Ergot alkaloid transport across ruminant gastric tissues.

Ergot alkaloids cause fescue toxicosis when livestock graze endophyte-infected tall fescue. It is generally accepted that ergovaline is the toxic component of endophyte-infected tall fescue, but there is no direct evidence to support this hypothesis. The objective of this study was to examine relative and potential transport of ergoline and ergopeptine alkaloids across isolated gastric tissues in vitro. Sheep ruminal and omasal tissues were surgically removed and placed in parabiotic chambers. Equimolar concentrations of lysergic acid, lysergol, ergonovine, ergotamine, and ergocryptine were added to a Kreb's Ringer phosphate (KRP) solution on the mucosal side of the tissue. Tissue was incubated in near-physiological conditions for 240 min. Samples were taken from KRP on the serosal side of the chambers at times 0, 30, 60, 120, 180, and 240 min and analyzed for ergot alkaloids by competitive ELISA. The serosal KRP remaining after incubation was freeze-dried and the alkaloid species quantified by HPLC. The area of ruminal and omasal tissues was measured and the potential transportable alkaloids calculated by multiplying the moles of transported alkaloids per square centimeter of each tissue type by the surface area of the tissue. Studies were conducted to compare alkaloid transport in reticular, ruminal, and omasal tissues and to determine whether transport was active or passive. Ruminal tissue had greater ergot alkaloid transport potential than omasal tissue (85 vs 60 mmol) because of a larger surface area. The ruminal posterior dorsal sac had the greatest potential for alkaloid transport, but the other ruminal tissues were not different from one another. Alkaloid transport was less among reticular tissues than among ruminal tissues. Transport of alkaloids seemed to be an active process. The alkaloids with greatest transport potential were lysergic acid and lysergol. Ergopeptine alkaloids tended to pass across omasal tissues in greater quantities than across ruminal tissues, but their transport was minimal compared to lysergic acid and lysergol.

Anti-inflammatory effects of ergotamine in steers.

The objective of this experiment was to investigate whether the ergot alkaloid, ergotamine (ET), an alkaloid used to model fescue toxicosis in cattle, modifies the response of cattle to endotoxin (LPS) challenge. Steers (n = 16) were divided into the following treatment groups: control (C), ergotamine (ET), endotoxin (LPS), and ET + LPS. ET and ET + LPS groups received a single bolus intravenous injection of ET (40 microg. kg. body wt(-1)), whereas C and LPS steers received a single bolus injection of sterile vehicle. Thirty minutes after ET/vehicle administration, a single bolus intravenous injection of LPS (0.2 microg. kg. body wt(-1)) was given. Blood was collected at various time points for 48 hr post. Endotoxin increased rectal temperature (RT) and the circulating levels of tumor necrosis factor-alpha (TNF-alpha), cortisol, haptoglobin (Hp), thromboxane B(2) (TXB(2)). The circulating Hp, TNF-alpha, and TXB(2) increases were blunted by pretreatment with ET compared with ET + LPS. Ergotamine by itself increased circulating cortisol and RT, whereas it decreased serum prolactin (PRL). Therefore, whereas administration of LPS at 0.2 microg/kg to steers resulted in an expected response, the combination of ET + LPS attenuated major effects of LPS alone. Thus, acute administration of ET appeared to be anti-inflammatory as it decreased the inflammatory response to LPS, an effect likely driven at least in part by the ET-caused cortisol increase.

Urinary alkaloid excretion as a diagnostic tool for fescue toxicosis in cattle.

Fescue toxicosis research studies have often included serum prolactin as a physiologic index of the disorder. Serum prolactin has not been used as a clinical measure of fescue toxicosis because of variation associated with sex and physiologic condition of the animal and climatic and seasonal factors. The primary excretory route of the alkaloids responsible for this toxicosis is the urine. Three pasture experiments were conducted to examine serum prolactin and urinary ergot alkaloid variability among steers continuously grazing endophyte-infected (E+) or endophyte-free (E-) tall fescue and among steers that were switched from one pasture form to the other. A fourth grazing experiment was used to examine how to best to manage the steers prior to sampling for urinary ergot alkaloid excretion. Coefficients of variability for urinary alkaloid excretion were consistently lower (46-65%) than serum prolactin (64-142%). Urinary alkaloid excretion patterns changed within 12 hours following switching steers from E+ to E- pasture or vice versa, but serum prolactin was recalcitrant to change. Because it is less variable and more dynamic than serum prolactin, urinary alkaloid excretion can be used for health assessment of steers grazing E+ and E- pastures. Regression analysis established a quadratic relationship between alkaloid excretion and average daily weight gain, with a regression coefficient of 0.86. Urinary alkaloid analysis was useful in determining whether cattle were consuming toxic tall fescue.

Vaccination against ergot alkaloids and the effect of endophyte-infected fescue seed-based diets on rabbits.

Three sequential experiments were conducted with rabbits to 1) determine the effect of endophyte-infected (E+) tall fescue seed on rabbit performance and examine the effect of anti-ergot alkaloid immunization on rabbit performance and protectiveness against fescue toxicosis, 2) compare immunogens designed to elicit systemic anti-ergot alkaloid antibodies, and 3) select a superior adjuvant. In Exp. 1, rabbits (n = 6/treatment) fed E+ fescue seed diets (20%, 340 ppb total ergot alkaloids) had reduced (P < .05) intake and weight gain compared with endophyte-free (E-) controls, whereas apparent diet digestibility was not different between E+ and E-. Rabbits immunized against ergot alkaloids (E+ vac) with lysergol conjugated to human serum albumin (Ly-HSA) had greater (P < .05) intake than E+ rabbits during the wk 1 of a 3-wk dietary challenge. In Exp. 2, rabbits (n = 4/treatment) were immunized with Ly-HSA, with H100-B (ergot alkaloid hapten, H100-different protein carrier, B conjugate), or combinations of both with alum as adjuvant. Greatest (P < .001) anti-ergot alkaloid antibody (Ab) titer developed in the group immunized with H100-B. In Exp. 3, rabbits (n = 4/treatment) were immunized with the immunogen H100-B in conjunction with six adjuvants. Freund's incomplete adjuvant (FIA) in combination with DEAE-dextran and FIA alone gave highest anti-ergot titers. In summary, rabbit weight gain and intake were reduced by feeding E+ fescue seed diets, immunization against ergot alkaloids provided temporary improvement in intake, and H100-B conjugate with FIA or FIA + DEAE-dextran as adjuvants elicited a superior anti-ergot immune response. We believe that rabbits may serve as a model animal for fescue toxicosis research.

Urinary and biliary excretion of ergot alkaloids from steers that grazed endophyte-infected tall fescue.

Ergot alkaloids cause fescue toxicosis when livestock graze endophyte-infected (E+) tall fescue. Little is known about the bioavailability of the ergot alkaloid classes (lysergic acid, lysergic acid amides, or ergopeptine alkaloids) in livestock, and this hampers development of pharmacological strategies to ameliorate the toxicosis. One method used to determine bioavailability of ergot alkaloids is to examine urinary and biliary excretion patterns. Thus, our objectives were to compare ergot alkaloid excretion via urinary or biliary systems and to determine the rate of appearance or clearance of these alkaloids in cattle that were grazing E+ or endophyte-free (E-) tall fescue. In autumn 1996, bile and urine samples were collected from eight steers (203 kg), each grazing E+ and E- tall fescue, and total alkaloid excretion was quantified using competitive ELISA. Approximately 96% of the ergot alkaloids were excreted in urine. The same steers were used to examine the rate of appearance in, or clearance from, urine when switched from E+ to E-, or from E- to E+, pastures in comparison with steers that were continuously grazing E+ or E- tall fescue at 0, 2, 5, and 7 d. Steers were returned to their original pastures after 7 d, and urine was collected at 2, 5, and 7 d. Urinary alkaloid concentrations in steers switched from E- to E+ pastures were similar (P = .55) to those in steers that continuously grazed E+ tall fescue after 2 d. Steers switched from E+ to E- pastures had urinary alkaloid concentrations similar (P = .91) to those in steers that continuously grazed E- pastures after 2 d. In 1997, two trials were conducted in which steers (191 kg) were switched or remained on E+ or E- pastures, and urine was collected at 0, 12, 24, 48, and 96 h to estimate rate of alkaloid appearance or clearance. Steers switched from E- to E+ 1) had about 33% as much urinary alkaloids as steers grazing E+ pasture after 12 h, 2) were not different after 24 h (P = .76), 3) had twice those of the E+ steers at 48 h (P < .05), and 4) were not different after 96 h. Steers switched from E+ to E- tall fescue had approximately 33% less (P < .05) urinary alkaloids than those grazing E+ at 12 h, 67% less (P < .05) at 24 and 48 h, and were not different (P = .86) from steers continuously grazing E- pastures after 96 h. Urinary alkaloid excretion patterns were similar to ergot alkaloid solubility patterns from in vitro digestion of E+ tall fescue. We suggest that alkaloids, liberated from the forage by ruminal microorganisms, were rapidly absorbed as lysergic acid amides and biotransformed ergopeptine alkaloids.

Antibody binding of circulating ergot alkaloids in cattle grazing tall fescue.

Direct evidence linking alkaloids found in endophyte-infected tall fescue forage with the livestock disorder known as fescue toxicosis is lacking. Physiologic effects of fescue toxicosis include reduced serum prolactin concentration in cattle. A monoclonal antibody specific to the lysergic moiety of ergot alkaloids was developed in mice after creating an immunogen by linking lysergol to human serum albumin. The antibody was specific to the lysergic moiety and, therefore, it cross-reacted with ergot alkaloids, lysergic acid, and lysergol. The antibody did not cross-react with alkaloid derivatives that had bromated or hydrogenated lysergic ring moieties. Fescue toxicosis conditions were elicited in yearling Angus steers by permitting them to graze endophyte-infected tall fescue containing > 650 micrograms/kg of ergovaline for 60 days. Passive immunization of steers by infusion of the monoclonal antibody increased serum prolactin concentration by 7 ng/ml, beginning immediately after infusion. Control steers did not respond to treatment with bovine serum albumin. Active immunization of yearling Angus heifers with immunogens containing lysergol or ergonovine linked to human serum albumin resulted in an antibody response.

Delayed hypersensitivity testing as a clinical measure of cell-mediated immunity in the cat.

The purposes of this study were to examine the cell-mediated immune response of the normal cat to the modified live feline viral rhinitis, calicivirus, and parvovirus (FVRCP) vaccine (Felocell CVR, Norden, Lincoln, NE), and to evaluate the intradermal skin test as a clinical measure of the immune response of cats. Vaccine and diluent were injected intradermally on the dorsal pinna of 34 normal adult cats. Skin thickness measurements, lymphocyte counts, and Concanavalin A mitogenesis indices were evaluated in 18 of these cats. Skin biopsies were obtained in 16 cats. In normal cats, the FVRCP vaccine induced a delayed hypersensitivity response characterized by a mononuclear infiltrate most pronounced at 72 h. Five cats with either feline leukemia (FeLV) or feline immunodeficiency virus (FIV) were tested and had a significantly reduced response to the skin test. The skin test provides a clinically useful method of evaluating immune function in cats and may be useful in development of a prognostic index.

A qualitative assay for beta cell antibodies. Preliminary results in dogs with diabetes mellitus.

Purified beta cells from a radiation-induced transplantable rat insulinoma were used to detect beta cell antibodies in serum from untreated diabetic dogs. Serum from dogs in which anti-beta cell antibodies were induced by injecting a purified beta cell suspension subcutaneously was used as positive control. Following incubation with test sera, fluorescein-labeled anti-dog immunoglobulins were used to visualize binding between the beta cells and dog gamma globulins. Nine of the 23 diabetic dogs showed a strongly positive reaction which was characterized by a ring fluorescence, three showed a weak reaction and 11 were negative, i.e. they showed diffuse fluorescence. In contrast, 14 of the 15 healthy dogs showed diffuse fluorescence and one dog showed a weakly positive reaction. Thyroid, liver and kidney cells did not elicit ring fluorescence. Although females (spayed and intact) represented the majority of the diabetic dogs, there was no correlation between sex and the occurrence of antibodies in the diabetic dogs. There was also no correlation to the age of the dogs. In conclusion, we have developed a specific test for anti-beta cell antibodies. The test is reproducible and economical to perform on a large number of samples.

Mitogen and antigen-specific induction of lymphoblast transformation in cats with subclinical toxoplasmosis.

Lymphoblast transformation in whole blood was assessed by 3H-thymidine incorporation after stimulation by concanavalin A and Toxoplasma gondii secretory and intracellular antigens in samples from cats with experimentally induced toxoplasmosis. Toxoplasma gondii-specific immunoglobulin M, immunoglobulin G, and circulating antigens were also measured throughout the study period. Lymphocytes from all cats were responsive to concanavalin A pre- and post-inoculation with T. gondii. Suppression of mitogen-stimulated lymphoblast transformation during the course of infection was not observed. Both the secretory and intracellular antigens stimulated lymphoblast transformation in many cats from Week 2 to Week 52 post-inoculation. Lymphoblast transformation was stimulated more frequently by intracellular antigens (66.25%) than by secretory antigens (48.75%). Lymphoblast transformation was not induced by T. gondii antigens in any cat prior to appearance of T. gondii-specific antibodies in serum or during the oocyst shedding period. Cats with persistent antigenemia had the most consistent lymphoblast transformation results induced by T. gondii-specific antigens.

An immunofluorescence test to determine the occurrence of type II collagen in muscle biopsies from cattle with rectovaginal constriction.

Type II collagen occurs in the muscles of rectovaginal constriction (RVC) affected and carrier cattle but not in normal cattle. Muscle biopsies from known RVC affected and carrier cattle and normal cattle were examined for the presence of Type II collagen using affinity purified goat anti-collagen II serum in a fluorescent antibody test. Type II collagen was consistently found in RVC affected animals (22 of 23 samples score positive). Rectovaginal constriction carrier animals had variable staining for the Type II collagen (25 of 47 samples scored positive). Some positive staining was also observed in the control animals (8 of 34 samples scored positive). Because of the variable occurrence of Type II collagen, the value of fluorescent antibody staining to identify RVC carrier animals is uncertain.

Unexpected pattern of antibody response of hens following infectious bursal disease virus vaccine.

The antibody titers to infectious bursal disease virus (IBDV) of a group of hens were determined every 2 weeks during the laying period using a kinetic-based enzyme-linked immunosorbent assay (ELISA). When the titers of the flock were regressed against time, the flock titer decayed with statistically significant linearity. However, when the antibody titers of individual hens were measured, their titers regressed on time in a significant quartic curvilinear fashion. Since these hens were not reimmunized, this suggests that a anamnestic response was stimulated from an unknown external source.

Prevalence of Toxoplasma gondii infection in cats in Georgia using enzyme-linked immunosorbent assays for IgM, IgG, and antigens.

Serologic prevalence of Toxoplasma gondii infection was determined using enzyme-linked immunosorbent assays detecting immunoglobulin M (IgM), immunoglobulin G (IgG), and circulating T. gondii antigens (Ag) in 81 healthy cats and 107 cats with clinical signs referable to toxoplasmosis. A higher prevalence of infection was detected using the three assays together in healthy cats, clinically ill cats, and combined healthy and clinically ill cats than when IgG class antibody detection alone was used. IgM titers greater than or equal to 1:256 and IgG titers greater than or equal to 1:512 were present more frequently in cats with clinical signs of disease. Prevalence of present or prior infection as defined by these three assays combined increased with advancing age in both groups of cats.

Diagnosis of recent Toxoplasma gondii infection in cats by use of an enzyme-linked immunosorbent assay for immunoglobulin M.

Subclinical Toxoplasma gondii infection was induced in young and adult cats by oral administration of tissue cysts. An antibody-capture ELISA to detect anti-Toxoplasma IgM-class antibodies in the serum of cats was developed. The serologic response to experimental infection was followed in the 2 groups of cats by use of anti-Toxoplasma IgM and IgG detection. This study shows that anti-Toxoplasma IgM-class antibody titers develop early in the course of experimental infection in cats and that the combination of IgM- and IgG-class antibody titer measurement can aid in the detection of recent subclinical toxoplasmosis.

Enzyme-linked immunosorbent assay for the detection of circulating antigens of Toxoplasma gondii in the serum of cats.

An ELISA was developed to detect circulating antigens of Toxoplasma gondii in the serum of cats. For the experiment, toxoplasmosis was induced in a group of cats by oral administration of bradyzoites. An ELISA that detects anti-Toxoplasma IgG, an ELISA to detect circulating antigens, and fecal examinations were performed on samples from each cat for 1 year after inoculation. When coupled with IgG-class antibody measurement, antigen detection can aid in the diagnosis of some cases of subclinical feline toxoplasmosis.

Lymphocyte reactivity to mitogens and natural killer cell activity in crossbred swine during the reproductive cycle.

The effect of pregnancy and lactation on the cell-mediated immune response of first litter gilts was assessed using the response of circulating lymphocytes to in vitro mitogen stimulation and the cytotoxic activity of the circulating natural killer (NK) cells. Groups of gilts were sampled during the first, second and third trimester of gestation, weekly during lactation, at weaning and estrus following weaning of the piglets. No significant differences were found in the response of the cells from any of the groups to phytohemagglutinin A or concanavalin A stimulation. The natural killer cell activity, measured as cytotoxicity, decreased during gestation reaching a low point during the second and third week of lactation.

Leukocyte function in Pelger-Huët anomaly of dogs.

Leukocyte function was evaluated in five dogs with Pelger-Huët (P-H) anomaly and in five control dogs. No significant differences were found between groups in neutrophil adherence, random movement, chemotaxis, phagocytosis, or bacterial killing of Staphylococcus intermedius. Neutrophils migrated rapidly into inflammatory sites where progressive nuclear lobulation was noted over time. Antibody titers to exogenous antigens were similar in the P-H and control groups indicating B- and T-lymphocyte cooperation in humoral immunity. Lymphocyte blastogenesis to phytohemagglutinin also was similar in both groups suggesting the presence of a responsive T-lymphocyte population. These findings indicate that no apparent predisposition to infection or immunodeficiency exists in dogs with P-H anomaly.

Effect of levamisole on immune function and reproductive performance in first-litter gilts.

First-litter commercial cross-bred gilts were treated with levamisole (1.5, 2.5, or 3.5 mg/kg of body weight) weekly during the last 4 weeks of gestation, because similar treatment of dairy heifers had improved postpartum maternal health and neonatal survival. In the gilts, differences in reproductive performance were not found on the basis of pig survival at birth, pig survival at weaning, birth weight, or weaning weight. Also, differences between treated and control gilts were not found in response of circulating lymphocytes to mitogen stimulation (phytohemagglutinin A, concanavalin A, and pokeweed mitogen). In all gilts, the lymphocyte response to mitogen stimulation was decreased during the first week after farrowing.

Suppression of humoral immunity in chickens with carrageenans.

Carrageenans (CGN), sulphated polygalactans, have been reported to be cytotoxic for macrophages in vitro. On this basis, the effect of the 3 major CGN types on humoral immune responses in chickens was investigated. Carrageenan had no effect on body and lymphoid organ weights. Histologically, CGN produced a significant proliferation of reticuloendothelial cells in liver and spleen, but no changes were observed in lymphocyte populations of the bursa of Fabricius, thymus, or spleen. Intracoelomic pretreatment with high doses of CGN induced a marked suppression of primary responses to sheep red blood cells (SRBC) given by the same route. However, if SRBC were injected intravenously into chickens already treated intracoelomically with CGN, no evidence of suppression was demonstrated. Antibody responses to Brucella abortus (BA), a T-independent antigen, were not affected by intracoelomic treatment with CGN. Intravenous pretreatment with CGN did not alter antibody responses to SRBC and BA given by the same route.

Complement immunofluorescence in sera of dogs with pemphigus foliaceus.

Sera from 14 dogs with active pemphigus foliaceus were tested for the presence of circulating antiepithelial antibodies. These antibodies could not be detected in the dogs with indirect immunofluorescent staining or in vitro complement-staining methods.

A comparison between the effect of an avian reovirus and infectious bursal disease virus on selected aspects of the immune system of the chicken.

Reovirus 81-176 was inoculated subcutaneously into day-old specific-pathogen-free leghorns and evaluated for its effects on the immune system over a 3-week period. Structural criteria included organ weights of the bursa of Fabricius (BF) and spleen (SP), scoring of histological lesions in the BF, SP, and thymus, and hematological analyses of the circulating leukocytes. Alterations in the functional capacity of the immune system were measured using the graft-versus-host reaction, the response of peripheral blood lymphocytes (PBLs) to mitogens, the ability of circulating monocytes to phagocytize latex beads, and the serological responses to Newcastle disease virus, sheep red blood cells, and Brucella abortus antigens. For comparison, infectious bursal disease virus (IBDV) was similarly evaluated by most of the same tests. Structurally, reovirus 81-176 altered BF and SP organ weights, the total numbers of white blood cells in circulation, and the degree of follicular atrophy in the BF. Functionally, reovirus inoculation reduced both the response of PBLs to the phytohemagglutinin-P stimulation and monocyte uptake of latex beads. According to the protocols used here, no significant alteration in B-cell function could be detected in reovirus-infected chicks. With the exception of leukocyte hematology, IBDV-infected chicks had significantly altered responses in all tests used. By way of comparison, the effects of IBDV were more persistent and pronounced than were those seen with reovirus. The graft-versus-host reaction indicated an elevated and/or uninhibited response of T-cells in the blood of IBDV-infected chicks.