Preliminary study of Porcine circovirus type 2 and Torque teno sus virus coinfection frequencies in Brazilian pig herds

Torque teno sus virus (TTSuV) is emergent in swine herds. Recent studies have shown an increased frequency of TTSuV2 in Porcine circovirus type 2 (PCV2)-associated diseases (PCVAD), which are endemic in many swine-producing countries, including Brazil. Coinfection with several other viral and bacterial agents results in an increased incidence of more severe PCVAD. Given the limited information on TTSuV and PCV2 coinfection, especially in Brazilian swine herds, this study made a preliminary estimation of the occurrence of coinfection in swine herds by testing samples from different categories. Between 2008 and 2009, 111 samples of feces and 23 serum samples from 5 swine herds were tested for PCV2 and TTSuVs and the results analyzed for associations between these agents. No significant differences in coinfection frequency were observed for PCV2 1 + TTSuV1 or for PCV2 1 TTSuV2 between nursery piglets (P = 0.730), growing pigs (P = 0.331), or sows (P = 0.472). However, a significant difference was observed for PCV2 1 TTSuV1 1 TTSuV2 between nursery piglets and growing pigs (P = 0.004; Fisher's exact test). Phylogenetic studies agreed with the grouping of TTSuV1 and TTSuV2 into 2 different clades, with no distinct pattern of clustering of these isolates with the animal categories.

Colibacillosis in lambs is associated to type I heat-stable enterotoxin in a farm in Sao Paulo State, Brazil

Twenty seven (48.2%) culture supernatants of 56 Escherichia coli isolated from diarrheic lamb feces (7 to 10 days old) in Sao Paulo State, Brazil, presented positive results to suckling mice assay (fluid accumulation) but none caused cytopathic effects on Vero and CHO cells, indicating that these strains did not produced LT or VT toxins. PCR assays showed that these 27 E. coli strains harbored estA, that codifies for STa, but not for stx1, stx2 or cnf genes. The positive STa strains were checked for genes that codify for F41, F17 and K99 fimbriae, wich are considered colonization factors in ETEC. Only F17 was detect in two samples (7.4%). Twelve of 27 STa positive carried hlyA gene and presented hemolytic activity in blood Agar. Presence of rotavirus was not detected among the diarrheic feces. These data suggests that STa must be an important diarrheagenic factor to small ruminants in Sao Paulo State.

Molecular cloning and insecticidal effect of Inga laurina trypsin inhibitor on Diatraea saccharalis and Heliothis virescens

Native Inga laurina (Fabaceae) trypsin inhibitor (ILTI) was tested for anti-insect activity against Diatraea saccharalis and Heliothis virescens larvae. The addition of 0.1% ILTI to the diet of D. saccharalis did not alter larval survival but decreased larval weight by 51%. The H. virescens larvae that were fed a diet containing 0.5% ILTI showed an 84% decrease in weight. ILTI was not digested by the midgut proteinases of either species of larvae. The trypsin levels were reduced by 55.3% in the feces of D. saccharalis and increased by 24.1% in the feces of H. virescens. The trypsin activity in both species fed with ILTI was sensitive to the inhibitor, suggesting that no novel proteinase resistant to ILTI was induced. Additionally, ILTI exhibited inhibitory activity against the proteinases present in the larval midgut of different species of Lepidoptera. The organization of the ilti gene was elucidated by analyzing its corresponding genomic sequence. The recombinant ILTI protein (reILTI) was expressed and purified, and its efficacy was evaluated. Both native ILTI and reILTI exhibited a similar strong inhibitory effect on bovine trypsin activity. These results suggest that ILTI presents insecticidal properties against both insects and may thus be a useful tool in the genetic engineering of plants. (c) 2012 Elsevier Inc. All rights reserved.

Lactobacillus acidophilus CRL 1014 improved “gut health” in the SHIME® reactor

Abstract Background How to maintain “gut health” is a goal for scientists throughout the world. Therefore, microbiota management models for testing probiotics, prebiotics, and synbiotics have been developed. Methods The SHIME® model was used to study the effect of Lactobacillus acidophilus 1014 on the fermentation pattern of the colon microbiota. Initially, an inoculum prepared from human feces was introduced into the reactor vessels and stabilized over 2-wk using a culture medium. This stabilization period was followed by a 2-wk control period during which the microbiota was monitored. The microbiota was then subjected to a 4-wk treatment period by adding 5 mL of sterile peptone water with L. acidophilus CRL1014 at the concentration of 108 CFU/mL to vessel one (the stomach compartment). Plate counts, Denaturing Gradient Gel Electrophoresis (DGGE), short-chain fatty acid (SCFA) and ammonium analyses were carried out for monitoring of the microbial community from the colon compartments. Results A significant increase (p < 0.01) in the Lactobacillus spp. and Bifidobacterium spp. populations was observed during the treatment period. The DGGE obtained showed changes in the lactobacilli community from the colon compartments of the SHIME® reactor. The (SCFA) concentration increased (p < 0.01) during the treatment period, due mainly to significant increased levels of acetic, butyric, and propionic acids. However, ammonium concentrations decreased during the same period (p < 0.01). Conclusions This study showed the beneficial influence of L. acidophilus CRL 1014 on microbial metabolism and lactobacilli community composition for improving human health.

Advanced Glycation in macrophages induces intracellular accumulation of 7-ketocholesterol and total sterols by decreasing the expression of ABCA-1 and ABCG-1

Abstract Background Advanced glycation end products (AGE) alter lipid metabolism and reduce the macrophage expression of ABCA-1 and ABCG-1 which impairs the reverse cholesterol transport, a system that drives cholesterol from arterial wall macrophages to the liver, allowing its excretion into the bile and feces. Oxysterols favors lipid homeostasis in macrophages and drive the reverse cholesterol transport, although the accumulation of 7-ketocholesterol, 7alpha- hydroxycholesterol and 7beta- hydroxycholesterol is related to atherogenesis and cell death. We evaluated the effect of glycolaldehyde treatment (GAD; oxoaldehyde that induces a fast formation of intracellular AGE) in macrophages overloaded with oxidized LDL and incubated with HDL alone or HDL plus LXR agonist (T0901317) in: 1) the intracellular content of oxysterols and total sterols and 2) the contents of ABCA-1 and ABCG-1. Methods Total cholesterol and oxysterol subspecies were determined by gas chromatography/mass spectrometry and HDL receptors content by immunoblot. Results In control macrophages (C), incubation with HDL or HDL + T0901317 reduced the intracellular content of total sterols (total cholesterol + oxysterols), cholesterol and 7-ketocholesterol, which was not observed in GAD macrophages. In all experimental conditions no changes were found in the intracellular content of other oxysterol subspecies comparing C and GAD macrophages. GAD macrophages presented a 45% reduction in ABCA-1 protein level as compared to C cells, even after the addition of HDL or HDL + T0901317. The content of ABCG-1 was 36.6% reduced in GAD macrophages in the presence of HDL as compared to C macrophages. Conclusion In macrophages overloaded with oxidized LDL, glycolaldehyde treatment reduces the HDL-mediated cholesterol and 7-ketocholesterol efflux which is ascribed to the reduction in ABCA-1 and ABCG-1 protein level. This may contribute to atherosclerosis in diabetes mellitus.

Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR) protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg) of feces). Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT) solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

Survey on Chlamydophila psittaci in captive ramphastids in São Paulo State, Brazil

Chlamydophila psittaci (C. psittaci) has been detected in 460 avian species, among them the most frequent are the Psittaciformes, Columbiformes, Anseriformes and raptors. In Brazil, the main avian species recognized as healthy carriers belong to the order Psittaciformes and Columbiformes, but very few studies have been done in other bird families. Reports of the occurrence of this disease in the clinical form are rare in the Ramphastids; consequently, they are not commonly evaluated for this agent. The present study reports the investigation of C. psittaci in 25 captive ramphastids from a zoological park in São Paulo State, Brazil. Swabs samples from the cloaca were submitted to semi-nested polymerase chain reaction (semi-nested PCR) for direct detection of the microorganism. Additionally, blood samples obtained from these birds were submitted to the Complement Fixation Test (CFT) for detection of antibodies anti-C. psittaci. The presence of C. psittaci was not detected in the cloacal swab samples tested by the PCR. Nevertheless, 16% (4/25) of the bird's sera were positive by the CFT. Among the species with positive results, there are the saffron toucanet (Pteroglossus bailloni) and black-necked-aracari (Pteroglossus aracari), two species with no descriptions of the survey of C. psittaci published in the literature. Intermittent elimination of C. psittaci is a feature of chronically infected birds; however the absence of a positive-antigen sample did not guarantee that the bird is Chlamydophila-free. The serological results obtained show that the ramphastids tested were previously exposed to the pathogen and developed immune response, but showed no clinical signs of the disease and didn't eliminate regularly the organism in their feces in the moment of the sample collection.

Molecular typing of Clostridium perfringens isolated from swine in slaughterhouses from São Paulo State, Brazil

Clostridium perfringens is an anaerobic Gram-positive bacterium known as common pathogen for humans, for domestic and wildlife animals. Although infections caused by C. perfringens type C and A in swine are well studied, just a few reports describe the genetic relationship among strains in the epidemiological chain of swine clostridioses, as well as the presence of the microorganism in the slaughterhouses. The aim of the present study was to isolate C. perfringens from feces and carcasses from swine slaughterhouses, characterize the strains in relation to the presence of enterotoxin, alpha, beta, epsilon, iota and beta-2 toxins genes, using polymerase chain reaction (PCR) and comparing strains by means of Pulsed field gel electrophoresis (PFGE). Clostridium perfringens isolation frequencies in carcasses and finishing pig intestines were of 58.8% in both types of samples. According to the polymerase chain reaction assay, only alfa toxin was detected, being all isolates also negative to enterotoxin and beta2 toxin. Through PFGE technique, the strains were characterized in 35 pulsotypes. In only one pulsotype, the isolate from carcass sample was grouped with fecal isolate of the same animal, suggesting that the risk of cross-contamination was low. Despite the high prevalence of C. perfringens in swine carcasses from the slaughterhouses assessed, the risk of food poisoning to Brazilian pork consumers is low, since all strains were negative to cpe-gene, codifying enterotoxin.

Comparison of indigestible markers from in situ and in vivo incubation to predict apparent digestibility in hay- and corn-fed horses

Four castrated crossbred horses were used in a randomized block design to study the use of indigestible internal markers iNDF and iADF obtained in situ (from bovines) or in vivo (from equines). Treatments consisted of determining digestibility by the direct method comprising total feces collection (TC) and by the indirect method comprising internal markers iNDF and iADF obtained by in situ incubation in bovine rumen or in vivo by the mobile nylon bag (MNB) technique with horses. iNDF-IV and iADF-IV resulted in better marker recovery rate (RR) (91.50%), similar to TC. The in situ technique resulted in lower RR values for the two indigestible markers, averaging 86.50% (p < 0.05). Estimates of the nutrient coefficient of digestibility (CD) were adequately predicted by iADF-IV, for horses fed on hay exclusively, with rates 46.41, 48.16, 47.92 and 45.51% for dry matter (DM), organic matter (OM), FDN and gross energy, respectively. Results show that MNB may be used to obtain iADF in horses fed on coast-cross hay exclusively, whereas NDFi and ADFi were selected for horses fed on mixed diets to predict the coefficient of nutrient digestibility.

Effects of flavomycin on ruminal fermentation, In situ degradability and In vivo digestibility in bovine fed sugarcane diets

Problem statement: The aim of the present study was to characterize and differentiate the effects of addition of flavomycin or monensin on ruminal fermentation and degradability as well as on total digestibility in bovine. Approach: Twelve non-pregnant and non-lactating cows (736 kg of BW) were randomly assigned to three treatments: control, flavomycin (20 mg animal-1 day-1) and monensin (300 mg animal-1 day-1). The trial lasted 21 days. The last 10 days were used for external marker administration (15 g of chromic oxide animal-1 day-1). The last 5 days of the trial were used for feces collection and evaluation of corn grain, soybean meal or sugarcane ruminal degradability and the 21st day was used for ruminal fluid sampling. Results: Monensin increased 27.2%, on average, propionate molar proportion at 0, 4, 6, 8, 10 and 12 h after feeding, compared to control and flavomycin groups. When compared to control, flavomycin reduced the degradation rate of soybean meal CP in 31.0%, decreasing the effective degradability when passage rates of 5 and 8% h-1 were used. Dry matter intake, pH, total Short Chain Fatty Acids (tSCFA) or ammoniacal Nitrogen (NH3-N) concentration were not influenced by the addition of either antibiotics. Effective degradability of sugarcane NDF was not influenced by the use of either antibiotic; neither were the TDN nor the digestibility of DM, CP, EE, NFE, ADF, NDF, GE or starch of the diet. Conclusion/Recommendations: In the present study, it was possible to show the beneficial effects of monensin but not of flavomycin, on rumen fermentation