Occurrence of enterotoxin-encoding genes in Staphylococcus aureus causing mastitis in lactating goats

Staphylococcal enterotoxins are the leading cause of human food poisoning worldwide. Staphylococcus spp. are the main mastitis-causing agents in goats and frequently found in high counts in goat milk. This study aimed to investigate the occurrence of enterotoxin-encoding genes in Staphylococcus aureus associated with mastitis in lactating goats in Paraiba State, Brazil. Milk samples (n=2024) were collected from 393 farms. Staphylococcus aureus was isolated in 55 milk samples. Classical (sea, seb, sec, sed, see) and novel (seg, seh, sei) enterotoxin-encoding genes were investigated by means of polymerase chain reaction (PCR). From thirty-six tested isolates, enterotoxin-encoding genes were detected in 7 (19.5%) S. aureus. The gene encoding enterotoxin C (seC) was identified in six isolates, while seiwas observed in only one isolate. The genes sea, seb, sed, see, seg and seh were not observed amongst the S. aureus investigated in this study. In summary, S. aureus causing mastitis in goats can harbor enterotoxin-encoding genes and seC was the most frequent gene observed amongst the investigated isolates. This finding is important for surveillance purposes, since enterotoxin C should be investigated in human staphylococcal food poisoning outbreaks caused by consumption of goat milk and dairy products.

Accidental and experimental Closantel intoxication in Uruguayan sheep

Abstract An outbreak of Closantel intoxication in sheep in Uruguay is described. The outbreak occurred in a group of 1300 weaning lambs treated orally with a 10% solution of Closantel. One hundred forty eight lambs showed clinical signs of intoxication and 14 died. The clinical signs included mydriasis, nystagmus, and negative pupillary reflex, bilateral blindness, bump into objects, and lateral movement of the head. No macroscopic lesions were observed. The histological lesions of the retina were cytoplasmic vacuolization in ganglion cells and in cells of the inner and outer nuclear layers with different degrees of atrophy. Vacuolization and axonal degeneration were observed in the optic nerve, with multifocal areas of fibrosis and infiltration by lymphocytes and Gitter cells. To reproduce the intoxication, four sheep were given two, four and 10 times the therapeutic dose of Closantel (0.1g/kg of BW). Only the animals receiving 10 times the recommended dose showed clinical signs. The histological examination of the lesions in experimental sheep showed similar results to those described in the accidental outbreak, except for the absence of optic nerve fibrosis and inflammation, characterizing an acute phase. Axonal myelin sheaths loss, fibroblasts and collagen fibers were observed in the ultrastructural study of the optic nerve of accidental intoxicated animals. The optic nerve of experimentally intoxicated animals had vacuoles that separated the myelin sheaths of axons. To prevent outbreaks it is suggested to weigh the animals before Closantel administration to avoid errors in dose calculation.

Effects of Corynespora cassiicola on Lantana camara

The present study combines the examination of toxins produced by C. cassiicola and the effects of the fungus colonization on L. camara. C. cassiicola was cultivated on solid media and the crude extracts CAE and CE were produced. Both extracts were submitted to a seed germination and growth assay utilizing Physalis ixocarpa, Trifolium alexandrinum, Lolium multiflorum and Amaranthus hypochodriacus. The effect of the extracts on the ATP-synthesis in isolated spinach chloroplasts was also tested. Bioassay guided chromatographic fractionation identified the most active extract (CAE). From this extract ergosta-4,6,8(14),22-tetraen-3-one (C1) and fatty acids were isolated. The C1 compound reduce ATP synthesis in isolated spinach chloroplasts. The interference of fatty acids with ATP synthesis and also with weed growth provides one explanation of the phytogrowth-inhibitory properties of such fungal extracts. Histological observations involving fungus-plant interaction were made on L. camara plants inoculated with C. cassiicola conidia suspension. After inoculations, fragments of the leaf blades were prepared for observation by light and scanning electron microscopy. Fungal colonization of Lantana camara was typical of a necrotroph and penetration initiated a hypersensitive response. L. camara reacted to the pathogen penetration through thickening of the epidermis walls, cytoplasm granulation and a cicatrisation tissue.

Tolerance of sugarcane cultivars to chemical eradication

Knowledge of the minimum rate of glyphosate required to eradicate sugarcane ratoons can reduce the amount of herbicide used. To confirm this hypothesis, this study aimed to investigate the tolerance of different sugarcane cultivars to chemical eradication, at different glyphosate rates. The experiment was conducted in a randomized block design in a split-plot scheme, with four replications. The sugarcane cultivars (IACSP94-2094, IACSP94-2101, IACSP93-3046, IACSP94‑4004, IAC86-2480, and RB72454) were allocated in plots and the glyphosate rates (0, 1,440, 2,160, 2,880, 3,600, and 4,320 g ha-1), in the sub plots. The traits evaluated were signs of poisoning symptoms; total chlorophyll content, plant height, percentage of dead tillers, and dry weight of the plants. At 45 days after application (DAA), the glyphosate rate of 1,440 g a.e. ha-1 eradicated the cultivars IACSP94-2094 and IACSP94-2101, as well as RB72454 with application of 2,160 g a.e. ha‑1. Application of glyphosate 2,880 g a.e. ha-1 eradicated both IACSP93-3046 and IAC86-2480 and glyphosate 3,600 g a.e. ha-1 eradicated IACSP94-4004. The most tolerant cultivar was IACSP94‑4004, eradicated at the rate of 3,600 g. a.e. ha-1. This confirms the hypothesis that knowing the cultivar's tolerance leads, in practice, to a smaller amount of herbicide applied to the environment, which also reduces production costs.

Sorption of fomesafen in Brazilian soils

The study of the dynamics of a herbicide in the soil focus on the interactions with environmental components to obtain agronomic efficiency, ensuring selectivity to the culture and risk reduction of environmental impact. This study evaluated the sorption process of fomesafen in the Brazilian soils Ultisol, Cambisol, and Organosol. Besides soil, washed sand was used as an inert material for determination of the sorption ratio of fomesafen in the soil. The bioassay method was applied, using Sorghum vulgare plants as bio-indicator of herbicide presence. Plant poisoning evaluation and harvest for dry matter determination were carried out 21 days after sorghum sowing. To calculate C50, the nonlinear log-logistic model was applied and sorption ratios of the herbicide were obtained in different soils. The decreasing sorption ratio of formesafen in the soils was: Organosol > Ultisol > Cambisol. It was concluded that the contents of organic matter and clay in the soils were the attributes that most influenced fomesafen sorption.

APPLICATION HEIGHT IN HERBICIDES EFFICIENCY IN BEAN CROPS

ABSTRACTInadequate herbicide application can result in failures in weed control and/or poisoning of the crops, resulting in yield losses. In this research were assessed the effects of the sprayer nozzle boom height in the distribution of the spray solution for weed control, influencing intoxication of beans and crop yield. Experiments were conducted in laboratory and field conditions. In laboratory, the performance of flat spray tip TT 11002 was assessed at heights 0.20, 0.30, 0.40 and 0.50 meters with respect to the target surface. In the field the same heights were assessed in applications of herbicides fomesafen, fluazifop-P-butyl and fomesafen + fluazifop-P-butyl. There was an inverse relationship between the height of the spray boom and the coefficients of variation of the patterns. The mixture better efficiency in a tank of fluazifop-P-butyl + fomesafen was obtained with the height of 0.50 m from the target. This treatment resulted in better weed control, lower poisoning of the bean plants and better crop yield rates.

Does the Ingestion of Isoxaflutole Herbicide Affect the Midgut and Salivary Glands of Pentatomidae Predators?

Podisus nigrispinus is a generalist predator naturally occurring in agricultural and forestry systems that effectively contributes to the population balance of phytophagous insects, especially defoliating caterpillars. Histological changes were evaluated in the salivary glands and midgut of P. nigrispinus caused by ingestion of systemic herbicide isoxaflutole. These predator females were fed with leaves of eucalyptus plants, Tenebrio molitor pupae or water, contaminated or not by herbicide. Salivary glands and midguts were dissected, processed and analyzed under a light microscope. Activity level and cell morphology of the salivary glands and midgut showed differences among insects fed on plants, contaminated water or pupae. The epithelia of the salivary gland and midgut of individuals which had no contact with the herbicide showed homogeneous cytoplasm, nucleus with predominance of decondensed chromatin and evident nucleoli, intense cell activity features. As for the insects in contact with contaminated food, they presented undeveloped nucleus and condensed chromatin. The luminal contents of the salivary glands in the contaminated insects had become more acidophilus than in insects without poisoning, as well as having heterogeneous and granular secretion, being more evident in the bioassay in which the insects fed on contaminated water. There was a marked morphological change in the midgut cells in contaminated insects. High degree of apoptosis, disorganization and secretory vacuoles in the epithelial cytoplasm were observed. The apical portion of the midgut cells proved undeveloped, irregular and partially destroyed. It is concluded that isoxaflutole causes morphological changes in the digestive system of the predator P. nigrispinus.

Single-step purification of crotapotin and crotactine from Crotalus durissus terrificus venom using preparative isoelectric focusing

We describe the isolation of crotoxin, a presynaptic B-neurotoxin, as well as its subunits B (crotactine) and A (crotapotin) from lyophilized Crotalus durissus terrificus venom by a single-step preparative isoelectric focusing procedure. From 98 mg of dried venom protein 20.1 mg of crotactine and 13.1 mg of crotapotin were recovered in the first step of focalization and 4.2 mg in a second run. These values correspond to 35.7% of the total venom protein applied. Crotactine separated in the 9.3-7.0 pH range (tubes 1-6) and crotapotin in the 1.8-2.8 pH range (tubes 15-19) and both were homogeneous by SDS-PAGE and N-terminal amino acid analysis. Crotactine, a 12-kDa protein, presented hemolytic and phospholipase A2 activity. Thus, using isoelectric focusing we simultaneously purified both toxins in high yields. This method can be used as an alternative for the purification and characterization of proteins from other snake venoms under conditions in which biological activity is retained

Rare adverse events associated with oral poliovirus vaccine in Brazil

Oral poliovirus vaccine (OPV) developed by A. Sabin has been effectively used to control poliomyelitis in Brazil, and the last case with the isolation of a wild poliovirus strain occurred in March 1989. Although the vaccine controlled the circulation of wild strains and poliomyelitis cases associated with these strains were not detected during the last eight years, rare cases classified as vaccine-associated paralytic poliomyelitis (VAPP) have been detected. Molecular characterization studies of poliovirus strains isolated from VAPP cases and from healthy contacts have confirmed that the isolates are derived from the Sabin vaccine strains and also detected genomic modifications known or suspected to increase neurovirulence such as mutations and recombination. The molecular characterization of polioviruses isolated during the last eight years from paralysis cases classified as Guillain-Barré (GBS) syndrome and transverse myelitits (TM), and from facial paralysis (FP) cases also confirmed the vaccine origin of the strains and demonstrated mutations known to increase neurovirulence. Analysis of the epidemiologic data of these GBS, TM and FP cases demonstrated that in most of them the last OPV dose was given months or years before the onset of the disease and the isolation of the polioviruses. The temporal association between the isolation of these strains and the GBS, TM and FP suggested that the Sabin vaccine-derived poliovirus strains could also rarely trigger the diseases.

E. coli a-hemolysin: a membrane-active protein toxin

Alpha-Hemolysin is synthesized as a 1024-amino acid polypeptide, then intracellularly activated by specific fatty acylation. A second activation step takes place in the extracellular medium through binding of Ca2+ ions. Even in the absence of fatty acids and Ca2+ HlyA is an amphipathic protein, with a tendency to self-aggregation. However, Ca2+-binding appears to expose hydrophobic patches on the protein surface, facilitating both self-aggregation and irreversible insertion into membranes. The protein may somehow bind membranes in the absence of divalent cations, but only when Ca2+ (or Sr2+, or Ba2+) is bound to the toxin in aqueous suspensions, i.e., prior to its interaction with bilayers, can a-hemolysin bind irreversibly model or cell membranes in such a way that the integrity of the membrane barrier is lost, and cell or vesicle leakage ensues. Leakage is not due to the formation of proteinaceous pores, but rather to the transient disruption of the bilayer, due to the protein insertion into the outer membrane monolayer, and subsequent perturbations in the bilayer lateral tension. Protein or glycoprotein receptors for a-hemolysin may exist on the cell surface, but the toxin is also active on pure lipid bilayers.

Partial sequence and toxic effects of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera

A neurotoxic peptide, granulitoxin (GRX), was isolated from the sea anemone Bunodosoma granulifera. The N-terminal amino acid sequence of GRX is AKTGILDSDGPTVAGNSLSGT and its molecular mass is 4958 Da by electrospray mass spectrometry. This sequence presents a partial degree of homology with other toxins from sea anemones such as Bunodosoma caissarum, Anthopleura fuscoviridis and Anemonia sulcata. However, important differences were found: the first six amino acids of the sequence are different, Arg-14 was replaced by Ala and no cysteine residues were present in the partial sequence, while two cysteine residues were present in the first 21 amino acids of other toxins described above. Purified GRX injected ip (800 µg/kg) into mice produced severe neurotoxic effects such as circular movements, aggressive behavior, dyspnea, tonic-clonic convulsion and death. The 2-h LD50 of GRX was 400 ± 83 µg/kg.

Modulation of fibronectin expression in the central nervous system of Lewis rats with experimental autoimmune encephalomyelitis

Fibronectin (FN), a large family of plasma and extracellular matrix (ECM) glycoproteins, plays an important role in leukocyte migration. In normal central nervous system (CNS), a fine and delicate mesh of FN is virtually restricted to the basal membrane of cerebral blood vessels and to the glial limitans externa. Experimental autoimmune encephalomyelitis (EAE), an inflammatory CNS demyelinating disease, was induced in Lewis rats with a spinal cord homogenate. During the preclinical phase and the onset of the disease, marked immunolabelling was observed on the endothelial luminal surface and basal lamina of spinal cord and brainstem microvasculature. In the paralytic phase, a discrete labelling was evident in blood vessels of spinal cord and brainstem associated or not with an inflammatory infiltrate. Conversely, intense immunolabelling was present in cerebral and cerebellar blood vessels, which were still free from inflammatory cuffs. Shortly after clinical recovery minimal labelling was observed in a few blood vessels. Brainstem and spinal cord returned to normal, but numerous inflammatory foci and demyelination were still evident near the ventricle walls, in the cerebral cortex and in the cerebellum. Intense expression of FN in brain vessels ascending from the spinal cord towards the encephalon preceded the appearance of inflammatory cells but faded away after the establishment of the inflammatory cuff. These results indicate an important role for FN in the pathogenesis of CNS inflammatory demyelinating events occurring during EAE.

Binding sites and actions of Tx1, a neurotoxin from the venom of the spider Phoneutria nigriventer, in guinea pig ileum

Tx1, a neurotoxin isolated from the venom of the South American spider Phoneutria nigriventer, produces tail elevation, behavioral excitation and spastic paralysis of the hind limbs after intracerebroventricular injection in mice. Since Tx1 contracts isolated guinea pig ileum, we have investigated the effect of this toxin on acetylcholine release, as well as its binding to myenteric plexus-longitudinal muscle membranes from the guinea pig ileum. [125I]-Tx1 binds specifically and with high affinity (Kd = 0.36 ± 0.02 nM) to a single, non-interacting (nH = 1.1), low capacity (Bmax 1.1 pmol/mg protein) binding site. In competition experiments using several compounds (including ion channel ligands), only PhTx2 and PhTx3 competed with [125I]-Tx1 for specific binding sites (K0.5 apparent = 7.50 x 10-4 g/l and 1.85 x 10-5 g/l, respectively). PhTx2 and PhTx3, fractions from P. nigriventer venom, contain toxins acting on sodium and calcium channels, respectively. However, the neurotoxin PhTx2-6, one of the isoforms found in the PhTx2 pool, did not affect [125I]-Tx1 binding. Tx1 reduced the [3H]-ACh release evoked by the PhTx2 pool by 33%, but did not affect basal or KCl-induced [3H]-ACh release. Based on these results, as well as on the homology of Tx1 with toxins acting on calcium channels (w-Aga IA and IB) and its competition with [125I]-w-Cono GVIA in the central nervous system, we suggest that the target site for Tx1 may be calcium channels.

Effects of 60Co gamma radiation on crotamine

Ionizing radiation can change the molecular structure and affect the biological properties of biomolecules. This has been employed to attenuate animal toxins. Crotamine is a strongly basic polypeptide (pI 10.3) from Crotalus durissus terrificus venom composed of 42 amino acid residues. It induces skeletal muscle spasms leading to a spastic paralysis of hind limbs in mice. The objective of the present study was to carry out a biochemical study and a toxic activity assay on native and irradiated crotamine. Crotamine was purified from C.d. terrificus venom by Sephadex G-100 gel filtration followed by ion-exchange chromatography, and irradiated at 2 mg/ml in 0.15 M NaCl with 2.0 kGy gamma radiation emitted by a 60Co source. The native and irradiated toxins were evaluated in terms of structure and toxic activity (LD50). Irradiation did not change the protein concentration, the electrophoretic profile or the primary structure of the protein although differences were shown by spectroscopic techniques. Gamma radiation reduced crotamine toxicity by 48.3%, but did not eliminate it.

Biological activity of Serratia marcescens cytotoxin

Serratia marcescens cytotoxin was purified to homogeneity by ion-exchange chromatography on a DEAE Sepharose Fast Flow column, followed by gel filtration chromatography on a Sephadex G100 column. The molecular mass of the cytotoxin was estimated to be about 50 kDa. Some biological properties of the cytotoxin were analyzed and compared with well-characterized toxins, such as VT1, VT2 and CNF from Escherichia coli and hemolysin produced by S. marcescens. The sensitivity of the cell lines CHO, HeLa, HEp-2, Vero, BHK-21, MA 104 and J774 to the cytotoxin was determined by the cell viability assay using neutral red. CHO and HEp-2 were highly sensitive, with massive cellular death after 1 h of treatment, followed by BHK-21, HeLa, Vero and J774 cells, while MA 104 was insensitive to the toxin. Cytotoxin induced morphological changes such as cell rounding with cytoplasmic retraction and nuclear compactation which were evident 15 min after the addition of cytotoxin. The cytotoxic assays show that 15 min of treatment with the cytotoxin induced irreversible intoxication of the cells, determined by loss of cell viability. Concentrations of 2 CD50 (0.56 µg/ml) of purified cytotoxin did not present any hemolytic activity, showing that the cytotoxin is distinct from S. marcescens hemolysin. Antisera prepared against S. marcescens cytotoxin did not neutralize the cytotoxic activity of VT1, VT2 or CNF toxin, indicating that these toxins do not share antigenic determinants with cytotoxin. Moreover, we did not detect gene sequences for any of these toxins in S. marcescens by PCR assay. These results suggest that S. marcescens cytotoxin is not related to any of these toxins from E. coli.