Analysis of Escherichia coli isolated from bovine mastitic milk

Mastitis has been recognized for some time as the most costly disease in dairy herds. From February to November 2004, 670 samples of bovine mastitic milk from which 231 Escherichia coli strains were isolated, were collected from two Brazilian states. The strains were screened for the presence of Shiga toxin-producing (stx 1 and stx 2) and intimin (eae) genes. Twenty (8.6%) strains were detected by PCR to harbor the Shiga toxin genes (8 the stx 1 gene, 12 the stx 2 gene and none both of them). Two (0.8%) of the Escherichia coli strains studied were eae positive non Shiga toxin-producing. The strains were also examined for resistance to 12 antimicrobial agents. The predominantly observed resistance was to tetracycline (92.2%), streptomycin (90.4%), nalidixic acid (88.3%), amikacin (86.5%) and cephalothin (84.8%). Multidrug resistance was found among 152 isolates (65.8%).

Focal symmetrical encephalomalacia in sheep

Focal symmetrical encephalomalacia (FSE) is the most prominent lesion seen in the chronic form of enterotoxemia by Clostridium perfringens type D. This paper reports FSE in sheep in Brazil. Six deaths occurred within a seven days period in a flock of 70, four to 30-month-old Santa Inês sheep in the state of Paraíba in the Brazilian semiarid. The flock was grazing a paddock of irrigated sprouting Cynodon dactylon (Tifton grass), and supplemented, ad libitum, with a concentrate of soybean, corn and wheat. Nervous signs included blindness and recumbence. A 19 month-old sheep was examined clinically and necropsied after a clinical course of three days. Gross lesions were herniation of the cerebellar vermis and multifocal, bilateral, symmetric brownish areas in the internal capsule, thalamus and cerebellar peduncles. Histologic lesions were multifocal, bilateral malacia with some neutrophils, swelling of blood vessels endothelium, perivascular edema, and hemorrhages. The flock was vaccinated, before the outbreak, with only one dose of Clostridium perfringens type D vaccine. Two factors are suggested to be important for the occurrence of the disease: insufficient immunity due to the incorrect vaccination; and high nutritional levels by the supplementation with highly fermentable carbohydrates.

Necrotizing enteritis associated with Clostridium perfringensType B in chinchillas (Chinchilla lanigera)

Four 3-4 month-old chinchillas (Chinchilla lanigera) from a commercial flock of 395 chinchillas, were found dead with evidence of previous diarrhea and prolapsed rectum. A fifth 8 month-old chinchilla died 8 hours after being found recumbent, apathetic, diarrheic and with a prolapsed rectum. Two chinchillas were necropsied and observed gross lesions consisted of extensive hemorrhagic enteritis, mild pulmonary edema and enlarged and yellow liver; this latter finding was particularly prominent in the chinchilla presenting longer clinical course. Histologically there was necrotizing enteritis associated with abundant bacterial rods aggregates in the intestinal surface epithelium and within the lamina propria. In the lungs there were small amounts of pink proteinaceous material (edema) in the interstitium and marked vacuolar hepatocellullar degeneration (lipidosis) in the liver. Anaerobic cultures from the intestinal contents of one of the affected chinchillas yielded Clostridium perfringens. Genotyping of this C. perfringens isolate was achieved by multiplex polymerase chain reaction (mPCR) as C. perfringenstype B due to detection of alpha, beta and epsilon-toxin genes. These findings suggest C. perfringens type B as an important cause of sudden or acute death in chinchillas.

Sawfly larval poisoning in cattle: Report on new outbreaks and brief review of the literature

Sawfly larval poisoning (SLP) is an acute hepatotoxicosis documented in livestock in Australia, Denmark and in countries of South America. It is caused by the ingestion of the larval stage of insects of the suborder Symphyta, order Hymenoptera, commonly known as "sawfly". Three species of sawfly are reportedly involved in the toxicosis. The insect involved in Australian SLP is Lophyrotoma interrupta (Pergidae), in Denmark the cause of SLP is the ingestion of the larvae Arge pullata (Argidae), and in South American countries documented outbreaks of SLP were caused by the ingestion of yet another sawfly, Perreyia flavipes (Pergidae). In all geographical areas where it occurred, SLP causes important livestock losses. In cattle, as well as in other affected species, the disease has a short clinical course and in many outbreaks affected cattle can be found dead. When observed, clinical signs include apathy, recumbence, tremors, paddling movements and death in 24-48 hours. Neurological signs such aggressiveness attributable to hepatic encephalopathy are also observed. In cases with a more protracted course icterus and photodermatitis may develop. Gross findings included ascites, petechiae and ecchymosis over serosal surfaces of thoracic and abdominal cavities, and an enlarged liver that displays accentuation of the lobular pattern and edema of the gall bladder wall. Sawfly larval body fragments and heads are consistently found in the fore stomachs and occasionally abomasum of affected cattle. Main microscopic lesions are restricted to the liver and consist of centrolobular (periacinar) to massive hepatocellular necrosis. In most lobules necrotic areas extended up to the portal triads where only a few viable hepatocytes remain. Mild to moderate lymphocyte necrosis is seen in lymphatic tissues. Cases occur in the winter months when the larval stages of the sawfly are developing. D-amino acid-containing peptides have been found to be the toxic principle in each sawfly involved in SLP. The octapeptide lophyrotomin is the major toxin in the in the larvae of Australian and Danish sawflies and is present in small amounts in the larvae of South American sawfly. The heptadecapeptide pergidin is the main toxin in the South American sawfly while small amounts of pergidin have been found in the other two species of toxic sawfly. During the winter of 2011 (July-August) four outbreaks of SLP were diagnosed in the State of Rio Grande do Sul, Brazil. The findings in those outbreaks are reported here and a brief review of the literature regarding SLP around the world is provided.

Type C botulism in swine fed on restaurant waste

The paper addresses the epidemiologic data of the death of pigs during the period of 2002 to 2009 following the ingestion of botulinum neurotoxin type C. This neurotoxin was present in food residues originating from restaurant and hotel kitchens, stored in barrels without shelter from the sun and administered in a collective trough without prior thermal treatment. Animals which died at different ages showed clinical signs of botulism characterized by flaccid paralysis, weight loss, anorexia, weakness, lack of coordination, locomotion difficulties with the evolution of lateral recumbency with involuntary urination and defecation. No alterations were observed at postmortem and histological examination. The bioassay with serum neutralization in mice was carried out on samples of intestinal contents from pigs affected and revealed the presence of large quantities of botulinum toxin type C.

Kinetics of epsilon antitoxin antibodies in different strategies for active immunization of lambs against enterotoxaemia

Enterotoxaemia, a common disease that affects domestic small ruminants, is mainly caused by the epsilon toxin of Clostridium perfringens type D. The present study tested four distinct immunization protocols to evaluate humoral response in lambs, a progeny of non-vaccinated sheep during gestation. Twenty-four lambs were randomly allocated into four groups according to age (7, 15, 30 and 45 days), receiving the first dose of epsilon toxoid commercial vaccine against clostridiosis with booster after 30 days post vaccination. Indirect ELISA was performed after the first vaccine dose and booster to evaluate the immune response of the lambs. Results showed that for the four protocols tested all lambs presented serum title considered protective (≥0.2UI/ml epsilon antitoxin antibodies) and also showed that the anticipation of primovaccination of lambs against enterotoxaemia conferred serum title considered protective allowing the optimization of mass vaccination of lambs.

Induction of immune response in broiler chickens immunized with recombinant FliC and challenged by Salmonella Typhimurium

The study examined (1) the immune response in broiler chickens after oral immunization with recombinant flagellin (rFliC) from Salmonella Typhimurium conjugated with sodium alginate microparticles, and the immune response enhancement in association with recombinant cholera toxin B subunit protein (rCTB) and pool of Lactobacillus spp. (PL). The immune responses were evaluated by dosage of IgY serum and IgA from intestinal fluid and immunostaining of CD8+ T lymphocytes in the cecum. The immunized animals were challenged with Salmonella Typhimurium (ST) 21 days after treatment. In all immunized groups, a significant increase (p<0.05) was observed in IgA levels (μg/mL), especially three weeks after immunization. The serum IgY levels (μg/mL) were little affected by the treatments and differed significantly among groups only in the second post-immunization week (p<0.05). After the challenge, the number of CD8+ T cells differed significantly between the treatments and negative control. Retrieval of Salmonella Typhimurium was not detected at 48 hours after the challenge in T2 (rFliC+rCTb), T3 (rFliC+PL) and T4 (rFliC+rCTB PL). The rFliC administered orally with or without rCTB and Lactobacillus spp. produces significant induction of humoral immune response, and the immunized chickens were more effective in eliminating Salmonella after challenge.

Enterohemorrhagic Escherichia coli O157: H7 from healthy dairy cattle in Mid-West Brazil: occurrence and molecular characterization

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 represents the major Shiga toxin-producing E. coli (STEC) strain related to large outbreaks and severe diseases such as hemorrhagic colitis (HC) and the potentially lethal hemolytic uremic syndrome (HUS). The aim of this study was to report the occurrence and molecular characterization of O157:H7 isolates obtained by rectal swab from 52 healthy dairy cattle belonging to 21 farms in Mid-West of Brazil. Detection of 16SrRNA, stx1, stx2, rfbO157, fliCh7, eae, ehxA, saa, cnf1, chuA, yjaA and TSPE4.C2 genes was performed by PCR. The isolates were further characterized by serotyping. Two hundred and sixty E. coli isolates were obtained, of which 126 were characterized as STEC. Two isolates from the same cow were identified as serotype O157:H7. Both isolates presented the stx2, eae, ehxA, saa and cnf1 virulence factor genes and the chuA gene in the phylogenetic classification (virulent group D), suggesting that they were clones. The prevalence of O157:H7 was found to be 1.92% (1/52 animals), demonstrating that healthy dairy cattle from farms in the Mid-West of Brazil are an important reservoir for highly pathogenic E. coli O157:H7.

Brazilian soybean Glycine max (L.) Merr. cultivars adapted to low latitude regions: seed composition and content of bioactive proteins

Among the goals of the Brazilian soybean improvement programmes, the breeding strategies for cultivars adapted to low latitudes have been included to extend crop areas and to increase production. Seeds of nine Brazilian soybean cultivars adapted to low latitudes were investigated regarding to their composition, and amino acid and antinutritional/toxic protein contents. Protein (394.5 ± 13.1 to 445.3 ± 8.0 g kg-1 dry matter) and oil (200.6 ± 1.2 to 232.3 ± 4.7 g kg-1 dry matter) contents showed low correlation to each other (r = -0.06). The total carbohydrate (141.7 ± 6.1 to 211.1 ± 15.0 g kg-1 dry matter) and ash contents (48.2 ± 4.2 to 52.2 ± 0.5 g kg-1 dry matter) were similar to data available for other soybean cultivars. All soybean cultivars presented low levels of tryptophan and sulphur amino acids. The lectin (1,152 to 147,456 HU kg-1 flour), trypsin inhibitor (34.45 ± 2.28 to 77.62 ± 2.63 g trypsin inhibited kg-1 flour), toxin (6,210 ± 134 to 34,650 ± 110 LD50 kg-1 flour) and urease (0.74 ± 0.02 to 1.22 ± 0.10 g kg¹ flour) presented variations in their contents amongst the cultivars. Compared to other soybean cultivars, urease was higher, the acute toxicity lower and the lectin and trypsin inhibitor contents similar to data available. In general, soybean cultivars showed similar biochemical composition to those developed in different geographic regions. The relevance of these findings to the agronomic features and to choice of soybean cultivars to be used as food or feed is discussed.

Induction of neutralizing antibodies in mice immunized with scorpion toxins detoxified by liposomal entrapment

The possibility of producing neutralizing antibodies against the lethal effects of scorpion toxins was evaluated in the mouse model by immunization with an immunogen devoid of toxicity. A toxic fraction (5 mg) from the venom of the scorpion Tityus serrulatus was entrapped in sphingomyelin-cholesterol liposomes. The liposomes were treated for 1 h at 37oC with a 1% (w/w) trypsin solution in 0.2 M sodium carbonate buffer, pH 8.3. This treatment led to a strong reduction in venom toxicity. Immunization was performed as follows: mice were injected sc with 20 µg of the liposome-entrapped toxic fraction on days 1 and 21 and a final injection (20 µg) was administered ip on day 36. After injection of the immunogen, all mice developed an IgG response which was shown to be specific for the toxic antigen. The antibodies were measured 10 days after the end of the immunization protocol. In an in vitro neutralization assay we observed that pre-incubation of a lethal dose of the toxic fraction with immune serum strongly reduced its toxicity. In vivo protection assays showed that mice with anti-toxin antibodies could resist the challenge with the toxic fraction, which killed, 30 min after injection, all non-immune control mice

Detection of cytotoxic activity on Vero cells in clinical isolates of Serratia marcescens

Cytotoxin production was studied in 60 Serratia marcescens strains isolated from hospitalized patients. Association of cytotoxic activity with serotype, source of isolation and presence of plasmids was also evaluated. Thirteen of the 60 S. marcescens strains produced a cytotoxic effect on Vero cells. These strains were isolated from distinct clinical sources and classified into seven different serotypes (O1:H7; O4:NM; O10:NT; O19:NM; O6,14:H4; O6,14:NM and O6,14:H1). No relationship was observed between cytotoxic activity and clinical source or serotypes of the strains. Plasmids from five cytotoxin-producing S. marcescens strains were transferred to E. coli K12/711. The transconjugants did not exhibit cytotoxicity, indicating that the cytotoxic effect is not plasmid-mediated among these strains. Although a cytotoxic activity was demonstrated in filtrates of some S. marcescens strains, further studies should be performed to assess the role of this toxin in pathogenesis

Cyanobacterial toxins in Portugal: effects on aquatic animals and risk for human health

Toxic cyanobacteria are common in Portuguese freshwaters and the most common toxins are microcystins. The occurrence of microcystin-LR (MCYST-LR) has been reported since 1990 and a significant number of water reservoirs that are used for drinking water attain high levels of this toxin. Aquatic animals that live in eutrophic freshwater ecosystems may be killed by microcystins but in many cases the toxicity is sublethal and so the animals can survive long enough to accumulate the toxins and transfer them along the food chain. Among these, edible mollusks, fish and crayfish are especially important because they are harvested and sold for human consumption. Mussels that live in estuarine waters and rivers where toxic blooms occur may accumulate toxins without many significant acute toxic effects. In this study data are presented in order to understand the dynamics of the accumulation and depuration of MCYST-LR in mussels. The toxin is readily accumulated and persists in the shellfish for several days after contact. In the crayfish the toxin is accumulated mainly in the gut but is also cleared very slowly. In carps, although the levels of the toxins found in naturally caught specimens were not very high, some toxin was found in the muscle and not only in the viscera. This raises the problem of the toxin accumulation by fish and possible transfer through the food chain. The data gathered from these experiments and from naturally caught specimens are analyzed in terms of risk for human consumption. The occurrence of microcystins in tap water and the incidence of toxic cyanobacteria in fresh water beaches in Portugal are reported. The Portuguese National Monitoring Program of cyanobacteria is mentioned and its implications are discussed.

Guanylin peptides: cyclic GMP signaling mechanisms

Guanylate cyclases (GC) serve in two different signaling pathways involving cytosolic and membrane enzymes. Membrane GCs are receptors for guanylin and atriopeptin peptides, two families of cGMP-regulating peptides. Three subclasses of guanylin peptides contain one intramolecular disulfide (lymphoguanylin), two disulfides (guanylin and uroguanylin) and three disulfides (E. coli stable toxin, ST). The peptides activate membrane receptor-GCs and regulate intestinal Cl- and HCO3- secretion via cGMP in target enterocytes. Uroguanylin and ST also elicit diuretic and natriuretic responses in the kidney. GC-C is an intestinal receptor-GC for guanylin and uroguanylin, but GC-C may not be involved in renal cGMP pathways. A novel receptor-GC expressed in the opossum kidney (OK-GC) has been identified by molecular cloning. OK-GC cDNAs encode receptor-GCs in renal tubules that are activated by guanylins. Lymphoguanylin is highly expressed in the kidney and heart where it may influence cGMP pathways. Guanylin and uroguanylin are highly expressed in intestinal mucosa to regulate intestinal salt and water transport via paracrine actions on GC-C. Uroguanylin and guanylin are also secreted from intestinal mucosa into plasma where uroguanylin serves as an intestinal natriuretic hormone to influence body Na+ homeostasis by endocrine mechanisms. Thus, guanylin peptides control salt and water transport in the kidney and intestine mediated by cGMP via membrane receptors with intrinsic guanylate cyclase activity.

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.

Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice

The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins.