J. C. Rodriguez-Lecompte

Effect of supplementing direct-fed microbials on broiler performance, nutrient digestibilities, and immune responses

Direct-fed microbials (DFM) are used to improve livestock health and performance. The effects of 2 DFM products, a blend of 3 Bacillus strains (DFMB) and a Propionibacteriumspp. (DFMP), on broiler performance, nutrient utilization, and immune responses were investigated. Day-old (n = 120) male broilers were divided into 24 groups of 5 birds and fed 3 wheat-based diets in mash form (8 groups per diet) from d 1 to 22. The control diet was fed without or with 7.5 × 10(4) cfu/g of either DFMB or DFMP. From d 19 to 21 fecal samples were collected for determination of total tract apparent retention (TTAR) of nutrients and AMEn. On d 21, feed intake and BW were determined. On d 22, 5 birds per treatment were killed by cervical dislocation to collect jejunal and ileal contents for determination of digesta viscosity and apparent ileal digestibility (AID) of nutrients, respectively, and ileum, cecal tonsil, and spleen tissues for Toll-like receptors (TLR) and cytokine expressions. Compared with the control, DFM did not affect BW gain and feed intake but DFMP reduced G:F (P < 0.01). Compared with the control (2,875 kcal/kg), birds fed on DFMB and DFMP had higher AMEn (2,979 and 2,916 kcal/kg, respectively; P < 0.05), whereas both DFM reduced the AID of DM (P < 0.001) and CP (P < 0.01). Furthermore, DFMP reduced TTAR of NDF (29.0 vs. 18.4%; P < 0.001), whereas both DFM increased TTAR of DM and fat (P < 0.001). Supplementing DFMP downregulated ileal expression of TLR-2b, IL-2, IL-4, IL-6, IL-10, and IL-13, whereas DFMB downregulated TLR-2b, IL-2, IL-4, and IL-6 in all 3 tissues, IL-10 in the spleen, and upregulated IL-13 in the spleen. In conclusion, the DFM did not improve performance but increased the AMEn of diet by possibly increasing DM and fat retention. Overall, both DFM showed an antiinflammatory effect in the ileum, but DFMB had more effects on local and systemic immunity than DFMP.

Innate immune response of pullets fed diets supplemented with prebiotics and synbiotics

Prebiotics and synbiotics are considered to be among the most promising replacements for in-feed antibiotic growth promoters (AGP) in poultry feed. The current study was designed to study the effect of Bacitracin methylene disalicylate (BMD) (Control), yeast-derived carbohydrates (YDC), and a blend of YDC and probiotics [Lactobacillus acidophilus, Lactobacillus casei, Streptococcus faecium, Bacillus subtilis, and YDC] (SNB) in the performance and innate immune response of pullets. Feed intake and BW were measured on a weekly basis. At the end of the study (d 21), 10 birds/treatment were sacrificed by cervical dislocation and ileum, cecal tonsil, and spleen samples were collected for gene expression analysis. No significant difference (P > 0.05) in feed intake and G:F was observed among treatments. In the second and third wk age, higher BW gain was observed in SNB treatment compared to control and both control and YDC treatments, respectively. Expression of TLR2b was upregulated in YDC and SNB in the ileum, and in SNB in the spleen (P < 0.05). Expression of TLR4 was downregulated in SNB in the cecal tonsil. Expression of TLR21 was downregulated in YDC in the ileum, while it was upregulated in SNB in the spleen (P < 0.05). In the ileum, YDC resulted in downregulated IL-12p35, CxCLi2, and IL-13, and SNB resulted in upregulated IL-6, interferon (IFN)-γ, and IL-4 (P < 0.05). In the cecal tonsil, YDC resulted in upregulated IL-12p35, IL-2, IL-13 and IL-10, and SNB resulted in downregulated IL-2 and upregulated IL-13 and IL-10 (P < 0.05). In the spleen, YDC resulted in dowregulated IL-2 and CxCLi2, and SNB resulted in upregulated IL-6, IFN-γ, IL-4, and IL-10 (P < 0.05). In conclusion, no change in performance was observed. Innate immune response analysis showed SNB with a more potent effect compared to YDC where the former showed a balanced T-helper (Th)-1/Th-2 response locally and a more Th-2-dependent response systemically; SNB might provide a more beneficial immune modulation with maintaining immune homeostasis, which was observed in a strong IL-10 response.

Functional and molecular responses of the blue mussel Mytilus edulis' hemocytes exposed to cadmium - An in vitro model and transcriptomic approach

Abstract The bivalve mollusk, Mytilus edulis, is used as a sentinel species in several monitoring programs due to its ability to bio‐accumulate contaminants. Its immune system consists of hemocytes and humoral components, which constitute the main part of the hemolymph. The present study is aimed at understanding the effects of Cd on the differentially expressed genes involved in the phagocytosis of M. edulis’ hemocytes. Our approach focuses on an in vitro model by exposing hemocytes to different concentrations of Cd ranging from 10−9 M to 10−3 M. Phagocytosis and cell viability as functional markers were measured using flow cytometry. The molecular mechanisms regulated by Cd were investigated using RNA‐seq and DGE analysis. Results showed that viability and phagocytosis of hemocytes exposed to 10−3 M of Cd were significantly decreased after 21 h of exposure. RNA sequencing data showed that 1112 transcripts (out of 352,976 contigs) were differentially regulated by the highest concentration of Cd. Among these identified transcripts, 1028 and 84 were up and down‐regulated respectively. The induction of super oxide dismutase (SOD), glutathion‐s‐transferase (GST), cytochrome P450 2C8 (CYP2C8), multidrug resistance protein (MRP1) and heat shock protein 70 (HSP70) suggests that Cd can regulate key molecular mechanisms. In addition, several toll‐like receptors (TLR) as well as genes involved in phagocytosis (actin and CDC42) and apoptosis (caspase 8 and XIAP/IAP) were induced by Cd. Thus, our model highlights the effect of Cd on the phagocytic function of M. edulis’ hemocytes along with the regulation of gene expression involved in innate immunity, detoxification and apoptosis. Further investigations need to be pursued to unravel the effects of Cd on the molecular mechanisms identified in this study. HighlightsTranscriptomic analysis of hemocytes exposed to cadmium.In vitro model of Mytilus edulis hemocytes exposed to cadmium.Molecular mechanisms of hemocytes exposed to cadmium.

Organic trace mineral supplementation enhances local and systemic innate immune responses and modulates oxidative stress in broiler chickens

The effect of organic trace mineral supplementation on performance, intestinal morphology, immune organ weights (bursa of Fabricius and spleen), expression of innate immune response related genes, blood heterophils/lymphocytes ratio, chemical metabolic panel, natural antibodies (IgG), and oxidative stress of broiler chickens was studied. A total of 1,080 day-old male broilers were assigned to 1 of 3 dietary treatments, which included basal diet with Monensin (control), control diet supplemented with bacitracin methylene disalicylate (BMD), and BMD diet supplemented with organic trace minerals (OTM). No difference in feed conversion ratio was observed among treatments; ileum histomorphological analysis showed a lower crypt depth, higher villi height/crypt depth ratio, and lower villi width in the OTM treatment compared to control. Furthermore, OTM treatment resulted in higher uric acid and lower plasma malondehaldehyde (MDA), indicating lower oxidative stress. Gene expression analysis showed that OTM treatment resulted in up-regulations of TLR2 bin the ileum, and TLR2b, TLR4, and IL-12p35 in the bursa of Fabricius, and down-regulation of TLR2b and TLR4 in the cecal tonsils. In the spleen, OTM treatment resulted in up-regulation of IL-10. In conclusion, OTM supplementation to broiler diets may have beneficial effects on intestinal development, immune system status, and survival by improving ileum histomorphological parameters, modulation of Toll-like receptors and anti-inflammatory cytokines, and decreasing level of MDA, which in conjunction could enhance health status.

Gut microbiota modulates type I interferon and antibody-mediated immune responses in chickens infected with influenza virus subtype H9N2

Commensal gut microbes play a critical role in shaping host defences against pathogens, including influenza viruses. The current study was conducted to assess the role and mechanisms of action of commensal gut microbiota on the innate and antibody-mediated responses of layer chickens against influenza virus subtype H9N2. A total of 104 one-day-old specific pathogen free chickens were assigned to either of the four treatments, which included two levels of antibiotics treatment (ABX- and ABX+) and two levels of H9N2 virus infection (H9N2- and H9N2+). At day 17 of age, chickens in the H9N2+ group were infected via the oral-nasal route with 400 μl of 107 TCID50/ml (200 μl/each route). Oropharyngeal and cloacal swabs at days 1, 3, 5, 7 and 9 post-infection (p.i.) for virus shedding, tissue samples at 12 h, 24 h and 36 h p.i. for mRNA measurement, and serum samples at days 7 and 14 p.i. for hemagglutination inhibition (HI) assay and IgG antibodies were collected. Virus shedding analysis showed that antibiotic treated (depleted)-H9N2 virus infected chickens showed a significantly higher oropharyngeal virus shedding at all time points, and cloacal shedding at days 3 and 5 p.i. compared to control treated (undepleted)-H9N2 infected chickens. Analysis of mRNA expression showed that infection of depleted chickens with H9N2 virus resulted in significantly down-regulated type I interferon responses both in the respiratory and gastrointestinal tracts compared to undepleted-H9N2 infected chickens. However, antibody-mediated immune response analysis showed a significantly higher HI antibody titre and IgG levels in the serum of chickens depleted with antibiotics and infected with H9N2 virus compared to undepleted-H9N2 infected chickens. In conclusion, findings from the current study suggest that the gut microbiota of chickens plays an important role in the initiation of innate responses against influenza virus infection, while the antibody-mediated immune response remains unaffected.

Whole-genome sequence analysis of antimicrobial resistance genes in Streptococcus uberis and Streptococcus dysgalactiae isolates from Canadian dairy herds

The objectives of this study are to determine the occurrence of antimicrobial resistance (AMR) genes using whole-genome sequence (WGS) of Streptococcus uberis (S. uberis) and Streptococcus dysgalactiae (S. dysgalactiae) isolates, recovered from dairy cows in the Canadian Maritime Provinces. A secondary objective included the exploration of the association between phenotypic AMR and the genomic characteristics (genome size, guanine–cytosine content, and occurrence of unique gene sequences). Initially, 91 isolates were sequenced, and of these isolates, 89 were assembled. Furthermore, 16 isolates were excluded due to larger than expected genomic sizes (>2.3 bp × 1,000 bp). In the final analysis, 73 were used with complete WGS and minimum inhibitory concentration records, which were part of the previous phenotypic AMR study, representing 18 dairy herds from the Maritime region of Canada (1). A total of 23 unique AMR gene sequences were found in the bacterial genomes, with a mean number of 8.1 (minimum: 5; maximum: 13) per genome. Overall, there were 10 AMR genes [ANT(6), TEM-127, TEM-163, TEM-89, TEM-95, Linb, Lnub, Ermb, Ermc, and TetS] present only in S. uberis genomes and 2 genes unique (EF-TU and TEM-71) to the S. dysgalactiae genomes; 11 AMR genes [APH(3′), TEM-1, TEM-136, TEM-157, TEM-47, TetM, bl2b, gyrA, parE, phoP, and rpoB] were found in both bacterial species. Two-way tabulations showed association between the phenotypic susceptibility to lincosamides and the presence of linB (P = 0.002) and lnuB (P < 0.001) genes and the between the presence of tetM (P = 0.015) and tetS (P = 0.064) genes and phenotypic resistance to tetracyclines only for the S. uberis isolates. The logistic model showed that the odds of resistance (to any of the phenotypically tested antimicrobials) was 4.35 times higher when there were >11 AMR genes present in the genome, compared with <7 AMR genes (P < 0.001). The odds of resistance was lower for S. dysgalactiae than S. uberis (P = 0.031). When the within-herd somatic cell count was >250,000 cells/mL, a trend toward higher odds of resistance compared with the baseline category of <150,000 cells/mL was observed. When the isolate corresponded to a post-mastitis sample, there were lower odds of resistance when compared with non-clinical isolates (P = 0.01). The results of this study showed the strength of associations between phenotypic AMR resistance of both mastitis pathogens and their genotypic resistome and other epidemiological characteristics.

Antimicrobial Susceptibility Patterns of Environmental Streptococci Recovered from Bovine Milk Samples in the Maritime Provinces of Canada

Determination of antimicrobial susceptibility of bovine mastitis pathogens is important for guiding antimicrobial treatment decisions and for the detection of emerging resistance. Environmental streptococci are ubiquitous in the farm environment and are a frequent cause of mastitis in dairy cows. The aim of the study was to determine patterns of antimicrobial susceptibility among species of environmental streptococci isolated from dairy cows in the Maritime Provinces of Canada. The collection consisted of 192 isolates identified in milk samples collected from 177 cows originating from 18 dairy herds. Results were aggregated into: (1) Streptococcus uberis (n = 70), (2) Streptococcus dysgalactiae (n = 28), (3) other Streptococci spp. (n = 35), (4), Lactococcus spp. (n = 32), and (5) Enterococcus spp. (n = 27). Minimum inhibitory concentrations (MICs) were determined using the Sensititre microdilution system and mastitis plate format. Multilevel logistic regression models were used to analyze the data, with antimicrobial susceptibility as the outcome. The proportion of susceptible S. uberis ranged from 23% (for penicillin) to 99% (for penicillin/novobiocin), with a median of 82%. All S. dysgalactiae were susceptible to all antimicrobials except for penicillin (93% susceptible) and tetracycline (18% susceptible). The range of susceptibility for other Streptococcus spp. was 43% (for tetracycline) to 100%, with a median percent susceptibility of 92%. Lactococcus spp. isolates displayed percent susceptibilities ranging from 0% (for penicillin) to 97% (for erythromycin), median 75%. For the antimicrobials tested, the minimum inhibitory concentrations were higher for Enterococcus spp. than for the other species. According to the multilevel models, there was a significant interaction between antimicrobial and bacterial species, indicating that susceptibility against a particular antimicrobial varied among the species of environmental streptococci and vice versa. Generally, susceptibility decreased with increasing within-herd average somatic cell count, isolates recovered in mid-lactation were more susceptible than isolates recovered in early lactation, and isolates recovered in samples collected post-clinical mastitis were more susceptible than isolates recovered from non-clinical lactating quarters. The results of this research support continued susceptibility of environmental streptococci to beta-lactam antimicrobials. A departure from the expected susceptibility to beta-lactams was the apparent reduced susceptibility of S. uberis to penicillin.

Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on growth performance and local innate immune response of broiler chickens challenged with Clostridium perfringens

ABSTRACT This study evaluated the effect of yeast-derived products on growth performance, gut lesion score, intestinal population of Clostridium perfringens, and local innate immunity of broiler chickens challenged with C. perfringens. One-day-old broiler chickens were randomly assigned to eight dietary treatments providing six replicate pens of 55 birds each per treatment. Dietary treatments consisted of Control diets without and with C. perfringens challenge, and diets containing bacitracin methylene disalicylate (BMD, 55 g/tonne), nucleotides (150 g/tonne), yeast cell wall (YCW, 300 g/tonne), and a commercial product Maxi-Gen Plus (1 kg/tonne) fed to chickens challenged with C. perfringens. Diets containing 10% distillers dried grains with solubles without and with C. perfringens challenge were also used. Birds were orally challenged with C. perfringens (108 colony-forming units (cfu)/bird) on day 14. On day 21, intestinal samples were collected for gene expression analysis. Pathogen challenge significantly (P < 0.05) impaired feed intake, body weight gain, and feed conversion ratio (FCR) shortly after the challenge (14–21 days). Increased C. perfringens counts and intestinal lesion scores were observed for challenged birds except the BMD-containing diet. Over the entire trial (1–35 days), no difference in growth performance was observed except the BMD diet which improved FCR over the Control, challenged group. Birds receiving nucleotides showed increased expression of toll-like receptors and cytokines interleukin (IL)-4 and IL-18 compared to the Control, challenged group. Expression of macrophage mannose receptor and IL-18 was upregulated in birds receiving YCW. Increased expression of cytokines and receptors involved in innate immunity in broilers receiving nucleotides and YCW suggests the immunomodulatory properties of these products under pathogen challenge conditions.

A simple method for screening bacterial colonies for mutagenized sites in plasmid DNA

Because of the multiple-step process that is involved in the detection of mutagenized restriction enzyme sites in plasmid DNA, a simple and accurate method was developed to analyse the plasmid DNA of site-directed mutagenesis experiments from bacterial colonies. The desired mutated part is located between the Eco RI restriction site on pUC19. Two mutagenic primers were designed to replace only one nucleotide on segments A and B of the bi-segmented genome of infectious bursal disease virus (IBDV). Two restriction sites were created for those mutations in each segment, Fsp I and Dra I, respectively. Following a protocol from the site-directed mutagenesis kit, the mutated plasmids were used to transform, and were propagated and maintained in DH5 alpha competent cells. Colonies were picked from the master plate, and used as DNA template for PCR. The PCR technique included the design of two pairs of primers, one for each segment, which were to amplify a region up to 1000 bp. Samples were pre-incubated for 3 min at 94 degrees C to induce bacterial lysis before starting the nucleic acid amplification. The PCR products 918 bp from segment A and 650 bp from segment B were digested with Fsp I and Dra I at 37 degrees C for 1 h. Products were resolved on 0.9% agarose gel which contained ethidium bromide. This method is simpler, faster and more accurate than the traditional method of mini-prep plasmid isolation and colony blot hybridization to identify the mutated plasmids.

A study of the effectiveness of a needle-free injection device compared with a needle and syringe used to vaccinate calves against bovine viral diarrhea and infectious bovine rhinotracheitis viruses.

The aim of this study was to compare the effectiveness of a needle-free injection device (NF) with a needle and syringe (NS) when used to vaccinate calves against bovine viral diarrhea virus (BVDV) and infectious bovine rhinotracheitis virus (IBRV). The study was conducted in two independent phases. Ninety-six crossbred beef calves were vaccinated in the spring and 98 beef calves in the autumn. The calves were vaccinated using a NF or NS at 2 months of age (day 0) and again on day 119, with a modified-live virus vaccine containing IBRV, BVDV (types 1 and 2), parainfluenza-3 virus, and bovine respiratory syncytial virus. In each herd 10 calves were left unvaccinated to determine whether exposure to either BVDV or IBRV occurred. Visible vaccine residue at the surface of the skin/hair was apparent immediately following vaccination with NF in 30% of the spring-born calves following both the primary and booster vaccination. In the autumn, visible vaccine residues occurred in 19% and 8% of NF-vaccinated calves following the primary and booster vaccination. Post-vaccination skin reactions recorded on days 21, 42, 119 and 140 occurred with greater frequency in NF-vaccinated calves than NS-vaccinated ones. Blood samples were collected on days 0, 21, 42, 119, and 140 and tested for antibodies to BVDV and IBRV. Vaccination technique had no significant effect on BVDV or IBRV antibody concentrations at any time point. NF was as effective as NS vaccination in eliciting BVDV and IBRV antibody responses.