depended on trypsin in the medium and incubation at 41 C. Viral antigens could be demonstrated in the cytoplasm by immunofluorescence, and extracellular virus was detected by an ELISA and negative electron microscopy. The cell-free virus had characteristics of TCV: shape, surface projections, buoyant density of 1.18 to 1.20 g/ml in sucrose, and hemagglutination of rat RBC. The one-step growth curve was complete by postinoculation hours 14 to 16, and maximal titers reached 9 to 9.5 log10 TCID50/ml during 5 passages, after which the titer remained stable. Electron microscopic examination of infected cell monolayers revealed budding of typical coronavirus particles through intracytoplasmic membranes and accumulation of complete virus in cytoplasmic vesicles. Late in the infection, aggregated progeny vial particles were detected near the outer surface of infected cells. One-day-old turkey poults inoculated orally with tissue culture-adapted TCV isolates developed mild to severe diarrhea.

Leukotoxin activity from culture supernatants of Pasteurella haemolytica serotype 1 in logarithmic growth phase caused rapid (less than 5 min) release of intracellular K+, uptake of extracellular Ca2+, and swelling of cultured bovine lymphoma cells (BL3 cells). Release of 51CrO(4)(2-) and lactate dehydrogenase (LDH) from BL3 cells began after 15 minutes of incubation with leukotoxin at 37 C and was completed between 60 and 120 minutes of incubation. In addition, leukotoxin exposure of BL3 cells resulted in cell aggregation and adherence to glass surfaces. Scanning electron microscopy indicated that after 10 minutes of leukotoxin exposure, BL3 cells increased in size, and large membrane defects developed between 20 and 60 minutes of exposure. The rate of release of LDH from leukotoxin-exposed BL3 cells was proportional to the amount of leukotoxin added. At high cell concentrations, the activity of LDH released at completion was directly proportional to the amount of leukotoxin added. Leukotoxin-induced release of LDH required a divalent cation, whereas K+ release and cell swelling did not. The addition of Ca2+, Mn2+, and Ba2+ resulted in increased leukotoxin-induced release of LDH. Divalent cation concentrations of 0.5 to 2.5 mM resulted in 50% of maximal stimulation. Ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid blocked increased release of LDH caused by Ca2+ addition, but had no effect on K+ release or cell swelling. Leukotoxin action on BL 3 cells (K+ release, cell swelling, Ca2+ uptake, and release of LDH) was prevented by incubation at 4 C.

Serum from dogs with surgically induced renal impairment was incorporated into the medium for erythroid bone marrow cultures. A significant correlation was found between serum activities of erythropoietin and numbers of erythroid colony-forming units grown in culture. Serum creatinine concentrations had no correlation, and serum parathyroid hormone activities had a negative correlation with numbers of erythroid colony-forming units that was below the level of significance. Purified 1-84 parathyroid hormone added to bone marrow cultures was found to be stimulatory to erythroid colony-forming unit growth in higher concentrations, but decreased the number of burst-forming units. Unmeasured substances in the canine serum appeared to have a greater effect on the canine erythroid bone marrow cultures than did creatinine or parathyroid hormone values.

Anaplasma marginale was propagated in a tick cell line derived from Dermacentor variabilis embryos. The rickettsial organism was identified and monitored in culture by transmission electron microscopy and the indirect immunofluorescence technique, using specific monoclonal antibodies. Inoculation of the embryonic tick cell line with midguts of infected adult ticks (culture 1), nymphal ticks (culture 2) and adult ticks that were infected as nymphs and dissected as adults (culture 3) resulted in 3 continuous cultures of A marginale. Culture 1 had been maintained through 22 passages over a 11-month period; cultures 2 and 3 had been maintained for 18 passages over a 9-month period. Growth of A marginale in the cell line began in the area of the nuclear membrane at approximately 4 days after inoculation or transfer. Thereafter, the organisms were observed in inclusions scattered throughout the cytoplasm of the host cells. Maximal growth of the organism occurred at 7 to 14 days, after which numbers of inclusions rapidly decreased to minimal or undetectable levels. The organism began new cycles of growth with each 1:5 to 1:10 split and transfer of the host cells. Electron microscopy of recently infected cells revealed a morphology of the organism that closely resembled that observed in marginal bodies of infected erythrocytes. After several passages, A marginale organisms had a varied morphology and resembled the organism described in midgut cells of naturally infected ticks. Substitution of adult bovine serum for fetal bovine serum and adjustment of the pH of the medium from 6.9 to 7.4 resulted in several-fold increases in amount of growth and reduced the period required to reach maximal growth to a predictable time of 5 to 7 days. The importance and potential of this method of continuous laboratory propagation of A marginale are discussed.

The use of cultured tissue has not yet become wide-spread in research involving equine disease, and this may be attributable in part to the scarcity of published reports concerning tissue culture methods for this species. We report here the isolation of equine microvascular endothelium (EMVE) from fresh omental tissue of horses and ponies. Fresh donor tissue was minced, subjected to collagenase digestion, and filtered. Cells were layered on 5% bovine serum albumin for gravity sedimentation, the bottom layer was collected, and the cells were plated onto fibronectin-coated flasks. Medium consisted of Dulbecco modified Eagle medium with 10% whole fetal bovine serum (wFBS) and 20 micrograms of endothelial cell growth supplement/ml. The EMVE grew readily in culture, had the cobble-stone morphologic feature at confluence, stained positively for factor VIII-related antigen, and metabolized acetylated low-density lipoprotein. Fibroblast and smooth muscle cell contamination was minimal in primary cell cultures, which were successfully passed and maintained in culture for 3 to 5 serial passages, using various media and substrates. Preliminary studies were undertaken to determine optimal growth conditions with a range of variables: serum concentration, extracellular matrix components, and growth factors. Optimal conditions were achieved with a minimum of 10% wFBS, and with either fibronectin or laminin as extracellular matrix substrates. The EMVE grew adequately in Dulbecco modified Eagle medium plus 10% wFBS, and the added growth factors or serum supplements did not appear necessary for growth of EMVE.

The infectivity and immunogenicity of Anaplasma marginale grown in a tick cell culture from embryonic Dermacentor variabilis ticks were assessed in splenectomized and intact calves, respectively. Culture 1 consisted of the cell line inoculated with midguts of adult ticks infected with the Mississippi isolate of A marginale and dissected 5 to 10 days after repletion and detachment from an experimentally infected calf. Cultures 2 and 3 consisted of the cell line inoculated with midguts of ticks infected with the Virginia isolate of the organism. Inoculum for culture 2 was derived from nymphal ticks dissected 5 to 10 days after repletion and detachment from the infected calf; inoculum for culture 3 was midguts from adult ticks that were fed as nymphs, allowed to molt in the laboratory and dissected 21 to 24 days after molting. In trial 1, cultures 1, 2, and 3 were maintained at pH 6.9 and incubated at 28 C; in trial 2, cultures 1 and 3 were maintained at pH 7.4 and incubated at either 28 C or 37 C. Cultures 1, 2, and 3 failed to induce infection when injected IV and SC into 6 calves in 2 separate trials. Prechallenge sera from these calves reacted with 2 purified Anaplasma antigens in the ELISA, but failed to react in the complement-fixation test. Results of a trial to use cultures 1 and 3 in combination with an oil-in-water adjuvant to immunize intact calves against A marginale were inconclusive. However, prechallenge sera from immunized calves reacted with the 2 purified Anaplasma initial body antigens in the ELISA but failed to react in the complement-fixation text. When reacted against electrophoretically separated A marginale initial body proteins disrupted by sodium dodecyl sulfate, prechallenge serums from calves used in infectivity and immunization trials reacted with a majority of the antigens precipitated by an animal experimentally infected by inoculation of infected blood. This offers additional evidence that A marginale was maintained in the tick culture for up to 11 months and that the organism in culture antigens similar, if not identical, to the erythrocytic stage of the rickettsial agent. The importance of the laboratory culture of A marginale is discussed.