Effect of live Eimeria vaccination or salinomycin on growth and immune status in broiler chickens receiving in-feed inclusion of gelatin and vitamin E.

This experiment determined if 2% of gelatin, to improve the levels of proline and glycine in the diet, and 70 mg/kg of vitamin E supplementation would relieve the impaired performance of male Cobb broilers vaccinated for coccidiosis. Half of the chicks were vaccinated via water (live oocysts), while the other half received medication (salinomycin) in the feed until 35 d of age. The effects of coccidiosis vaccine on performance and mRNA levels of genes involved in mucin synthesis, cytokines, trefoil family factor-2 (TFF2), and metabolic processes (CD36) in the jejunum of broilers were measured. Vaccination negatively affected performance in the first 21 d; however, the inclusion of gelatin and vitamin E reduced this negative response. Additionally, supplementation with these nutrients led to an improvement in broilers receiving the coccidiostat (P < 0.05). From 21 to 35 d, birds treated with gelatin and coccidiosis vaccine experienced better body weight gain than birds without gelatin and vitamin E (P < 0.05). Vaccinated chickens had decreased body weight and decreased anti-inflammatory cytokine expression. Furthermore, they had increased inflammatory cytokine expression, mucin 2 expression, and TFF2 compared to salinomycin-fed broilers (P < 0.05). Transcripts for IL-1B, IFN-y, MUC2, TFF2 were decreased while mRNAs for IL-4 and IL-10 increased in salinomycin-fed broilers compared to vaccinated broilers (P < 0.05). In conclusion, broilers vaccinated against coccidiosis increase their pro-inflammatory immune status and mucin expression compared to broilers receiving salinomycin. These events may contribute to lower performance in vaccinated broiler chicks. Moreover, vitamin E and gelatin can minimize the vaccine's negative immune effects and promote better performance.

Changes in the ceca microbiota of broilers vaccinated for coccidiosis or supplemented with salinomycin.

The objective of this study was to characterize differences in the cecal microbiota of chickens vaccinated for coccidiosis or receiving salinomycin in the diet. In this study, 140 male 1-day-old broiler chickens were divided in 2 groups: vaccine group (live vaccine) vaccinated at the first day and salinomycin group (125 ppm/kg since the first day until 35 d of age). Each treatment was composed for 7 replicates of 10 birds per pen. At 28 d, the cecal content of one bird per replicate was collected for microbiota analysis. The genetic sequencing was conducted by the Miseq Illumina platform. Vaccine group showed lower body weight, weight gain, and poorer feed conversion in the total period (P < 0.05). Bacterial 16S rRNA genes were classified as 3 major phyla (Bacteroidetes, Firmicutes, and Proteobacteria), accounting for more than 98% of the total bacterial community. The microbiota complexity in the cecal was estimated based on the α-diversity indices. The vaccine did not reduce species richness and diversity (P > 0.05). The richness distribution in the salinomycin group was larger and more uniform than the vaccinated birds. Salinomycin group was related to the enrichment of Bacteroidetes, whereas Firmicutes and Proteobacteria phyla were in greater proportions in the vaccine group. The last phylum includes a wide variety of pathogenic bacteria. The vaccine did not decrease the species richness but decreased the percentage of Bacteroidetes, a phylum composed by genera that produce short-chain fatty acids improving intestinal health. Vaccine group also had higher Proteobacteria phylum, which may help explain its poorer performance.

Assessing the quality of fresh Whitemouth croaker (Micropogonias furnieri) meat based on micro-organism and histamine analysis using NGS, qPCR and HPLC-DAD.

AIMS: Quality evaluation of fresh whitemouth croaker (Micropogonias furnieri) by histamine determination using the HPLC-DAD method and quantification of histamine-forming bacteria using NGS and qPCR. METHODS AND RESULTS: The histamine content of fresh whitemouth croaker was detected by high performance liquid chromatography with diode array detector with a concentration ranging from 258·52 to 604·62 mg kg-1 being observed. The number of histidine decarboxylase (hdc gene) copies from Gram-negative bacteria and the bacteria Morganella morganii and Enterobacter aerogenes were quantified by quantitative polymerase chain reaction. All samples were positive, with copy numbers of the hdc gene ranging from 4·67 to 12·01 log10 per g. The microbial community was determined by sequencing the V4 region of the 16S rRNA gene using the Ion Torrent platform. The bioinformatics data generated by frog software showed that the phylum Proteobacteria was the most abundant, with the family Moraxellaceae being more prevalent in samples collected in the summer, whereas the Pseudomonadaceae was more present in the winter. CONCLUSIONS: All fish muscle samples analysed in this study presented histamine values higher than those allowed by CODEX Alimentarius. Additionally, a wide variety of spoilage micro-organisms capable of expressing the enzyme histidine decarboxylase were detected. Thus, improvements in handling and processing are required to minimize the prevalence of histamine-producing bacteria in fish. SIGNIFICANCE AND IMPACT OF THE STUDY: Global fish production in 2016 was 171 million tons, with the largest consumer being China, followed by Indonesia and the USA. In Brazil, 1·3 million tons of fish are consumed per year, with whitemouth croaker being the main fish landed. Notably, cases associated with histamine poisoning are quite common. According to the European Food Safety Authority and European Centre for Disease Prevention and Control, a total of 599 HFP outbreaks were identified in the European Union during the period 2010-2017. In the USA, there were 333 outbreaks with 1383 people involved between 1998 and 2008.

Frequency of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in non-clinical Enterococcus faecalis and Enterococcus faecium strains / Frequência das repetições palindrômicas curtas agrupadas e regularmente interespaçadas (CRISPRs) em cepas não-clínicas de Enterococcus faecalis e Enterococcus faecium

Abstract The fidelity of the genomes is defended by mechanism known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems. Three Type II CRISPR systems (CRISPR1- cas, CRISPR2 and CRISPR3-cas) have been identified in enterococci isolates from clinical and environmental samples. The aim of this study was to observe the distribution of CRISPR1-cas, CRISPR2 and CRISPR3-cas in non-clinical strains of Enterococcus faecalis and Enterococcus faecium isolates from food and fecal samples, including wild marine animals. The presence of CRISPRs was evaluated by PCR in 120 enterococci strains, 67 E. faecalis and 53 E. faecium. It is the first report of the presence of the CRISPRs system in E. faecalis and E. faecium strains isolated from wild marine animal fecal samples. The results showed that in non-clinical strains, the CRISPRs were more frequently detected in E. faecalis than in E. faecium. And the frequencies of CRISPR1-cas and CRISPR2 were higher (60%) in E. faecalis strains isolated from animal feces, compared to food samples. Both strains showed low frequencies of CRISPR3-cas (8.95% and 1.88%). In conclusion, the differences in the habitats of enterococcal species may be related with the results observe in distribution of CRISPRs systems.

Resumo A fidelidade dos genomas ​​é defendida por mecanismos conhecidos como sistemas de repetições palindrômicas curtas agrupadas e regularmente interespaçadas (CRISPRs). Três tipos de sistemas CRISPR II (CRISPR1-cas, CRISPR2 e CRISPR3-cas) têm sido identificados em cepas de enterococos isolados de amostras clínicas e ambientais. O objetivo deste estudo foi observar a distribuição dos CRISPR1-cas, CRISPR2 e CRISPR3-cas em cepas não-clínicas de Enterococcus faecalis e Enterococcus faecium isoladas de amostras alimentícias e fecais, incluindo animais marinhos selvagens. A presenca dos CRISPRs foi determinada por PCR em 120 cepas de enterococos, sendo 67 E. faecalis e 53 E. faecium. É o primeiro relato da presença do sistema CRISPRs nas estirpes E. faecalis e E. faecium isoladas de amostras fecais de animais marinhos selvagens. Os resultados mostraram que em cepas não-clínicas, os CRISPRs foram mais frequentemente detectados em E. faecalis do que em E. faecium. E as frequências de CRISPR1-cas e CRISPR2 foram maiores (60%) em cepas de E. faecalis isoladas de fezes de animais, quando comparadas à amostras de alimentos. Ambas as cepas apresentaram baixas freqüências de CRISPR3-cas (8,95% e 1,88%). Em conclusão, as diferenças nos habitats das espécies de enterococos podem estar relacionadas com os resultados observados na distribuição dos sistemas CRISPRs.

Comparison between a commercial blend of functional oils and monensin on the performance and microbiota of coccidiosis-challenged broilers.

The aim of the study was to evaluate the effects of a cashew nut shell oil and commercial castor oil blend (CNSL-Castor oil) on the performance and microbiota of broiler chickens with and without coccidiosis challenge. A total of 864 one-day-old male chicks (Cobb) were randomly distributed to receive 6 treatments (8 pens/treatment; 18 chicks/pen) in a 3 × 2 factorial, with 3 additives (control [non-additives], 100 ppm sodium monensin, or 0.15% CNSL-Castor oil blend), and 2 levels of coccidiosis challenge at 14 D of age (unchallenged or inoculated by gavage with 1 mL of solution containing oocysts sporulated with Eimeria tenella, Eimeria acervulina, and Eimeria maxima). No differences in productive performance were observed among treatments in the pre-challenge period and in unchallenged birds (P > 0.05). Seven-days post-challenge, birds receiving monensin performed better than birds in the positive control group (non-additive and challenge) or in the CNSL-Castor oil group (P > 0.05). However, 14 D post-challenge, birds supplemented with CNSL-Castor oil presented higher weight gain and better feed conversion (P > 0.05), without any change in feed intake (P > 0.05). During the accumulated period (1 to 42 D of age), the live weight, weight gain, and feed intake did not differ between the CNSL-Castor oil and monensin groups, both of which presented higher values than the positive control. Lactobacillus spp. and Clostridium perfringens numbers were increased in the challenged birds (P < 0.05). CNSL-Castor oil supplementation reduced Clostridium cluster XIV, C. perfringens, and S. aureus, compared with the monensin and control groups (P > 0.05). In addition, the CNSL-Castor oil group presented the highest number of Lactobacillus spp. copies, followed by the monensin and positive control groups (P > 0.05). Thus, monensin and CNSL-Castor oil effectively minimized the impact of coccidiosis at different times. While monensin acts as an antimicrobial, CNSL-Castor oil modulates the intestinal microbiota with antimicrobial action against gram-positive bacteria, mainly C. perfringens and S. aureus.

Effect of functional oils on the immune response of broilers challenged with Eimeria spp.

Infection with Eimeria sp. results in the activation of multiple facets of the host immune system; the use of phytogenics can modulate the inflammatory response and improve the performance of the challenged animal. The aim of this study was to evaluate the effect of a commercial blend of cashew nut shell liquid (CNSL) and castor oil on the immune response of broilers challenged with coccidiosis. A total of 864 one-day-old male chicks (Cobb 500) were randomly distributed into six treatment groups (8 pens/treatment and 18 chicks/pen) in a three-by-two factorial design with three additives: control (non-additive), 100 ppm of monensin or 0.15% CNSL-castor oil. Challenge status was determined twice at 14 days of age. Unchallenged birds were inoculated by gavage with oocysts sporulated with Eimeria tenella, Eimeria acervulina and Eimeria maxima. Although the positive control (non-additive and challenged) and CNSL-castor oil treatment groups exhibited similar variation in weight gain (ΔBWG) compared to unchallenged birds fed without additives, the variation observed in birds fed diets containing CNSL-castor oil was associated with a higher maintenance requirement and not feed efficiency. In the second week after infection, ΔBWG of the CNSL-castor oil treatment group did not significantly change compared to the other treatment groups. At days 7 and 14 post-challenge, there was a higher excretion of oocysts in the control group, whereas the CNSL-castor oil and monensin groups did not differ. The CNSL-castor oil group exhibited increased gene expression of interferon (IFN), interleukin 6 (IL-6) and tumor necrosis factor (TNF), while the control group exhibited increased expression of cyclooxygenase (COX) and IL-1. The heterophils/lymphocyte ratio was low for the monensin treatment group. The unchallenged birds that received monensin treatment presented higher gene expression of IFN, COX and IL-1 compared to the other treatments, while the CNSL-castor oil group exhibited reduced gene expression, except for TNF. The commercial blend of cashew nut liquid and castor oil modulated the inflammatory response against Eimeria spp. In the absence of the parasite, there was no stimulation of genes involved in the inflammatory response, demonstrating that the blend is an effective tool in specifically modulating the immune system of birds afflicted with coccidiosis.

Frequency of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in non-clinical Enterococcus faecalis and Enterococcus faecium strains.

The fidelity of the genomes is defended by mechanism known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems. Three Type II CRISPR systems (CRISPR1- cas, CRISPR2 and CRISPR3-cas) have been identified in enterococci isolates from clinical and environmental samples. The aim of this study was to observe the distribution of CRISPR1-cas, CRISPR2 and CRISPR3-cas in non-clinical strains of Enterococcus faecalis and Enterococcus faecium isolates from food and fecal samples, including wild marine animals. The presence of CRISPRs was evaluated by PCR in 120 enterococci strains, 67 E. faecalis and 53 E. faecium. It is the first report of the presence of the CRISPRs system in E. faecalis and E. faecium strains isolated from wild marine animal fecal samples. The results showed that in non-clinical strains, the CRISPRs were more frequently detected in E. faecalis than in E. faecium. And the frequencies of CRISPR1-cas and CRISPR2 were higher (60%) in E. faecalis strains isolated from animal feces, compared to food samples. Both strains showed low frequencies of CRISPR3-cas (8.95% and 1.88%). In conclusion, the differences in the habitats of enterococcal species may be related with the results observe in distribution of CRISPRs systems.

Frequency of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in non-clinical Enterococcus faecalis and Enterococcus faecium strains

Abstract The fidelity of the genomes is defended by mechanism known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems. Three Type II CRISPR systems (CRISPR1- cas, CRISPR2 and CRISPR3-cas) have been identified in enterococci isolates from clinical and environmental samples. The aim of this study was to observe the distribution of CRISPR1-cas, CRISPR2 and CRISPR3-cas in non-clinical strains of Enterococcus faecalis and Enterococcus faecium isolates from food and fecal samples, including wild marine animals. The presence of CRISPRs was evaluated by PCR in 120 enterococci strains, 67 E. faecalis and 53 E. faecium. It is the first report of the presence of the CRISPRs system in E. faecalis and E. faecium strains isolated from wild marine animal fecal samples. The results showed that in non-clinical strains, the CRISPRs were more frequently detected in E. faecalis than in E. faecium. And the frequencies of CRISPR1-cas and CRISPR2 were higher (60%) in E. faecalis strains isolated from animal feces, compared to food samples. Both strains showed low frequencies of CRISPR3-cas (8.95% and 1.88%). In conclusion, the differences in the habitats of enterococcal species may be related with the results observe in distribution of CRISPRs systems.

Resumo A fidelidade dos genomas é defendida por mecanismos conhecidos como sistemas de repetições palindrômicas curtas agrupadas e regularmente interespaçadas (CRISPRs). Três tipos de sistemas CRISPR II (CRISPR1-cas, CRISPR2 e CRISPR3-cas) têm sido identificados em cepas de enterococos isolados de amostras clínicas e ambientais. O objetivo deste estudo foi observar a distribuição dos CRISPR1-cas, CRISPR2 e CRISPR3-cas em cepas não-clínicas de Enterococcus faecalis e Enterococcus faecium isoladas de amostras alimentícias e fecais, incluindo animais marinhos selvagens. A presenca dos CRISPRs foi determinada por PCR em 120 cepas de enterococos, sendo 67 E. faecalis e 53 E. faecium. É o primeiro relato da presença do sistema CRISPRs nas estirpes E. faecalis e E. faecium isoladas de amostras fecais de animais marinhos selvagens. Os resultados mostraram que em cepas não-clínicas, os CRISPRs foram mais frequentemente detectados em E. faecalis do que em E. faecium. E as frequências de CRISPR1-cas e CRISPR2 foram maiores (60%) em cepas de E. faecalis isoladas de fezes de animais, quando comparadas à amostras de alimentos. Ambas as cepas apresentaram baixas freqüências de CRISPR3-cas (8,95% e 1,88%). Em conclusão, as diferenças nos habitats das espécies de enterococos podem estar relacionadas com os resultados observados na distribuição dos sistemas CRISPRs.

Molecular detection of virulence factors among food and clinical Enterococcus faecalis strains in South Brazil.

The present report aimed to perform a molecular epidemiological survey by investigating the presence of virulence factors in E. faecalis isolated from different human clinical (n = 57) and food samples (n = 55) in Porto Alegre, Brazil, collected from 2006 to 2009. In addition, the ability to form biofilm in vitro on polystyrene and the ß-haemolytic and gelatinase activities were determined. Clinical strains presented a higher prevalence of aggregation substance (agg), enterococcal surface protein (esp) and cytolysin (cylA) genes when compared with food isolates. The esp gene was found only in clinical strains. On the other hand, the gelatinase (gelE) and adherence factor (ace) genes had similar prevalence among the strains, showing the widespread occurrence of these virulence factors among food and clinical E. faecalis strains in South Brazil. More than three virulence factor genes were detected in 77.2% and 18.2% of clinical and food strains, respectively. Gelatinase and ß-haemolysin activities were not associated with the presence of gelE and cylA genes. The ability to produce biofilm was detected in 100% of clinical and 94.6% of food isolates, and clinical strains were more able to form biofilm than the food isolates (Student's t-test, p < 0.01). Results from the statistical analysis showed significant associations between strong biofilm formation and ace (p = 0.015) and gelE (p = 0.007) genes in clinical strains. In conclusion, our data indicate that E. faecalis strains isolated from clinical and food samples possess distinctive patterns of virulence factors, with a larger number of genes that encode virulence factors detected in clinical strains.

Molecular detection of virulence factors among food and clinical Enterococcus faecalis strains in South Brazil

The present report aimed to perform a molecular epidemiological survey by investigating the presence of virulence factors in E. faecalis isolated from different human clinical (n = 57) and food samples (n = 55) in Porto Alegre, Brazil, collected from 2006 to 2009. In addition, the ability to form biofilm in vitro on polystyrene and the β-haemolytic and gelatinase activities were determined. Clinical strains presented a higher prevalence of aggregation substance (agg), enterococcal surface protein (esp) and cytolysin (cylA) genes when compared with food isolates. The esp gene was found only in clinical strains. On the other hand, the gelatinase (gelE) and adherence factor (ace) genes had similar prevalence among the strains, showing the widespread occurrence of these virulence factors among food and clinical E. faecalis strains in South Brazil. More than three virulence factor genes were detected in 77.2% and 18.2% of clinical and food strains, respectively. Gelatinase and β-haemolysin activities were not associated with the presence of gelE and cylA genes. The ability to produce biofilm was detected in 100% of clinical and 94.6% of food isolates, and clinical strains were more able to form biofilm than the food isolates (Student's t-test, p < 0.01). Results from the statistical analysis showed significant associations between strong biofilm formation and ace (p = 0.015) and gelE (p = 0.007) genes in clinical strains. In conclusion, our data indicate that E. faecalis strains isolated from clinical and food samples possess distinctive patterns of virulence factors, with a larger number of genes that encode virulence factors detected in clinical strains.

Biofilm formation on polystyrene under different temperatures by antibiotic resistant Enterococcus faecalis and Enterococcus faecium isolated from food.

The ability of antibiotic resistant E. faecalis and E. faecium isolated from food to form biofilm at different temperatures in the absence or presence of 0.75% glucose was evaluated. A synergistic effect on biofilm at 10 °C, 28 °C, 37 °C and 45 °C and glucose was observed for E. faecalis and E. faecium.

Biofilm formation on polystyrene under different temperatures by antibiotic resistant Enterococcus faecalis and Enterococcus faecium isolated from food

The ability of antibiotic resistant E. faecalis and E. faecium isolated from food to form biofilm at different temperatures in the absence or presence of 0.75% glucose was evaluated. A synergistic effect on biofilm at 10 °C, 28 °C, 37 °C and 45 °C and glucose was observed for E. faecalis and E. faecium.

Species distribution and antimicrobial susceptibility of enterococci isolated from broilers infected experimentally with Eimeria spp and fed with diets containing different supplements

Resistant bacteria in animal can be spread to environment and to humans. Poultry feed and infections caused by Eimeria spp. are important factors in determining the intestinal microbial communities. The aim of this study was to verify the prevalence of species and antimicrobial susceptibility of Enterococcus isolated from broilers fed with different supplements and infected experimentally with Eimeria spp. Broilers were divided in eight groups, fed with diets supplemented with a combination of antimicrobial, ionophore-coccidiostatics, probiotic, essential oil. At 14 days old all birds, except the control, received a solution containing oocysts of Eimeria spp. Samples of cloacal swabs from broilers were collected. A total of 240 Enterococcus sp. strains were isolated, confirmed genus by PCR, classified as species, tested for antimicrobial susceptibility and screened by PCR for the presence of tet(L), tet(M) and erm(B) genes. The overall distribution of species isolated from fecal samples was E. faecalis (40 percent), followed by E. casseliflavus/E. gallinarum (10.8 percent), E. mundtii (10.8 percent), E. faecium (10.8 percent), E. columbae (5.8 percent) and E. gallinarum (4.2 percent). Changes in the composition or frequency of Enterococcus species were observed in all dietary supplementation. Antimicrobial susceptibility tests showed resistance phenotypes a range of antibiotics, especially used in humans such as, streptomycin, penicillin, rifampicin and vancomycin. There was no correlation between different supplementation for broilers and antimicrobial resistance and the presence of tet(M), tet(L) and erm(B) genes. Dietary supplementation had effect on the Enterococcus sp. colonization, but did not have significant effect on the phenotype and genotype of antimicrobial resistance in enterococci.

Species distribution and antimicrobial susceptibility of enterococci isolated from broilers infected experimentally with Eimeria spp and fed with diets containing different supplements.

Resistant bacteria in animal can be spread to environment and to humans. Poultry feed and infections caused by Eimeria spp. are important factors in determining the intestinal microbial communities. The aim of this study was to verify the prevalence of species and antimicrobial susceptibility of Enterococcus isolated from broilers fed with different supplements and infected experimentally with Eimeria spp. Broilers were divided in eight groups, fed with diets supplemented with a combination of antimicrobial, ionophore-coccidiostatics, probiotic, essential oil. At 14 days old all birds, except the control, received a solution containing oocysts of Eimeria spp. Samples of cloacal swabs from broilers were collected. A total of 240 Enterococcus sp. strains were isolated, confirmed genus by PCR, classified as species, tested for antimicrobial susceptibility and screened by PCR for the presence of tet(L), tet(M) and erm(B) genes. The overall distribution of species isolated from fecal samples was E. faecalis (40%), followed by E. casseliflavus/E. gallinarum (10.8%), E. mundtii (10.8%), E. faecium (10.8%), E. columbae (5.8%) and E. gallinarum (4.2%). Changes in the composition or frequency of Enterococcus species were observed in all dietary supplementation. Antimicrobial susceptibility tests showed resistance phenotypes a range of antibiotics, especially used in humans such as, streptomycin, penicillin, rifampicin and vancomycin. There was no correlation between different supplementation for broilers and antimicrobial resistance and the presence of tet(M), tet(L) and erm(B) genes. Dietary supplementation had effect on the Enterococcus sp. colonization, but did not have significant effect on the phenotype and genotype of antimicrobial resistance in enterococci.