Experimentally induced infectious bovine rhinotracheitis virus infection during early pregnancy: effect on the bovine corpus luteum and conceptus

Pairs of heifers were inoculated IV with infectious bovine rhinotracheitis virus on postbreeding days (PBD) 7, 14, 21, or 28, and were euthanatized 13 to 15 days after inoculation. Reproductive tracts were examined for cytopathologic changes (light microscopy), virus (cell culture), and viral antigen (immunohistochemical evaluation). Heifers inoculated on PBD 7 or 14 had mild oophoritis characterized by foci of necrosis and mononuclear cell accumulations in the corpus luteum. Most of these heifers also had a few necrotic follicles in at least one ovary. Heifers inoculated on PBD 21 or 28 did not have corpus luteum lesions, but necrotic follicles were numerous in both ovaries. Viral antigen was observed in all ovarian lesions, and infectious virus was isolated from a few of the affected tissues. The uteri of all heifers inoculated on PBD 21 or 28 and 1 heifer inoculated on PBD 7 contained normal-appearing concepti. The uterus of the other PBD 7 heifer contained a degenerating conceptus that was infected with infectious bovine rhinotracheitis virus, as determined by viral isolation, immunohistochemical evaluation, and electron microscopy. Heifers inoculated on PBD 14 were not pregnant at necropsy, but histologic evidence was found that the postbreeding estrous cycle had been longer than normal, indicating that early embryonic death had occurred.     

Standardization and kinetics of in vitro bovine blood lymphocyte stimulation with bovine rotavirus

Two groups of 3-month old calves were immunized intramuscularly with attenuated bovine rotavirus and boosted 21 and 42 days later. The first group of three calves were vaccinated with live virus emulsified with incomplete Freund's adjuvant (IFA) and the second group was immunized with live virus suspended in phosphate buffered saline (PBS). Three other calves, serving as controls, were inoculated with PBS emulsified with IFA. The specific cell-mediated and antibody responses of the animals were studied. Preliminary analysis of in vitro peripheral blood lymphocyte transformation to bovine rotavirus determined optimal conditions as: 96 h culture period, 5 X 10(5) cells per culture in RPMI 1640 medium containing 10% heat-inactivated bovine fetal serum and the use of inactivated virus in the cell culture at a concentration of 5 X 10(6) median tissue culture infective dose before inactivation. Specific blastic stimulation was observed on calves immunized with the rotavirus emulsified with IFA after the second and third vaccine inoculation with stimulation index values varying from 2.00 to 5.73. Serum neutralizing antibody titers of 1/25,600 were also induced in the same calves. Calves immunized with rotavirus-PBS suspension developed a mean antibody titer of 1/1,600, but showed no specific lymphocyte stimulation. No increase in specific immune responses was detected in the control animals.     

Inhibitory effect of African swine fever virus on lectin-dependent swine lymphocyte proliferation

The incubation of swine peripheral blood mononuclear cells (PBMC) with African swine fever (ASF) virus preparations strongly inhibited the proliferative response of lymphocytes to PHA and other lectins. The inhibition, which persisted after inactivation of the virus by UV radiation, was dependent upon the dose and the time that virus preparations were present in cultures. When virus preparations were fractionated by ultracentrifugation, the inhibitory activity resulted to be soluble, whereas no activity was found in the sedimented viral fraction. However, the preincubation during 4 days of this sedimented fraction with swine PBMC, before the addition of the mitogen, restored the inhibitory activity. The results obtained suggest that the inhibition is mediated by one or more soluble factors released by swine PBMC after coincubation with ASF virus in a time dependent process. These factors show a molecular weight between 40 and 80 kDa by gel filtration chromatography. The inhibitory activity described in the present paper is an indication of inhibition of lymphocyte function produced by ASF virus which can help to understand how this virus escapes from the host immune system.     

In vitro stimulation of bovine peripheral blood lymphocytes: effect of short-term storage of blood prior to lymphocyte culture

Storage of peripheral blood from Mycobacterium bovis-sensitized cattle from 1 to 48 hours at 4, 22, and 37 C was shown not to alter markedly the lymphocyte blastogenic response to M bovis-purified protein derivative. Concanavalin A-induced lymphocyte mitogenic responses were unaffected by storage of blood for 1, 24, or 48 hours at 22 C and 37 C; however, storage of blood for 48 hours at 4 C significantly lowered (P less than 0.05) mitogenic responses to concanavalin A, as compared with responses to blood kept at 22 C. Mononuclear cell recovery from stored blood at all temperatures was markedly less than that from freshly drawn blood samples. Cell recoveries were most affected by storage of blood at 4 C and 37 C.     

Ultrasonographic appearance of the bovine conceptus from days 10 through 20

The bovine conceptus was monitored in 19 heifers by intrarectal ultrasonic imaging from the day an embryonic vesicle was first detected (mean, day 11.7 +/- 0.4; range, days 10 to 17) until detection of the embryo proper (mean, day 20.3 +/- 0.3). Fifteen heifers maintained the conceptus and 4 heifers apparently lost the conceptus. In the heifers that maintained the conceptus, 73% of the embryonic vesicles were classified as spherical (mean diameter, 2.8 +/- 0.2 mm) and 27% were classified as oblong (mean dimensions, 2.0 +/- 0.0 mm and 4.5 +/- 1.0 mm) on the day of first detection. All vesicles were in the uterine horn ipsilateral to the corpus luteum. The vesicles increased in length from the day of first detection. On the average, the vesicle occupied all of the ipsilateral uterine horn on day 16.9 +/- 0.6 and all of the contralateral horn on day 19.6 +/- 0.9. During elongation, the vesicle remained at an approximate height of 2 mm throughout its length, but developed a localized enlargement or bulge on mean day 19.7 +/- 0.2. The embryo proper was detected within the bulge in all 15 heifers. A heartbeat (mean, 188 +/- 4.8 beats/min) was detected on the first day of detection of the embryo proper (8 heifers) or on the following day (7 heifers). The mean length of the interovulatory interval in the 4 heifers that apparently lost the embryonic vesicle was not significantly different from that of nonbred heifers. The vesicles were lost (not ultrasonographically detectable) on days 17 (2 heifers) and 19 (2 heifers).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrasonographic appearance of the bovine conceptus from days 20 through 60

The ultrasonographic appearance of the conceptus on days 20 to 60 was determined by daily examinations in 15 heifers. The mean length of the embryo proper increased from the day of first detection (day 20.3 +/- 0.3; 3.8 +/- 0.3 mm) to day 60 (66.1 +/- 1.7 mm). The growth curve was quadratic, with an increasing growth rate after approximately day 50. The embryo initially had the appearance of a short, straight line and developed a C shape on day 25.4 +/- 0.8 and an L shape on day 32.7 +/- 1.1. The average day of first detection of various structures was as follows: allantois, 23.2 +/- 0.3; spinal cord, 29.1 +/- 0.5; forelimbs, 29.1 +/- 0.3; amnion, 29.5 +/- 0.5; optic area, 30.2 +/- 0.4; hindlimbs, 31.2 +/- 0.3; placentomes, 35.2 +/- 1.0; optic lens, 40.0 +/- 0.6; split hooves, 44.6 +/- 0.7; fetal movements, 44.8 +/- 0.8; and ribs, 52.8 +/- 0.5. Embryonic death, as indicated by cessation of heartbeats, occurred in one heifer on day 26. The embryonic mass increased in echogenicity and was maintained with a gradually decreasing fluid volume until the occurrence of estrus 17 days after death.     

Effects of Hemophilus somnus on the pregnant bovine reproductive tract and conceptus following cervical infusion

Three strains of Hemophilus somnus were infused into the posterior cervix of six pregnant cows. The organism persisted in the cervicovaginal region for eight to 87 days, and at parturition H. somnus was isolated from chorioallantois in four of six cows; placentitis developed, and fetal membranes were retained. All calves were born alive and no H. somnus was recovered from them. One cow died 14 days after parturition. The death was attributed to severe necrotizing metritis; H. somnus was not isolated from the uterus at death, but was isolated from the placenta at parturition and cervicovaginal mucus two days days later.     

Inhibitory effect of trichothecene mycotoxins on bovine platelets stimulated by platelet activating factor.

Several species of fungi, which infect cereals and grains, can produce a class of compounds, known as trichothecene mycotoxins, which is characterized by a substituted epoxy-trichothecene ring structure. Cattle are susceptible to intoxication from feeds contaminated with T-2 toxin, one of the more potent trichothecene mycotoxins, while swine refuse to ingest feed contaminated with T-2 toxin. The bovine platelet has been used as a model cell system to evaluate the effects of T-2 toxin and its natural metabolites, HT-2 toxin and T-2 tetraol, on cell function in vitro. Due to the lipophilic nature of these mycotoxins, a biologically active phospholipid was used to stimulate the platelets in the presence and absence of the toxins. The mycotoxin T-2 toxin and its major metabolite HT-2 toxin inhibited platelet activating factor-stimulated bovine platelets, suspended in homologous plasma, in a concentration but not time dependent manner. Significant inhibition of platelet function (p less than 0.01) occurred with 135 ng T-2 toxin per 10(6) platelets and with 77 ng HT-2 toxin per 10(6) platelets. These mycotoxins exerted an additive inhibitory effect on the platelet aggregation response. In contrast, the minor metabolite T-2 tetraol had no inhibitory effect on platelet function and had no influence on the responses of T-2 toxin or HT-2 toxin when the mycotoxins were present together in the platelet suspensions.(ABSTRACT TRUNCATED AT 250 WORDS)     

甘草查尔酮A对LPS诱导的奶牛蹄真皮细胞炎性反应的抑制作用

采用10,50,100 mg/L甘草查尔酮A处理蹄真皮炎性细胞24 h后,测定细胞上清液中TNF-a、IL-1β、IL-6水平及SOD、MDA含量,Western blot法检测ERK、JNK、p38、IκBα及p65蛋白表达水平.结果 显示,甘草查尔酮A可显著降低TNF-a、IL-1β、IL-6水平,提高SOD活性并...     

Inhibitory effects of epigallocatechin gallate on the propagation of bovine coronavirus in Madin-Darby bovine kidney cells

Epigallocatechin gallate (EGCg) is the main active component of tea polyphenol and shows several biological activities, such as antimicrobial, antitumor‐promoting, anti‐inflammatory and anti‐oxidative activities. In the present study, the inhibitory effect of EGCg on bovine coronavirus (BCV) propagation in Madin‐Darby bovine kidney (MDBK) cells was investigated. EGCg at concentrations of less than 10 µg/mL did not show any cytotoxicity to MDBK cells. BCV propagation was significantly inhibited by pretreatment of the virus with EGCg (0.5–10 µg/mL) before virus inoculation in dose‐dependent, incubation time‐dependent and temperature‐dependent manners. The antiviral effect of pretreating MDBK cells with EGCg on BCV propagation was much weaker than that of pretreating BCV with EGCg. The hemagglutination activity of BCV was also reduced by EGCg in a dose‐dependent manner. These results demonstrate that EGCg possesses a distinct anti‐BCV activity and strongly suggest that EGCg interferes with the adsorption of BCV to MDBK cells by the interaction of EGCg with BCV particles. EGCg may therefore be a useful candidate for controlling BCV infection more effectively.     

Mitogen and mixed lymphocyte culture responses of isolated bovine lymphocyte subpopulations

Examinations were made of the mitogen and mixed lymphocyte culture (MLC) responses of subpopulations of bovine lymphocytes isolated by density sedimentation following sequential E-rosetting with aminoethylisothiouronium bromide- and neuraminidase-treated sheep erythrocytes (EAET, EN). These procedures consistently separated 3 populations of E-rosetting T cells from a 4th population of non-E-rosetting cells (B and null cells). The isolated B and null cell population did not respond to phytohemagglutinin, concanavalin A, or pokeweed mitogen and responded minimally in mixed lymphocyte culture. Conversely, isolated T cells responded well to each of these stimuli. Moreover, differences in responsiveness were found among the 3 T-cell populations isolate by differential rosetting. T cells with receptors for both EAET and EN rosette formation were the most responsive to the mitogens used and demonstrated maximum activity in MLC. T cells with only EAET receptors had equivalent activity in MLC and less activity in response to mitogens. Cells with only EN receptors were the least active T-cell population in these assays. The different reactivities of these T-cell populations in lymphoproliferative assays indicated that they represent distinct subsets of bovine T cells.     

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.     

Alterations in bovine lymphocyte function during the periparturient period

Lymphocytes from 8 Holstein heifers were evaluated during the periparturient period for mitogen-induced lymphocyte blastogenic responses. Phytohemagglutinin P- and concanavalin A-induced lymphocyte blastogenesis increased 2 and 3 weeks before parturition, respectively. However, by the first week after parturition, lymphocyte blastogenesis was markedly impaired.     

The antibody response to bovine lymphocyte alloantigens

Antibodies were raised against lymphocyte cell-surface antigens by multiple immunisations with purified lymphocytes or by the exchange of skin allografts. Eighteen of 21 cattle immunised with lymphocytes raised a detectable cytotoxic antibody response. The serum antibody from 10 responders recognized only common lymphocyte antigens, those antigens which are present on all peripheral blood lymphocytes. One animal responded only to B lymphocyte antigens while 7 others responded to both classes of antigens. The amount of antibody produced varied greatly between individuals; antibody titres ranged from 1 to 1028. Antibody raised early in the response was sensitive to treatment with 2-mercaptoethanol (2-ME) suggesting that IgM was the predominant class of immunoglobulin. Subsequently antibody became resistant to this treatment suggesting the appearance of IgG. The antibody responses following the exchange of skin grafts were very similar in all 12 cattle studied. High titred antibody to common lymphocyte antigens was detected in the serum 14 days after grafting. The early antibody activity was sensitive to 2-ME treatment but became totally resistant within 14 days. Total peak antibody titres ranged from 128-2048. Antibody to B lymphocyte antigens was identified in 8 of the 12 cattle. The responses to B lymphocyte antigens were similar to those against the more widely distributed common lymphocyte antigens with respect to time of antibody appearance, time of peak titre and sensitivity to 2-ME. Peak titres ranged from 2 to 32. The change in antibody specificity with time was also studied. Sera from 11 of the 18 cattle which had responded against lymphocytes showed an increase or broadening in reaction frequency as immunisations increased, suggesting the production of antibody to secondary specificities. In the cattle which had been skin grafted, the broadest reaction patterns were seen 14 to 21 days after grafting. The broadest reaction patterns were seen when the antibody responses were at their highest titre levels and narrowed as titres decreased.     

Regulation of phytohemagglutinin-induced bovine lymphocyte blastogenesis by leukotrienes

Leukotriene (LT) B4, a 5-lipoxygenase metabolite of arachidonic acid, is a potent inducer of suppressor cells in phytohemagglutinin-stimulated cultures of bovine peripheral blood mononuclear cells. In contrast, LTC4 and LTD4 have little activity. Incubation of T lymphocytes with LTB4 at concentrations as low as 1 X 10(-12)M rendered these lymphocytes suppressive of [3H]thymidine incorporation in subsequent phytohemagglutinin-stimulated cultures of fresh autologous lymphocytes. This LTB4-induced cell was radiosensitive to irradiation at 2,000 rads. Leukotriene B4 may have an important part in immunoregulation during hypersensitivity reactions.