A Study on the Efficiency of Synbiotics for Prevention and Control of Diarrhoea in 21 Dogs Submitted to Radiotherapy

A synbiotic is a formulation containing both probiotics and prebiotics. This study aims to evaluate the effect of supplementation with a synbiotic containing Enterococcus faecium strain E1707 (NCIMB 10415) in preventing or controlling diarrhoea and other gastrointestinal signs in boarded canine radiotherapy patients. A double-blind, randomized, placebocontrolled clinical trial was carried out in 21 adult dogs undergoing radiotherapy and boarded for a duration period of 2 to 3 weeks to treat their cancers. Dogs were randomly divided between two groups: A and B, the synbiotic and placebo group, respectively. The content of the sachets was added to the food once daily. Faecal score was assessed daily, and dogs were also monitored for the development of diarrhoea and other gastrointestinal signs such as weight loss, reduced appetite and vomiting. The results from descriptive statistics seem to favour group B, however these findings were not validated with inferential statistics due to insufficient statistical sample power. Because of this, it is not possible to make conclusions about the benefits of synbiotic as supportive treatment for dogs undergoing radiotherapy. All results should be considered to be preliminary, until they are elucidated by further animal inclusion.

Analysis of Pan-European attitudes to the eradication and control of bovine viral diarrhoea

At present, national-level policies concerning the eradication and control of bovine viral diarrhoea (BVD) differ widely across Europe. Some Scandinavian countries have enacted strong regulatory frameworks to eradicate the disease, whereas other countries have few formal policies. To examine these differences, the attitudes of stakeholders and policy makers in 17 European countries were investigated. A web-based questionnaire was sent to policy makers, government and private sector veterinarians, and representatives of farmers' organisations. On total, 131 individuals responded to the questionnaire and their responses were analysed by applying a method used in sociolinguistics: frame analysis. The results showed that the different attitudes of countries that applied compulsory or voluntary frameworks were associated with different views about the attribution or blame for BVD and the roles ascribed to farmers and other stakeholders in its eradication and control.

The glycosylation pattern of baculovirus expressed envelope protein E2 affects its ability to prevent infection with bovine viral diarrhoea virus

We have investigated the role of glycosylation of the envelope glycoprotein E2 of bovine viral diarrhoea virus (BVDV), produced in insect cells, in BVDV infection. When amino acids predicated to code for the C-terminal N-linked glycosylation site were mutated the resulting protein was less efficient than wild type protein at preventing infection of susceptible cells with BVDV. In addition, mutational analysis showed that a further two predicted N-terminal N-linked glycosylation sites of E2 are required for efficient production of recombinant protein. (c) 2005 Elsevier B.V. All rights reserved.

Prebiotics and probiotics: potential strategies for reducing travellers' diarrhoea in athletes competing abroad

Travellers’ diarrhoea (TD) is the most common gastrointestinal illness to affect athletes competing abroad. Consequences of this debilitating condition include difficulties with training and/or participating in competitions which the athlete may have spent several years preparing for. Currently, there are no targeted strategies to reduce TD incidence in athletes. General methods used to reduce TD risk, such as avoidance of contaminated foods, chemoprophylactics and immunoprophylactics, have disadvantages. Since most causative agents of TD are microbial, strategies to minimise TD risks may be better focused on the gut microbiota. Prebiotics and probiotics can fortify the gut microbial balance, thus potentially aiding the fight against TD-associated microorganisms. Specific probiotics have shown promising actions against TD-associated microorganisms through antimicrobial activities. Use of prebiotics has led to an improved intestinal microbial balance which may be better equipped to combat TD-associated microorganisms. Both approaches have shown promising results in general travelling populations; therefore, a targeted approach for athletes has the potential to provide a competitive advantage.

Investigation of the faecal microbiota associated with canine chronic diarrhoea.

Diarrhoea is a common problem in dogs and can result in disturbance of the normal intestinal microbiota. However, little is known about the gastrointestinal microbiota of dogs with chronic diarrhoea and controlled canine studies of dietary management are scarce. The aims of this study were to investigate the predominant faecal microbiota of chronic diarrhoea dogs and to examine the effect(s) of a fibre blend on the canine faecal microbiota. A 3-week fibre supplementation feeding study was performed in nine chronic diarrhoea and eight control dogs. Atopobium cluster, Lactobacillus-Enterococcus group and Clostridium cluster XIV were the predominant bacterial groups in all dogs. Chronic diarrhoea dogs had significantly higher Bacteroides counts at baseline and significantly lower Atopobium cluster counts following fibre supplementation compared with control dogs. Atopobium cluster levels increased significantly in control dogs, while counts of sulphate-reducing bacteria decreased significantly and Clostridium clusters I and II counts increased significantly in chronic diarrhoea dogs during fibre supplementation. Microbial profiles (detected by denaturing gradient gel electrophoresis) demonstrated interindividual variation, with greater similarity seen between the chronic diarrhoea and control dogs' profiles after fibre supplementation compared with baseline. In conclusion, fibre supplementation induced changes in the canine faecal microbiota, with greater resemblance between the microbiota of chronic diarrhoea and control dogs after this dietary modulation.

A double-blind, placebo-controlled, randomized human study assessing the capacity of a novel galacto-oligosaccharide mixture in reducing travellers' diarrhoea.

Background/Objectives: Prebiotics have attracted interest for their ability to positively affect the colonic microbiota composition, thus increasing resistance to infection and diarrhoeal disease. This study assessed the effectiveness of a prebiotic galacto-oligosaccharide mixture (B-GOS) on the severity and/or incidence of travellers' diarrhoea (TD) in healthy subjects. Subjects/Methods: The study was a placebo-controlled, randomized, double blind of parallel design in 159 healthy volunteers, who travelled for minimum of 2 weeks to a country of low or high risk for TD. The investigational product was the B-GOS and the placebo was maltodextrin. Volunteers were randomized into groups with an equal probability of receiving either the prebiotic or placebo. The protocol comprised of a 1 week pre-holiday period recording bowel habit, while receiving intervention and the holiday period. Bowel habit included the number of bowel movements and average consistency of the stools as well as occurrence of abdominal discomfort, flatulence, bloating or vomiting. A clinical report was completed in the case of diarrhoeal incidence. A post-study questionnaire was also completed by all subjects on their return. Results: Results showed significant differences between the B-GOS and the placebo group in the incidence (P<0.05) and duration (P<0.05) of TD. Similar findings occurred on abdominal pain (P<0.05) and the overall quality of life assessment (P<0.05). Conclusions: Consumption of the tested galacto-oligosaccharide mixture showed significant potential in preventing the incidence and symptoms of TD.

Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus

Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress.

Aeromonas detection and their toxins from drinking water from reservoirs and drinking fountains

Aeromonads are inhabitants of aquatic ecosystems and are described as being involved in intestinal disturbances and other infections. A total of 200 drinking water samples from domestic and public reservoirs and drinking fountains located in Sao Paulo (Brazil), were analyzed for the presence of Aeromonas. Samples were concentrated by membrane filtration and enriched in APW. ADA medium was used for Aeromonas isolation and colonies were confirmed by biochemical characterization. Strains isolated were tested for hemolysin and toxin production. Aeromonas was detected in 12 samples (6.0%). Aeromonas strains (96) were isolated and identified as: A. caviae (41.7%), A. hydrophila (15.7%), A. allosacharophila (10.4%), A. schubertii (1.0%) and Aeromonas spp. (31.2%). The results revealed that 70% of A. caviare, 66.7% of A. hydrophila, 80% of A. allosacharophila and 46.6% of Aeromonas spp. were hemolytic. The assay for checking production of toxins showed that 17.5% of A. caviae, 73.3% of A. hydrophila, 60% of A. allosacharophila, 100% of A. schubertii, and 33.3% of Aeromonas spp. were able to produce toxins. The results demonstrated the pathogenic potential of Aeromonas, indicating that the presence of this emerging pathogen in water systems is a public health concern.

Dynamics of Aeromonas species isolated from wastewater treatment system

Aeromonas are widely distributed in the aquatic environment, and are considered to be emerging organisms that can produce a series of virulence factors. The present study was carried out in a sanitary sewage stabilization pond treatment system, located in Lins, State of Sao Paulo, Brazil. Most probable number was applied for estimation of the genus Aeromonas. Colony isolation was carried out on blood agar ampicillin and confirmed by biochemical characterization. Aeromonas species were isolated in 72.4% of influent samples, and in 55.2 and 48.3% of effluent from anaerobic and facultative lagoons, respectively. Thirteen Aeromonas species were isolated, representing most of the recognized species of these organisms. Even though it was possible to observe a tendency of decrease, total elimination of these organisms from the studied system was not achieved. Understanding of the pathogenic organism`s dynamics in wastewater treatment systems with a reuse potential is especially important because of the risk it represents.

Molecular detection of enterotoxins in environmental strains of Aeromonas hydrophila and Aeromonas jandaei

Aeromonas species are widely distributed in aquatic environments and recent studies include the genus in the emergent pathogens group because of its frequent association with local and systemic infections in immunocompetent humans. Aiming to search for virulence genes in environmental strains of Aeromonas hydrophila and Aeromonas jandaei, we designed specific primers to detect act/hly A/aer complex and alt genes. Primers described elsewhere were used to detect ast. Eighty-seven strains previously identified using phenotypic and genotypic tests as A. hydrophila (41) and A. jandaei (46) were analysed for the presence of the virulence genes using PCR. DNA fragments of expected size were purified and directly sequenced. Among the 41 strains of A. hydrophila 70.7% (29), 97.6% (40) and 26.8% (11) possessed act/hly A/aer complex, ast and alt genes, respectively. Among the 46 strains of A. jandaei, 4.4% (2), 0% (0) and 32.6% (15) were positive for act/hly A/aer complex, ast and alt genes, respectively. Sequencing allowed for the confirmation of amplified products using BLAST. The present work proposes a specific and rapid diagnostic method to detect the main virulence determinants of Aeromonas, a genus potentially pathogenic to humans.

Detection of metallo-beta-lactamases-encoding genes in environmental isolates of Aeromonas hydrophila and Aeromonas jandaei

Aims: To determine the prevalence and expression of metallo-beta-lactamases (MBL)-encoding genes in Aeromonas species recovered from natural water reservoirs in southeastern Brazil. Methods and Results: Eighty-seven Aeromonas isolates belonging to Aeromonas hydrophila (n = 41) and Aer. jandaei (n = 46) species were tested for MBL production by the combined disk test using imipenem and meropenem disks as substrates and EDTA or thioglycolic acid as inhibitors. The presence of MBL genes was investigated by PCR and sequencing using new consensus primer pairs designed in this study. The cphA gene was found in 97.6% and 100% of Aer. hydrophila and Aer. jandaei isolates, respectively, whereas the acquired MBL genes bla(IMP), bla(VIM) and bla(SPM-1) were not detected. On the other hand, production of MBL activity was detectable in 87.8% and 10.9% of the cphA-positive Aer. hydrophila and Aer. jandaei isolates respectively. Conclusions: Our results indicate that cphA seems to be intrinsic in the environmental isolates of Aer. hydrophila and Aer. jandaei in southeastern Brazil, although, based on the combined disk test, not all of them are apparently able to express the enzymatic activity. Significance and Impact of the Study: These data confirm the presence of MBL-producing Aeromonas species in natural water reservoirs. Risk of water-borne diseases owing to domestic and industrial uses of freshwater should be re-examined from the increase of bacterial resistance point of view.

A Broad-spectrum mer Operon in a Multi-drug Resistant Strain of the Fish Pathogen, Aeromonas salmonicida.

Aeromonas salmonicida AS03, a potential fish pathogen, was isolated from Atlantic salmon, Salmo salar, in 2003. This strain was found to be resistant to ≥1000 mM HgCl2 and ≥32 mM phenylmercuric acetate as well as multiple antimicrobials. Mercury (Hg) and antibiotic resistance genes are often located on the same mobile genetic elements, so the genetic determinants of both resistances and the possibility of horizontal gene transfer were examined. Specific PCR primers were used to amplify and sequence distinctive regions of the mer operon. A. salmonicida AS03 was found to have a pDU1358-like broad-spectrum mer operon, containing merB as well as merA, merD, merP, merR and merT, most similar to Klebsiella pneumonaie plasmid pRMH760. To our knowledge, the mer operon has never before been documented in Aeromonas spp. PCR and gene sequencing were used to identify class 1 integron associated antibiotic resistance determinants and the Tet A tetracycline resistance gene. The transposase and resolvase genes of Tn1696 were identified through PCR and sequencing with Tn21 specific PCR primers. We provide phenotypic and genotypic evidence that the mer operon, the aforementioned antibiotic resistances, and the Tn1696 transposition module are located on a single plasmid or conjugative transposon that can be transferred to E. coli DH5α through conjugation in the presence of low level Hg and absence of any antibiotic selective pressure. Additionally, the presence of low-level Hg or chloramphenicol in the mating media was found to stimulate conjugation, significantly increasing the transfer frequency of conjugation above the transfer frequency measured with mating media lacking both antibiotics and Hg. This research demonstrates that mercury indirectly selects for the dissemination of the antibiotic resistance genes of A. salmonicida AS03.

A broad-spectrum mer operon in a multi-drug resistant strain of the fish pathogen, Aeromonas salmonicida

Aeromonas salmonicida AS03, a potential fish pathogen, was isolated from Atlantic salmon, Salmo salar, in 2003. This strain was found to be resistant to ≥1000 mM HgCl2 and ≥32 mM phenylmercuric acetate as well as multiple antimicrobials. Mercury (Hg) and antibiotic resistance genes are often located on the same mobile genetic elements, so the genetic determinants of both resistances and the possibility of horizontal gene transfer were examined. Specific PCR primers were used to amplify and sequence distinctive regions of the mer operon. A. salmonicida AS03 was found to have a pDU1358-like broad-spectrum mer operon, containing merB as well as merA, merD, merP, merR and merT, most similar to Klebsiella pneumonaie plasmid pRMH760. To our knowledge, the mer operon has never before been documented in Aeromonas spp. PCR and gene sequencing were used to identify class 1 integron associated antibiotic resistance determinants and the Tet A tetracycline resistance gene. The transposase and resolvase genes of Tn1696 were identified through PCR and sequencing with Tn21 specific PCR primers. We provide phenotypic and genotypic evidence that the mer operon, the aforementioned antibiotic resistances, and the Tn1696 transposition module are located on a single plasmid or conjugative transposon that can be transferred to E. coli DH5α through conjugation in the presence of low level Hg and absence of any antibiotic selective pressure. Additionally, the presence of low-level Hg or chloramphenicol in the mating media was found to stimulate conjugation, significantly increasing the transfer frequency of conjugation above the transfer frequency measured with mating media lacking both antibiotics and Hg. This research demonstrates that mercury indirectly selects for the dissemination of the antibiotic resistance genes of A. salmonicida AS03.