Ignacio Badiola

Role of the High-Affinity Zinc Uptake znuABC System in Salmonella enterica Serovar Typhimurium Virulence

ABSTRACT The Salmonella enterica serovar Typhimurium znuABC genes encoding a high-affinity zinc uptake system and its regulatory zur gene have been cloned. Salmonella serovar Typhimurium zur and znuC knockout mutants have been constructed by marker exchange. The 50% lethal dose of the znuC mutant increased when either orally or intraperitoneally inoculated in BALB/c mice, while virulence of the zur mutant decreased only when mice were intraperitoneally challenged.

The high‐affinity zinc‐uptake system znuACB is under control of the iron‐uptake regulator (fur) gene in the animal pathogen Pasteurella multocida

The Pasteurella multocida znuACB genes encoding a high-affinity zinc-uptake system have been identified and cloned. In contrast to what happens in Escherichia coli, znuA is not physically linked to znuCB. Through lacZ transcriptional fusions it has been demonstrated that zinc negatively regulates both znuA and znuCB operons. Nevertheless, and contrary to that determined so far for all other znuACB bacterial systems known, P. multocida znuACB genes are not under control of the zur gene, which is absent in this bacterial species, but rather are under its iron-uptake regulator (fur) gene. Furthermore, construction of defective mutants has demonstrated that P. multocida znuA and znuCB transcriptional units are required for virulence of this organism in a mouse model.

Effect of dietary supplementation with glutamine and a combination of glutamine-arginine on intestinal health in twenty-five-day-old weaned rabbits

The effect of dietary supplementation with 1% l-glutamine and a combination of 1% l-glutamine and 0.5% l-arginine on intestinal health was examined in weaned rabbits. A basal diet was formulated to meet nutrient recommendations. Another 2 diets were formulated by adding 1% (as-fed basis) Gln or a mixture of 1% (as-fed basis) Gln + 0.5% (as-fed basis) Arg (Gln-Arg) to the basal diet. In Exp. 1, a total of 357 rabbits were blocked by litter and assigned at random to the experimental diet to determine mortality (119 per diet) and growth performance (35 per diet; from weaning at 25 to 56 d of age). Rabbits were fed the experimental diets for a 2-wk period and thereafter received a commercial diet. Rabbits weaned at 25 d (blocked by litter and assigned at random to diets) were slaughtered at 35 d and used to determine apparent ileal digestibility of DM, CP, and AA (Exp. 2, a total of 60 rabbits), intestinal morphology, N-aminopeptidase and myeloperoxidase intestinal activity, the expression of PPARgamma at the ileum and kidney, serum immunoglobulin in healthy and sick rabbits (Exp. 3, a total of 24 rabbits), and ileal and cecal microbial composition by PCR-RFLP (Exp. 4, a total of 45 rabbits). Dietary treatment did not affect ADG, ADFI, or G:F, during the entire fattening period. Supplementation with Gln reduced mortality during the first 2 wk and the whole fattening period from 18.5 to 8.4% (P = 0.023) and from 31.9 to 20.2% (P = 0.039), respectively, whereas no effect was detected for Arg supplementation. Among all the variables studied, the reduction on mortality due to Gln was related to a reduced intestinal colonization (Eimeria lesions) and changes on microbial ecosystem in the ileum and cecum, reducing the frequency of detection of Clostridium spp. (from 86.7 to 33.3%, P = 0.003) at the ileum, and Helicobacter spp. at the ileum (from 86.7 to 46.7%, P = 0.003) and at the cecum (from 86.7 to 46.7, P = 0.028), whereas no effect was detected for Arg supplementation. In conclusion, 1% l-Gln supplementation to postweaned rabbit diets decreased fattening mortality and modified the intestinal microbiota (although no consistent effects were observed on mucosal histology or inflammatory and systemic immune response). Diets containing a combination of 1% Gln and 0.5% Arg were of little additional benefit.

Wheat-barley-rye- or corn-fed growing pigs respond differently to dietary supplementation with a carbohydrase complex

Thirty-six pigs (22 kg of BW) were used to evaluate a carbohydrase preparation, with xylanase and β-glucanase as main activities, added to either wheat-barley-rye- (WBR) or corn-based diets on performance, intestinal environment, and nutrient digestibility. Pigs were offered 1 of 4 different dietary treatments for 27 d according to a factorial arrangement of treatments (a 2 × 2) with 2 cereal types (WBR or corn) and 2 levels of supplemental carbohydrase (0 or 0.01%). Pig growth and feed intake were individually measured every week until the end of the experiment when pigs were slaughtered to obtain samples of digesta and tissues. Cereal type affected performance only during wk 1, in which WBR improved ADG (590 vs. 440 g/d; P = 0.008) and G:F (0.61 vs. 0.43; P = 0.045) compared with corn. The WBR also increased the viscosity of the digestive contents in stomach (1.95 vs. 1.23 mPa·s; P = 0.001) and ileum (6.53 vs. 2.80 mPa·s; P = 0.001) and resulted in greater cecal starch digestibility (95.7 vs. 93.9%; P = 0.012). However, trends for a reduction in digestibility were observed for glucose in the nonstarch polysaccharide (NSP) fraction in the ileum (64.4 vs. 75.8%; P = 0.074) and galactose in the NSP fraction in the cecum (1.4 vs. 1.8%; P = 0.055). The use of the enzyme preparation increased ADFI during wk 2 (1,328 vs. 1,215 g/d; P = 0.028), and increased villus height (423 vs. 390 µm; P = 0.045) and tended to reduce relative pancreas weight (0.16 vs. 0.17% BW; P = 0.079) at d 27. The enzyme also improved cecal starch digestibility (95.5 vs. 94.1%; P = 0.043) and tended to improve ileal energy digestibility (61.3 vs. 53.7%; P = 0.090) and cecal glucose digestibility in the NSP fraction (76.0 vs. 54.5%; P = 0.055). However, it reduced the cecal digestibility of mannose in the NSP fraction (27.0 vs. 50.5%; P = 0.016). Interactions (P < 0.05) between cereal type and enzyme supplementation were observed for ADG and G:F during wk 2, BW and ADG during wk 3, and BW and ADFI over the whole trial; and also for villus-height-to-crypt-depth ratio and for cecal DM digestibility. In all instances, whereas the added enzyme had no effect in the case of the corn diet, improvements were observed with WBR. In conclusion, the multi-enzyme tested had different effects depending on the type of cereal present in the diet.

Characterization of the Pasteurella multocida hgbA Gene Encoding a Hemoglobin-Binding Protein

ABSTRACT Reverse transcriptase PCR analyses have demonstrated that open reading frames (ORFs) PM0298, PM0299, and PM0300 of the animal pathogen Pasteurella multocida constitute a single transcriptional unit. By cloning and overexpression studies in Escherichia coli cells, the product of ORF PM0300 was shown to bind hemoglobin in vitro; this ORF was therefore designated hgbA. In vitro and in vivo quantitative assays demonstrated that the P. multocida hgbA mutant bound hemoglobin to the same extent as the wild-type strain, although the adsorption kinetics was slightly slower for the hgbA cells. In agreement with this, the virulence of P. multocida hgbA cells was not affected, suggesting that other functional hemoglobin receptor proteins must be present in this organism. On the other hand, P. multocida mutants defective in PM0298 and PM0299 could be isolated only when a plasmid containing an intact copy of the gene was present in the cells, suggesting that these genes are essential for the viability of this bacterial pathogen. By adapting the recombinase-based expression technology in vivo to P. multocida, we also demonstrated that the transcriptional PM0298-PM0299-hgbA unit is iron regulated and that its expression is triggered in the first 2 h following infection in a mouse model. Furthermore, hybridization experiments showed that the hgbA gene is widespread in P. multocida strains regardless of their serotype or the animal from which they were isolated.

Virulence of Pasteurella multocida recA mutants

In order to determine the role of the RecA protein in the virulence of Pasteurella multocida, a recA mutant was constructed and used in studies of virulence and competition in relation to wild-type strain. To achieve this, firstly, the recA gene was isolated and sequenced, showing an Escherichia coli-like SOS box and encoding a protein of 354 amino acids which has the closest identity with the Haemophilus influenzae RecA protein. Further, the recA mutant was constructed, by inactivating this gene by single recombination of a suicide plasmid containing an internal region of the P. multocida recA gene, and shown to be more sensitive to UV radiation than the parental strain. The P. multocida mutant was slightly attenuated in virulence, as indicated by the LD(50), the time of death of infected animals, and a failure to compete with the wild-type strain in mixed infections. Compared to the parent strain, the mutant had a similar growth rate but a longer lag phase. These data suggest that the diminished virulence of the recA mutant as well as its failure in competition were more a consequence of the long lag phase rather than a direct effect of the inactivation of the recA gene on genes involved in virulence.

Bacillus cereus var. toyoi promotes growth, affects the histological organization and microbiota of the intestinal mucosa in rainbow trout fingerlings

In this preliminary study, we evaluated the effects of a gram-positive soil bacteria Bacillus cereus var. toyoi on the growth performance, digestive enzyme activities, intestinal morphology, and microbiota in rainbow trout Oncorhynchus mykiss fingerlings. Trout were maintained in a recirculation system and fed 2 diets: 1) a commercial trout feed deprived of the probiotic and 2) the same diet but with the spores of the probiotic bacteria dissolved in fish oil during the manufacturing of the feed (final concentration = 2 × 10(4) cfu/g). Each diet was tested in three 400-L cylindroconical tanks (125 fish per tank; initial density = 1.3 kg/m(3); 13.2°C) for a period of 93 d. The probiotic-supplemented diet promoted growth, and the final mean BW and standard length in fish fed the probiotic were 3.4% and 2.1%, respectively, which was greater than the control group (P < 0.05). Fish fed the probiotic showed a more homogeneous distribution in the final BW, with a greater frequency of individuals around the modal of the normal distribution of the population. This result is of practical importance because homogenous production lots can improve rearing practices, reducing hierarchical dominance situations arising from individuals of larger sizes. In addition, the probiotic-supplemented diet increased the level of leukocyte infiltration in the lamina propria of the intestinal mucosa, the number of goblet cells (P < 0.010), and villi height (P < 0.001) but did not affect villi width. The administration of the probiotic changed the intestinal microbiota as indicated by 16S rDNA PCR-restriction fragment length polymorphism. In this sense, fish fed the probiotic formed a well-defined cluster composed of 1 super clade, whereas compared control fish had a greater degree of diversity in their gut microbiota. These changes in gut microbiota did not affect the specific activity of selected pancreatic and intestinal digestive enzymes. These results indicate that the inclusion of the probiotic bacteria in trout feeds could be beneficial for the host by enhancing its intestinal innate immune function and promoting growth.

Diversity of Multi-Drug Resistant Avian Pathogenic Escherichia coli (APEC) Causing Outbreaks of Colibacillosis in Broilers during 2012 in Spain

Avian pathogenic Escherichia coli (APEC) are the major cause of colibacillosis in poultry production. In this study, a total of 22 E. coli isolated from colibacillosis field cases and 10 avian faecal E. coli (AFEC) were analysed. All strains were characterised phenotypically by susceptibility testing and molecular typing methods such as pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The presence of 29 virulence genes associated to APEC and human extraintestinal pathogenic E. coli (ExPEC) was also evaluated. For cephalosporin resistant isolates, cephalosporin resistance genes, plasmid location and replicon typing was assessed. Avian isolates belonged to 26 O:H serotypes and 24 sequence types. Out of 22 APEC isolates, 91% contained the virulence genes predictors of APEC; iutA, hlyF, iss, iroN and ompT. Of all strains, 34% were considered ExPEC. PFGE analysis demonstrated a high degree of genetic polymorphism. All strains were multi-resistant, including those isolated from healthy animals. Eleven strains were resistant to cephalosporins; six contained bla CTX-M-14, two bla SHV-12, two bla CMY-2 and one bla SHV-2. Two strains harboured qnrA, and two qnrA together with aac(6’)-Ib-cr. Additionally, the emergent clone O25b:H4-B2-ST131 was isolated from a healthy animal which harboured bla CMY-2 and qnrS genes. Cephalosporin resistant genes were mainly associated to the presence of IncK replicons. This study demonstrates a very diverse population of multi-drug resistant E. coli containing a high number of virulent genes. The E. coli population among broilers is a reservoir of resistance and virulence-associated genes that could be transmitted into the community through the food chain. More epidemiological studies are necessary to identify clonal groups and resistance mechanisms with potential relevance to public health.

Bacillus cereus var. toyoi promotes growth, affects the histological organization and microbiota of the intestinal mucosa in rainbow trout fingerlings1

In this preliminary study, we evaluated the effects of a gram-positive soil bacteria Bacillus cereus var. toyoi on the growth performance, digestive enzyme activities, intestinal morphology, and microbiota in rainbow trout Oncorhynchus mykiss fingerlings. Trout were maintained in a recirculation system and fed 2 diets: 1) a commercial trout feed deprived of the probiotic and 2) the same diet but with the spores of the probiotic bacteria dissolved in fish oil during the manufacturing of the feed (final concentration = 2 × 10(4) cfu/g). Each diet was tested in three 400-L cylindroconical tanks (125 fish per tank; initial density = 1.3 kg/m(3); 13.2°C) for a period of 93 d. The probiotic-supplemented diet promoted growth, and the final mean BW and standard length in fish fed the probiotic were 3.4% and 2.1%, respectively, which was greater than the control group (P < 0.05). Fish fed the probiotic showed a more homogeneous distribution in the final BW, with a greater frequency of individuals around the modal of the normal distribution of the population. This result is of practical importance because homogenous production lots can improve rearing practices, reducing hierarchical dominance situations arising from individuals of larger sizes. In addition, the probiotic-supplemented diet increased the level of leukocyte infiltration in the lamina propria of the intestinal mucosa, the number of goblet cells (P < 0.010), and villi height (P < 0.001) but did not affect villi width. The administration of the probiotic changed the intestinal microbiota as indicated by 16S rDNA PCR-restriction fragment length polymorphism. In this sense, fish fed the probiotic formed a well-defined cluster composed of 1 super clade, whereas compared control fish had a greater degree of diversity in their gut microbiota. These changes in gut microbiota did not affect the specific activity of selected pancreatic and intestinal digestive enzymes. These results indicate that the inclusion of the probiotic bacteria in trout feeds could be beneficial for the host by enhancing its intestinal innate immune function and promoting growth.

Changes in Bacterial Population of Gastrointestinal Tract of Weaned Pigs Fed with Different Additives

This study aimed to provide novel insights into the gastrointestinal microbial diversity from different gastrointestinal locations in weaning piglets using PCR-restriction fragment length polymorphism (PCR-RFLP). Additionally, the effect of different feed additives was analyzed. Thirty-two piglets were fed with four different diets: a control group and three enriched diets, with avilamycin, sodium butyrate, and a plant extract mixture. Digesta samples were collected from eight different gastrointestinal segments of each animal and the bacterial population was analysed by a PCR-RFLP technique that uses 16S rDNA gene sequences. Bacterial diversity was assessed by calculating the number of bands and the Shannon-Weaver index. Dendrograms were constructed to estimate the similarity of bacterial populations. A higher bacterial diversity was detected in large intestine compared to small intestine. Among diets, the most relevant microbial diversity differences were found between sodium butyrate and plant extract mixture. Proximal jejunum, ileum, and proximal colon were identified as those segments that could be representative of microbial diversity in pig gut. Results indicate that PCR-RFLP technique allowed detecting modifications on the gastrointestinal microbial ecology in pigs fed with different additives, such as increased biodiversity by sodium butyrate in feed.