Immunological evaluation of SW-HSA conjugate on goats.

Locoweeds cause significant livestock poisoning and economic loss all over the world. The purpose of this study was to investigate the immune effects of locoweed toxin, swainsonine (SW) and human serum albumin (HSA) conjugate (SW-HSA), on goats. Twenty-four Sannon goats were randomly separated into immune control group (eight goats), immune poisoning group I (six goats), immune poisoning group II (six goats) and poisoning control group (four goats). Immune control group, immune poisoning groups I and II were first vaccinated with SW-HSA conjugate. The poisoning control group, immune poisoning groups I and II were then fed with 10.0 g/kg BW/day dry powder of Oxytropis kansuensis Bunge everyday morning. The immune control group was supplied with an alfalfa-based diet. Blood samples of these experimental animals were collected at different time interval. Immunoassay was performed using indirect ELISA and E-rosette technique. The results show that, after second booster immunization: (1) anti-SW antibody level in some goats increased to 2(8), which proves that SW-HSA conjugate can induce experimental animals to produce high-level anti-SW antibody in their bodies; (2) the high-level antibody in their bodies could maintain 30 days, and decreased gradually after poisoning experiment (in our experiment, there was a return of the antibody level on day 21 after poisoning experiment); (3) the decreasing of the E-rosette rate of the immune poisoning group was delayed 14 days, which suggests that SW-HSA could low down the loss of the immunity of the goats; (4) swainsonine concentration in the blood was significantly lower (p<0.01) in the immune poisoning groups than that in the poisoning control group, and there was no significant difference (p>0.01) between the two immune poisoning groups within the poisoning experiment.

A chimeric protein of abrin and Abrus precatorius agglutinin that neutralizes abrin mediated lethality in mice.

Abrin, a type II ribosome inactivating protein from the Abrus precatorius plant, is extremely toxic. It has been shown to be 75 times more potent than its infamous sister toxin, ricin and their potential use in bio-warfare is a cause of major concern. Although several vaccine candidates are under clinical trials for ricin, none are available against abrin. The present study proposes a chimeric protein, comprising of 1-123 amino acids taken from the A chain of abrin and 124-175 amino acids from Abrus precatorius agglutinin A chain, as a vaccine candidate against abrin intoxication. The design was based on the inclusion of the immunogenic region of the full length protein and the minimal essential folding domains required for inducing neutralizing antibody response. The chimera also contains the epitope for the only two neutralizing antibodies; D6F10 and A7C4, reported against abrin till now. Active immunization with the chimera protected all the mice challenged with 45 X LD50 of abrin. Also, passive transfer of antibodies raised against the chimera rescued all mice challenged with 50 X LD50 of toxin. Hence the chimeric protein appears to be a promising vaccine candidate against abrin induced lethality.

Evaluation of humoral immune responses in cattle grazing endophyte-infected or endophyte-free fescue.

Anecdotal reports suggest cattle with fescue toxicosis may not respond to vaccination and thus, experience increased incidence of Bovine Respiratory Disease Complex (BRDC) when shipped to feedlots. Fescue toxicosis causes hypoprolactemia in cattle. Hypoprolactemia decreases humoral immune responses in mice. Therefore, a study was conducted to compare the magnitude of primary and secondary humoral immune responses against specific antigens in cattle grazing endophyte-infected or endophyte-free fescue. Angus steers were blocked by weight and allocated into four groups. Two groups grazed endophyte-infected (EI) fescue and the other two groups grazed endophyte-free (EF) fescue. All steers were injected IM on d 0 and 21 with lysozyme without adjuvant and concanavalin. A (Con A) with sheep red blood cells (SRBC) in incomplete adjuvant of Freund. Steers were bled on days 0, 21 and 35 post-vaccination. Average daily gains (ADG), alkaline phosphatase (ALP) activity, cholesterol concentrations, rectal temperatures, and serum prolactin concentrations were measured to confirm fescue toxicosis in steers grazing EI fescue. Antibodies to Con A and SRBC were determined by ELISA and hemagglutination assay, respectively. The ADG were decreased for the EI group during the first month. Rectal temperature were elevated and serum prolactin concentrations were decreased in the EI group. Cholesterol and ALP concentrations also were decreased in the EI group. Primary and secondary immune responses against Con A tended to be increased and were increased against SRBC in the EI group. Antibodies against lysozyme were not induced in either group. In conclusion, cattle grazing EI fescue mounted similar humoral immune responses to vaccination, despite hypoprolactemia, as cattle grazing EF fescue. Increases in bovine respiratory disease in cattle maintained on EI fescue probably is not associated with lack of humoral immune response to vaccination protocols as a result of fescue toxicosis.

A negative serological relationship between cases of grass sickness in Scotland and Clostridium perfringens type A enterotoxin.

In an attempt to compare the equine grass sickness as reported in Europe with that described in the Republic of Colombia, sera from horses experiencing grass sickness in Scotland were used in neutralisation tests with Clostridium perfringens type A enterotoxin. The sera, from acute and chronic cases of the disease, failed to neutralise either crude or partially-purified enterotoxin. Neither were precipitin lines formed when the sera were treated against the toxin in immunoelectrophoresis. These results suggest that grass sickness in Europe and the equine disease in Colombia have a different aetiology.

Evaluation of vaccination against methyllycaconitine toxicity in mice.

The purpose of this study was to determine whether larkspur toxins conjugated to protein carriers would promote active immunity in mice. Mice were injected with several larkspur toxin-protein conjugates or adjuvant alone to determine whether the resulting immunological response altered animal susceptibility to methyllycaconitine, the major toxic larkspur alkaloid. Although vaccinations increased the calculated lethal dose 50% (LD50) for intravenous methyllycaconitine toxicity, overlapping confidence intervals did not provide evidence of differences between the vaccinated and control groups. In the lycoctonine conjugate (LYC)-vaccinated group, mouse survival was related (P = 0.001) to serum titers for methyllycaconitine doses up to 4.5 mg/kg of body weight. When mice withlow antibody titers were removed from the vaccinated groups in which titer was related to survival, the recalculated LD50 estimates were 20% greater than the LD50 of the control group. However, the 95% confidence intervals of the recalculated LD50 groups overlapped with the control groups. Overall, these results suggest that vaccination altered methyllycaconitine toxicity in mice and that vaccination may be useful in decreasing the effects of larkspur toxins in animals. Additional studies are warranted to continue development of potential larkspur vaccines for livestock.

Sleep time following anesthesia in mouse lines selected for resistance or susceptibility to fescue toxicosis.

In previous work, a mouse line selected for resistance (R) to fescue toxicosis had higher activities of two hepatic Phase II detoxification enzymes than a mouse line selected for fescue toxicosis susceptibility (S). The primary objective of the present study was to determine whether those same lines also differed in hepatic Phase I enzyme activity, estimated from sleep time (ST) following sodium pentobarbital anesthesia. Additional objectives were to determine whether ST differences between lines were modulated by endophyte-infected fescue in the diet (with or without an enzyme inducer) and whether ST of individual mice was correlated with the effect of a toxin-containing diet on the postweaning growth of those mice. In Exp. I, 24 males from each line were randomly assigned to each of five diets: control (commercial rodent food meal); E+ (50% endophyte-infected fescue seed, 50% control); E+P (the E+ diet supplemented with 1,000 ppm phenobarbital); E- (50% endophyte-free fescue seed, 50% control); and E-P (the E- diet supplemented with 1,000 ppm phenobarbital). After 4 wk on these diets, ST was measured on all the mice. A second ST was recorded on each mouse by randomly sampling one-fourth of the population after 1, 2, 3, or 4 wk on a pelleted rodent food diet. Regardless of diet, R mice had shorter first and second ST than S mice (P < 0.01), suggesting higher hepatic Phase I microsomal enzyme activity. Mice on both phenobarbital-supplemented diets had shorter first ST than mice whose diets did not include that microsomal enzyme inducer (P < 0.01). In Exp. II, ST was measured on male and female R and S mice (n = 280) after they had been fed the E- diet for 2 wk, then the E+ diet for 2 wk, and then a pelleted rodent food diet for 2 wk. Growth response to the E+ diet was the percentage of reduction in gain on the E+ diet compared to gain on the E- diet the previous 2 wk. As in Exp. I, S mice slept longer than R mice (P < 0.01). The residual correlation between ST and gain reduction associated with the E+ diet equaled 0.04. Thus, an animal's apparent Phase I enzyme activity did not predict its growth rate depression on the toxin-containing diet. Based on these and previous studies, divergent selection for toxicosis response in mice was successful partially by causing divergence in activities of hepatic Phase I and II detoxification enzymes.

Differences between progeny of beef sires in susceptibility to fescue toxicosis.

Fifteen progeny of two Polled Hereford sires were fed endophyte-infected tall fescue seed (E+) to investigate variability in susceptibility to fescue toxicosis. One sire, bred in Missouri, was reputed to produce calves that were resistant to fescue toxicosis. The Control sire, from Virginia, had unknown merit for susceptibility. There were two phases in which E+ was included in the diet (2 and 4) and three in which it was not (1, 3, and 5). Voluntary feed intake, serum prolactin, cholesterol, and alkaline phosphatase concentrations; ability to maintain homeothermy (afternoon minus morning rectal temperature); and heat-transfer inefficiency (afternoon rectal minus afternoon surface temperatures) were monitored. Intake and prolactin concentration were decreased by E+, but sire groups did not differ in responses across phases. Cholesterol levels were lower during E+ phases, and the rate of increase in cholesterol concentration after withdrawal of E+ differed between groups. Alkaline phosphatase concentrations were lower during E+ phases; calves from the Control sire showed greater reduction than calves sired by the Missouri sire. Ability to maintain homeothermy was reduced by E+, but the reduction did not differ between sire groups. Heat-transfer inefficiences were not indicative of fescue toxicosis in this study. Evidence was not compelling to support the existence of differences between the sire groups for susceptibility to toxicosis. However, progeny of the Missouri bull had higher feed intake and lower rectal and body surface temperatures through nearly all phases of the trial. This may account for the reputation of the Missouri bull to sire calves with relatively high resistance to fescue toxicosis.

Equine grass sickness: serologic evidence of association with Clostridium perfringens type A enterotoxin.

Clostridium perfringens type A enterotoxin seroneutralization was carried out on sera from 50 horses recovered from grass sickness and from 100 other horses with no record of having had the disease. Of the affected horses, 70% had seroneutralizating titers higher than 1:64, half of these being equal or higher than 1:128. More than 88% of the horses with no record of grass sickness had titers lower than 1:64. These data support the theory of association between C perfringens type A toxins and grass sickness.

Effect of repeated locoweed feeding on peripheral lymphocytic function and plasma proteins in sheep.

Seven healthy, adult, crossbred yearling ewes were given (orally) 340 g of locoweed (Astragalus lentiginosus) every day for 10 weeks. Another 7 ewes were not fed the plant, but were housed similarly (controls). Blood samples were obtained once a week to evaluate the mitogen-induced lymphocytic responsiveness. For the locoweed-exposed ewes, there was decreased activity in the presence of phytohemagglutinin (a T-cell mitogen). This effect, although not statistically significant at all times, was consistent and became significant after 7 weeks. A similar response was observed in the blood cell cultures in the presence of pokeweed mitogen, but the differences were not statistically significant at most time points. For the locoweed-exposed ewes there also were gradual numerical decreases in total leukocyte and lymphocytes in peripheral blood. Peripheral leukocytes had cytoplasmic vacuolation. The results indicated that a selective effect may occur on cell-mediated immune responses. Serum proteins and gamma-globulins were not affected by locoweed treatment. Locoweed, certain species of Astragalus and Oxytropis, causes considerable economic loss to the livestock industry of western United States. Locoweed consumption by livestock can result in neurologic problems, emaciation, habituation, and reproductive alterations. The reproductive alterations include abortions, birth defects, and some interference with spermatogenesis and oogenesis. Signs of poisoning are CNS depression, rough dry coat, dull eyes, irregular gait, and excitement when stressed. The microscopic lesions are neurovisceral cytoplasmic vacuolations. Microscopic lesions are also observed in the fetuses and newborns of dams which were fed locoweed.(ABSTRACT TRUNCATED AT 250 WORDS)

Humanization and characterization of an anti-ricin neutralization monoclonal antibody.

Ricin is regarded as a high terrorist risk for the public due to its high toxicity and ease of production. Currently, there is no therapeutic or vaccine available against ricin. D9, a murine monoclonal antibody developed previously in our laboratory, can strongly neutralize ricin and is therefore a good candidate for humanization. Humanization of D9 variable regions was achieved by a complementarity-determining region grafting approach. The humanized D9 (hD9) variable regions were further grafted onto human heavy and light chain constant regions to assemble the complete antibody gene. A foot-and-mouth-disease virus-derived 2A self-processing sequence was introduced between heavy and light chain DNA sequences to cleave the recombinant protein into a functional full-length antibody molecule from a single open reading frame driven by a single promoter in an adenoviral vector. After expression in mammalian cells and purification, the hD9 was demonstrated to have equimolar expression of the full-length antibody heavy and light chains. More importantly, the hD9 exhibited high affinity to ricin with K(D) of 1.63 nM, comparable to its parental murine D9 (2.55 nM). In a mouse model, intraperitoneal (i.p.) administration of hD9, at a low dose of 5 µg per mouse, 4 hours after the i.p. challenge with 5×LD50 ricin was found to rescue 100% of the mice. In addition, administered 6 hours post-challenge, hD9 could still rescue 50% of the mice. The hD9 has the potential to be used for prophylactic or therapeutic purposes against ricin poisoning.

Identification of lamb flocks susceptible and resistant against Brachiaria poisoning / Identificação de rebanhos de cordeiros suscetíveis e resistentes a intoxicação por braquiária

This study was designed to assess the influence of genetic resistance against brachiaria poisoning in sheep. Two groups of sheep, one identified as susceptible (formed by two ewes and one ram) and the other as resistant against brachiaria poisoning (formed by three ewes and one ram) were selected. Sheep considered susceptible were those that presented clinical signs of brachiaria poisoning at any time of their life; resistant sheep were those that even raised on Brachiaria spp. pastures, did not developed any sign of the poisoning during their life. The offspring of the two flocks (15 lambs from the sensitive flock and 9 lambs from the resistant flock) were placed into brachiaria pasture (initially Brachiaria decumbens and B. brizantha,and only B. decumbens after weaning) and followed up during two years (2013-2014). The determination of protodioscin levels in B. decumbens pasture was performed only in 2014 and revealed significant amounts of the toxic principle. Eleven lambs of the susceptible group were affected to some degree of brachiaria poisoning and six died; no lamb of the resistant group was affected. Clinical signs consisted of varying degrees of subcutaneous edema of the face and, erythema and loss of hair of the ears, crusts on the skin of ears, around the eyes and on planum nasale, scar deformation of the ears, and bilateral ocular discharge; affected lambs also sought for shadowy shelters and they were poor doers. Several sheep recovered from the condition and then relapsed. Necropsy findings in six lambs included pale mucous membranes, emaciation, dermatitis, scar deformation of the ears, large yellow livers with marked lobular pattern, and moderate infestation by Haemonchus contortus...

Este estudo avaliou a resistência genética na ocorrência de intoxicação por braquiária em ovinos. Foram selecionados dois grupos de ovinos, um identificado como suscetível (formado por duas ovelhas e um carneiro) e o outro como resistente (formado por três ovelhas e um carneiro). Foram considerados suscetíveis ovinos que apresentaram sinais de intoxicação por Brachiaria spp. em algum ponto de suas vidas e resistentes aqueles ovinos que, mesmo criados em pastagem de braquiária, nunca desenvolveram qualquer sinal da intoxicação. A progênie desses dois grupos (15 cordeiros do grupo suscetível e 9 no grupo resistente) foi colocada numa pastagem de braquiária (inicialmente Brachiaria decumbens e B. brizantha e, após o desmame, apenas B. decumbens) e acompanhada durante dois anos (2013-2014). A determinação dos níveis de protodioscina em B. decumbens foi realizada apenas em 2014 e foram encontradas quantidades significativas do princípio tóxico. Onze cordeiros do grupo suscetível foram afetados por algum grau de intoxicação por braquiária; nenhum cordeiro do grupo resistente foi afetado. Os sinais clínicos consistiam de graus variáveis de edema subcutâneo da face e eritema e alopecia da pele das orelhas, crostas na pele das orelhas e ao redor dos olhos e no plano nasal, retração cicatricial das orelhas, fotofobia e corrimento ocular bilateral. Três cordeiros apresentaram desenvolvimento retardado. Vários cordeiros se recuperaram da condição, mas posteriormente quando foram colocados na pastagem apresentaram recidivas. Achados de necropsia em seis cordeiros incluíam mucosas pálidas, pobre condição corporal, dermatite, deformação cicatricial das orelhas, fígado aumentado de volume, amarelo e com padrão lobular evidenciado e graus moderados de infestação por Haemonchus contortus...

Effect of Senna occidentalis seeds on immunity in broiler chickens.

This study investigated possible immunotoxic effects of Senna occidentalis (So) seeds incorporated in broiler chicken rations at different concentrations (0.0%, 0.25%, 0.50% and 0.75%), for 28 or 42 days. We evaluated innate immune function (macrophage activities of spreading, phagocytosis, peroxide and nitric oxide production) and acquired immune function (humoral and cellular immune responses), as well as lymphoid organ weights and pathology. There was enhanced macrophage activity, as hydrogen peroxide production increased (P < 0.05) in cells of birds given 0.75%So, but there were no other pro-inflammatory effects. Birds receiving 0.75% of So in ration for 42 days gained less weight (P < 0.01), and showed a decrease in relative weight of the bursa of Fabricius (P < 0.05) and spleen (P < 0.01). In addition, morphological changes were also noted in these lymphoid organs, with depletion of lymphoid cells on the spleen and bursa of Fabricius, resulting in lower relative weight of both lymphoid organs. No impairment of humoral immune response against Newcastle disease and in cellular immune response after a phytohaemagglutinin challenge was found. It is probable that mitochondrial damage and related apoptosis may be responsible for the enhanced peroxide production and the reduced relative weight of the bursa of Fabricius and spleen.

Immediate hypersensitivity to leaf extracts of Callisia fragrans (inch plant) in a dog.

Contact with members of the plant family Commelinaceae, which includes wandering jew (Commelina spp. formerly called Tradescantia spp.) and inch plant (Callisia fragrans), can cause cell-mediated contact dermatitis in dogs. However, reports of canine IgE-mediated hypersensitivity to these plants have not been published. The purpose of this study was to discover whether IgE antibodies specific for extractable components of C. fragrans could be identified in serum from a dog that had anaphylactic shock after exposure to the plant and after skin patch testing with the sap from a leaf of C. fragrans. Separate aqueous extracts of leaves and flowers of C. fragrans were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Serum from a dog with no history or symptoms of any allergies showed no specific IgE antibodies against the leaf extract. Serum from a dog with clinical symptoms of delayed, but not immediate hypersensitivity to leaf sap from C. fragrans, showed only minor IgE recognition of a single 65 k component in sap extracted from leaves harvested in summer but not in winter. However, IgE antibodies to a serum dilution of 1:200 specific for several components of the leaf extract were seen in serum from the dog that had anaphylactic shock after exposure to sap. The molecular weights of these molecules were in the range 51 k to 83 k. The bands on the immunoblots did not match with prominently stained protein bands in the gel, but instead identified molecules in a lightly stained area of the gel with diffuse bands. Testing for glycans indicated that the carbohydrate side chains of glycoproteins contributed significantly to the immunoreactivity of the putative allergens. All three dog sera failed to show any immunoreactivity against the extract from the flowers of C. fragrans.

Lectin histochemistry of foam cells in tissues of cattle grazing Brachiaria spp.

Brachiaria decumbens and B. brizantha (signal grass), which occupy millions of acres in Brazil, are an important source of fodder for ruminants. Sporadic outbreaks of photosensitization in ruminants grazing on signal grass have been reported. Intoxicated animals showed the presence of foamy cells in the liver, spleen, intestinal submucosa and lymph nodes. These foamy cells are macrophages. They are very difficult to distinguish with haematoxylin and eosin stain, especially in the case of isolated cells. The purpose of the present study was to detect specific carbohydrate residues of storage material in the foamy cells in tissues of cattle exposed to Brachiaria spp. The characterization of glycoconjugates provides clues to the pathogenesis of these cells. Besides, the lectin peanut agglutinin was found to be an excellent marker to differentiate and quantify the foam cells, and could be used as a specific marker.

Acute plant poisoning and antitoxin antibodies.

Plant poisoning is normally a problem of young children who unintentionally ingest small quantities of toxic plants with little resulting morbidity and few deaths. In some regions of the world, however, plant poisonings are important clinical problems causing much morbidity and mortality. While deaths do occur after unintentional poisoning with plants such as Atractylis gummifera (bird-lime or blue thistle) and Blighia sapida (ackee tree), the majority of deaths globally occur following intentional self-poisoning with plants such as Thevetia peruviana (yellow oleander) and Cerbera manghas (pink-eyed cerbera or sea mango). Antitoxins developed against colchicine and cardiac glycosides would be useful for plant poisonings--anti-digoxin Fab fragments have been shown to be highly effective in T. peruviana poisoning. Unfortunately, their great cost limits their use in the developing world where they would make a major difference in patient management. Therapy for some other plant poisonings might also benefit from the development of antitoxins. However, until issues of cost and supply are worked out, plant antitoxins are going to remain a dream in many of the areas where they are now urgently required.

Susceptibility of Bovans chicks to low dietary levels of Ammi visnaga and Artemisia herba-alba.

Ammi visnaga seeds and Artemisia herba-alba shoots were fed to 7-d-old Bovans chicks at 2% and 10% of diet for 9 w. The 10% A visnaga seed was toxic but not lethal to chicks and caused a consistently reduced body weight gain, inefficient feed utilization, enterohepatonephropathy, anemia, and alterations of serum aspartate transaminase and creatine kinase activities and cholesterol, total lipid and uric acid concentrations. The depression in growth and damage to vital organs of chicks fed 10% A herba-alba shoots 2% A visnaga seed, or 2% A herba-alba shoots were less marked.

Cross-reactivity with Tagetes in Arnica contact eczema.

A 69-year-old patient, with known mercury and adhesive plaster allergy, developed facial dermatitis within 24 h of contact with arnica (Arnica). Skin testing showed positive reactions to arnica and, among various other plants of the Compositae, also to Tagetes sp. hybr. (marigold). Cross-reactivity between Tagetes and arnica has not previously been described.

Effect of subacute swainsonine (locoweed; Oxytropis sericea) consumption on immunocompetence and serum constituents of sheep in a nutrient-restricted state.

The subacute dose-response effects of swainsonine (SW) consumption on immunocompetence and serum constituents of sheep in a nutrient-restricted state were investigated. Sheep (23 wethers, 5 ewes) were assigned to 1 of 5 SW treatments (0, 0.2, 0.4, 0.8, or 1.6 mg swainsonine/ kg bw/d). Swainsonine was delivered by feeding locoweed (Oxytropis sericea) with grama grass and alfalfa hays for a 28-d treatment period followed by a 21-d recovery period without locoweed. Body weights were measured weekly and behavioral changes were monitored for clinical signs of SW toxicity. Venous blood was collected weekly for lymphoblastogenesis and serum constituent analyses. Clinical signs (sluggishness, decreased responsiveness) of swainsonine toxicity were observed from d 14 to 35 in the 0.8 and 1.6 mg treatments. Subacute oral exposure did not appear to affect lymphoblastogenic analyses. Acute and subacute alterations in various serum constituents did indicate subclinical effects of SW ingestion. Linear, quadratic and cubic dose-response relationships were detected for some serum constituents (e.g., alkaline phosphatase, aspartate aminotransferase). Subacute SW consumption at the levels investigated does not seem to affect the immunocompetence of nutrient restricted sheep. The lack of change in serum alkaline phosphatase at the 0.2 mg SW/kg bw/d dose indicates the potential for a no adverse effect level of SW consumption in nutrient restricted sheep. In combination with measurable SW in serum, rises in serum alkaline phosphatase and aspartate aminotransferase activities, and declines in serum Fe and cholesterol during subacute exposure to SW establish these markers as potential indicators of subclinical SW toxicosis.