Avian Pathology
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Evaluation of a selected lactic acid bacteriabased probiotic on Salmonella enterica serovar Enteritidis colonization and intestinal permeability in broiler chickens Omar F. Prado-Rebolledo, Jaime de Jesus Delgado-Machuca, Rafael J. Macedo-Barragan, Luis J. Garcia-Márquez, Jesus E. Morales-Barrera, Juan D. Latorre, Xochitl Hernandez-Velasco & Guillermo Tellez To cite this article: Omar F. Prado-Rebolledo, Jaime de Jesus Delgado-Machuca, Rafael J. Macedo-Barragan, Luis J. Garcia-Márquez, Jesus E. Morales-Barrera, Juan D. Latorre, Xochitl Hernandez-Velasco & Guillermo Tellez (2016): Evaluation of a selected lactic acid bacteriabased probiotic on Salmonella enterica serovar Enteritidis colonization and intestinal permeability in broiler chickens, Avian Pathology, DOI: 10.1080/03079457.2016.1222808 To link to this article: http://dx.doi.org/10.1080/03079457.2016.1222808
Accepted author version posted online: 22 Aug 2016.
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Date: 21 September 2016, At: 07:02
Publisher: Taylor & Francis & Houghton Trust Ltd Journal: Avian Pathology DOI: 10.1080/03079457.2016.1222808
Evaluation of a selected lactic acid bacteria-based probiotic on Salmonella enterica serovar Enteritidis colonization and intestinal permeability in broiler chickens Omar F. Prado-Rebolledo1, Jaime de Jesus Delgado-Machuca1, Rafael J. Macedo-Barragan1, Luis J. Garcia-Márquez2, Jesus E. Morales-Barrera3, Juan D. Latorre4, Xochitl Hernandez-Velasco5, and Guillermo Tellez4* 1
Facultad de Medicina Veterinaria y Zootecnia, Universidad de Colima, Colima 28100, México,
2
Centro Universitario de Investigación y Desarrollo Agrícola, Universidad de Colima, Colima
28100, México, 3Departamento de Producción Agrícola y Animal, Universidad Autónoma Metropolitana, México City 04960, México, 4Department of Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA, 5Departamento de Medicina y Zootecnia de Aves, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, México City 04919, México
*Corresponding Author: Dr. Guillermo Tellez Department of Poultry Science, Center of Excellence for Poultry Science, University of Arkansas, 1260 W. Maple, POSC 0-114, Fayetteville, AR 72701, USA. (479) 575-8495 (telephone) (479) 575-8490 (fax) E-mail:
[email protected]
Short title: Probiotics can improve gut barrier integrity
Abstract Two experiments were conducted to evaluate the effect of a lactic acid bacteria-based probiotic (FloraMax-B11®) against Salmonella enterica serovar Enteritidis intestinal colonization and intestinal permeability in broiler chickens. Experiment 1 consisted of two independent trials. In each trial, day-old broiler chicks were assigned to one of two groups; control + S. Enteritidis; or probiotic + S. Enteritidis. At 72 h post- S. Enteritidis challenge, hematology and ceca content were evaluated for S. Enteritidis colonization. In Experiment 2, day-old broiler chicks were assigned to one of four groups; negative control; probiotic; control + S. Enteritidis; or probiotic + S. Enteritidis. At 72 h post- S. Enteritidis challenge, chickens in all groups were given an oral gavage dose of fluorescein isothiocyanate dextran. In both trials of experiment 1, a significant reduction (P < 0.05) in colony forming units/gram of S. Enteritidis in ceca content and a reduction in the incidence of S. Enteritidis enriched ceca samples were observed in probiotic + S. Enteritidis chickens. In addtition, a significant heterophilia and lymphopenia was observed in control + S. Enteritidis chickens. In experiment 2, a decrease in numbers of S. Enteritidis in ceca were observed in probiotic + S. Enteritidis chickens when compared to control + S. Enteritidis. Also, an increase in serum fluorescein isothiocyanate dextran concentration was detected in control + S. Enteritidis. These results suggest that early infection with S. Enteritidis can increase intestinal permeability, but the adverse effects can be prevented by the administration of the probiotic tested.
Keywords: broiler, intestinal permeability, probiotic, Salmonella Enteritidis
Introduction In recent years, many investigators (Hammes & Hertel, 2002; Tellez et al., 2012) have demonstrated the use of probiotics as an alternative to antibiotic growth promoters. Although the use of probiotics date for long time, it is only during the last two decades that scientist have started to elucidate the mechanisms of action as well as the potential of probiotics, to improve health and performance parameters of meat-producing production animals (Dominguez-Bello & Blaser, 2008). Studies have shown how probiotic bacteria regulate production of proinflammatory and anti-inflammatory cytokines ( Lyte, 2011), exert anti-oxidant properties (Howarth & Wang, 2013), and enhance barrier integrity (Hsieh et al., 2015). In addition, several investigators have demonstrated the benefits of probiotics on innate immunity (Vanderpool et al., 2008; Molinaro et al., 2012) as well as on humoral immunity Howarth & Wang, 2013). Our laboratory has worked to identify probiotic candidates for use in poultry. FloraMax-B11® is a defined lactic acid bacteria-based probiotic that was confirmed to increase the resistance of poultry to salmonella infections ( Higgins et al., 2007; Menconi et al., 2011; Tellez et al., 2012). More recently, microarray analysis of gut mRNA expression showed differences in birds treated with this probiotic in genes associated with the NFκB complex (Higgins et al., 2011). The objective of this study was to understand how FloraMax-B11® affects Salmonella enterica serovar Enteritidis intestinal colonization and intestinal permeability in chickens.
Materials and methods Probiotic culture. FloraMax-B11® (Pacific Vet Group USA Inc., Fayetteville AR 72703) is a defined probiotic culture derived from poultry gastrointestinal origin that contains proprietary strains of LAB (Menconi et al., 2014).
Animal source and diets. One-day-old male broiler chickens, were randomly housed in heated brooder batteries. Chicks had ad libitum access to unmedicated broiler starter feed and water for the duration of each experiment. Experiments were conducted at the College of Veterinary Medicine of the University of Colima. All animal handling procedures were in compliance with the Institutional Animal Care and Use Committee of the University of Colima. In each experiment, a small number of chicks (n = 10) were humanely killed upon arrival with CO2 asphyxiation. Ceca-cecal tonsils, liver and spleen were aseptically cultured in tetrathionate enrichment broth (Catalog no. 210420, Becton Dickinson, Sparks, MD). Enriched samples were confirmed negative for members of Salmonella by streak plating the samples on Xylose Lysine Tergitol-4 (XLT-4, Catalog no. 223410, BD Difco™) selective media.
Bacterial strain and culture conditions. The challenge organism used in all experiments was a poultry isolate of Salmonella enterica serovar Enteritidis (SE), bacteriophage type 13A, obtained from the USDA National Veterinary Services Laboratory, Ames, IA. This isolate was resistant to 25 µg/mL of novobiocin (NO, catalog no.N-1628, Sigma) and was selected for resistance to 20 µg/mL of nalidixic acid (NA, catalog no.N-4382, Sigma) in our laboratory. For the present studies, 100 µL of SE from a frozen aliquot was added to 10 mL of tryptic soy broth (Catalog no. 22092, Sigma) and incubated at 37°C for 8 h, and passed three times every 8 h to ensure that all bacteria were in log phase. Post-incubation, bacterial cells were washed 3 times with sterile 0.9% saline by centrifugation at 1,864 × g for 10 minutes, reconstituted in saline, quantified by densitometry with a spectrophotometer (Spectronic 20D+, Spectronic Instruments Thermo Scientific), and diluted to an approximate concentration of 108 CFU per milliliter. Concentrations of SE were further verified by serial dilution and plating on brilliant green agar (BGA, Catalog
no. 70134, Sigma) with NO and NA for enumeration of actual CFU used to challenge the chickens.
Experiment 1. Effect of probiotic on hematological profile and intestinal colonization of SE. Two independent trials were conducted. In each trial, twenty chickens were randomly assigned to one of two groups; Control SE challenged (CONT+SE), or SE challenged plus probiotic (PROB+SE). The challenge with SE was administered to both groups of chicks on arrival at the laboratory by oral gavage (4 × 104 CFU/0.25 mL/bird) using an animal feeding needle. At 1h post challenge, probiotic was administered to PROB+SE chicks by oral gavage (4 x 107 CFU/0.25 mL/bird). Vehicle (sterile saline) was administered to chickens in CONT+SE group. In each trial, at 72 h post SE challenge, blood samples were collected after CO2 asphyxiation from the femoral vein of all chickens from each group. The recovered total leukocyte numbers were counted with a hemocytometer. Differential cell counts were conducted on blood smears from each chicken. Smears were air dried, fixed in methanol and stained with the Diff-Quik staining system (Curtin Matheson Scientific Co., Houston, TX). At least 200 cells on each slide were examined microscopically (1000 X magnification) and the proportions of cells were determined. Heterophil/lymphocyte ratio (Gross & Siegel, 1983) was calculated by dividing the number of heterophils in 1 mL of peripheral blood by the number of lymphocytes. In both trials, CCT were aseptically collected for SE recovery as explained below.
Experiment 2. Effect of probiotics on SE intestinal colonization using fluorescein isothiocyanate dextran (FITC-d) permeabilization assay. The experiment 2 was a repetition and extension of experiment 1. For this experiment, forty-eight day-of hatch broiler chickens
were randomly assigned to one of the four groups (n = 12/group); control no SE challenged (CONT), probiotic no SE challenged (PROB), control SE challenged (CONT+SE), or SE challenged plus probiotic (PROB+SE). The challenge with SE was administered to both groups of chicks on arrival at the laboratory by oral gavage (4 × 104 CFU/0.25 mL/bird) using an animal feeding needle. At 1h post challenge, probiotic was administered to the probiotic group of chicks by oral gavage (4 x 107 CFU in 0.25 mL). Vehicle (sterile saline) was administered to chickens with no SE challenge. All chickens in CONT+SE and PROB+SE were humanely killed 72 h postSE challenge to evaluate Salmonella recovery as describe below.
Recovery of salmonella. For experiments 1 and 2, Ceca-cecal tonsils (CCT) were homogenized and diluted with saline (1:4 by wt/vol) and tenfold dilutions were plated on BGA with NO and NA, incubated at 37°C for 24 h to enumerate total SE colony forming units. However, in both trials of experiment 1, following plating to enumerate total SE, the CCT samples were enriched in double strength tetrathionate enrichment broth and further incubated at 37°C for 24 h. Following this, enrichment samples were plated on BGA with NO and NA and incubated at 37°C for 24 h to confirm presence/absence of typical lactose-negative colonies of Salmonella.
Serum determination of FITC-d leakage. Intestinal leakage of fluorescein isothiocyanate dextran (FITC-d) (MW 3-5 KDa; Sigma-Aldrich Co., St. Louis, MO) and the measurement of its serum concentration was done in experiment 2 as a marker of paracellular transport and mucosal barrier dysfunction (Yan et al., 2009; Kuttappan et al., 2015; Vicuña et al., 2015a, b). At 72 h, post SE challenge, chickens in all groups were given an oral gavage dose of FITC-d (4.16 mg/kg). Following 2.5 h, they were killed with CO2 asphyxiation. Blood samples were collected from the
femoral vein kept at room temperature for 3 h and centrifuged (500 x g for 15 min) to separate the serum from the red blood cells. FITC-d levels of diluted serum samples (1:1 PBS) were measured at excitation wavelength of 485 nm and emission wavelength of 528 nm with a Synergy HT, Multi-mode microplate fluorescence reader (BioTek Instruments, Inc., Vermont, USA). Fluorescence measured was then compared to a standard curve with known FITC-d concentrations. Gut leakage for each bird was reported as μg of FITC-d/mL of serum (Kuttappan et al., 2015).
Data and statistical analysis. Log10 CFU/g of SE in cecal contents, peripheral blood counts and serum FITC-d concentration were subjected to analysis of variance as a completely randomized design, using the General Linear Models procedure of SAS (SAS Institute, 2002). Significant differences among the means were determined by Duncan’s multiple-range test at P < 0.05. The enrichment data were expressed as positive/total chickens (%), and the percent recovery of SE was compared using the chi-squared test of independence, testing all possible combinations to determine the significance (P ≤ 0.01) for these studies.
Results The results of the effect of FloraMax-B11® on SE intestinal colonization in neonatal broiler chickens in trials 1 and 2 of experiment 1 are summarized in table 1. In both trials, a significant reduction (P < 0.05) in Log10 CFU of SE in CCT samples and a decrease (P < 0.001) in the incidence of SE enriched CCT were observed in PROB+SE chickens when compared with CONT+SE chickens (Table 1).
Table 2 shows the results of the effect of a selected Lactobacillus-Based probiotic on total leukocyte counts (WBC), and percentages of heterophils, lymphocytes, eosinophils, and basophils of peripheral blood of neonate broilers chickens in both trials of Experiment 1. In both trials, 72 h following SE challenge, a significant (P < 0.05) heterophilia and lymphopenia were observed in CONT+SE chickens when compared with PROB+SE (Table 3). Consequently, an increase in the heterophils-to-lymphocyte ratio was also observed in CONT+SE, but not in PROB+SE. In both trials, significant increases in basophils were detected in CONT+SE when compared with PROB+SE chickens. In trial 2, eosinophilia was observed in CONT+SE when compared with PROB+SE chickens (Table 2). The results of the effect of FloraMax-B11® on SE intestinal colonization and gut leakage of FITCd as determined by its serum concentration in experiment 2 are summarized in table 3. In this experiment, a significant reduction in Log10 CFU of SE in CCT was observed in PROB+SE chickens when compared to CONT+SE. Also, a significant increase in serum FITC-d concentration was detected in CONT+SE chickens when compared with CONT; PROB, and PROB+SE (Table 3).
Discussion Salmonellosis remains one of the most comprehensive foodborne diseases that can be transmitted to humans through animal and plant products (Hernández-Reyes & Schikora, 2013). Experiments 1 and 2 of the present study, confirm previous reports from our laboratory using the same therapeutic SE challenge model. In the present study, the marked heterophilia and lymphopenia, observed in chickens challenged with SE resulted in a significant increase of heterophils-tolymphocyte ratio as has been previously reported (Al-Murrani et al., 2002). However, these
hematological changes were prevented by chickens that received the probiotic one h after SE challenge (Table 2). Further studies should consider the inclusion of control or probiotic chickens without SE challenge for the evaluation of hematological changes, as they were not included in the present study. In experiment 2, SE challenge increased the levels of serum FITC-d, a molecule which does not cross the intact gastrointestinal tract barrier. However, when conditions disturb tight junctions (TJ) between epithelial cells, FITC-d can enter circulation as demonstrated by an increase in paracellular permeability after oral administration of FITC-d (Kuttappan et al., 2015). Tight junctions act as intercellular cement between epithelial cells and regulate the permeability and dissemination of microorganisms and antigens (Ulluwishewa et al., 2011). Hence, any damage to this fragile epithelium may lead to chronic intestinal and systemic inflammation. Uptake and distribution within the host of salmonellae infections are associated with disruption of the TJ complex, loss of barrier function, bacterial translocation and initiation of polymorphonuclear cells (PMN) migration across the intestinal barrier. Therefore, appropriate gut barrier function is indispensable to maintain optimal health (Pastorelli et al., 2013). Bacterial endotoxin has been shown to activate aldose reductase (AR), a member of aldoketo reductase super family, and the nuclear factor kappa B (NFκB). Activation of NF-κB results in the expression of several inflammatory cytokines (Overman et al., 2012). It is well known that oxidative stress-induced inflammation is a major contributor to several diseases. AR catalytic activity plays a crucial role in some inflammatory diseases associated with disruption of TJ between epithelial cells (Pastel et al., 2012). Interestingly, microarray analysis with FloramaxB11® in broiler chickens challenged with SE showed a significant reduction in intestinal gene expression associated with the NF-κB complex and AR (Higgins et al., 2011). The results of Experiment 2 also suggest that the probiotic preserved the integrity of the intestinal epithelial cells
(IEC), which represent the second layer of innate defense mechanisms of the gastrointestinal tract (Johansson et al., 2010). These results are in agreement with studies demonstrating that probiotics prevent Salmonella translocation, suppressed the oxidant-induced intestinal permeability, and improve intestinal barrier function Segawa et al., 2011; Hsieh et al., 2015). Metchnikoff founded the research field of beneficial microorganisms for animals and humans (probiotics), aimed at modulating the intestinal microflora (Metchnikoff, 1907). At the moment, new molecular techniques are helping us to understand how the anti-inflammatory, cell integrity and anti-oxidant properties of probiotics can improve gut and barrier integrity. Given the recent international legislation and domestic consumer pressures to withdraw growth-promoting antibiotics and limit antibiotics available for treatment of bacterial infections, probiotics, and direct-fed microbials can offer clear alternative options. These results suggest that early infection with SE can induce paracellular transport leakage of FITC-d and mucosal barrier dysfunction, but the adverse effects can be prevented by the administration of the selective probiotic.
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Table 1. Effect of probiotics on Salmonella Enteritidis (SE) intestinal colonization in neonatal broiler chickens1. Experiment 1. Trial 1
Trial 2
Log10 CFU of SE cecal contents
SE incidence
CONT+SE PROB+SE 1
SE incidence
Ceca-cecal tonsils2
Log10 CFU of SE cecal contents
2.99 ± 0.20a
10/10 (100 %)
4.31 ± 0.69a
8/10 (80 %)
0.46 ± 0.03b
4/10 (40 %)*
1.61 ± 0.69b
3/10 (30 %)*
Ceca-cecal tonsils
Ceca-cecal tonsils were cultured to enumerate Log10 CFU of SE/g of ceca content, and the data is expressed as mean ± standard error of the mean. a–b Different superscripts within columns indicate significant differences P < 0.05; n = 10 chickens/group. 2 Ceca-cecal tonsils enrichment data is expressed as positive/total chickens for each tissue sampled (%). *Values in the same column are significantly different from the control value (P < 0.001).
Table 2. Effect of probiotics on total leukocyte counts (WBC), and percentages of heterophils, lymphocytes, eosinophils, and basophils of peripheral blood of neonate broilers chickens1. Experiment 1. Trial
1
Trial
2
CONT+SE
PROB+SE
CONT+SE
PROB+SE
34,260 ± 1,249a
27,160 ± 1,331b
33,590 ± 3,284a
24,860 ± 1,779b
Heterophils (%)
56.20 ± 1.39a
40.40 ± 3.14b
60.10 ± 2.21a
38.70 ± 1.45b
Lymphocytes (%)
41.50 ± 1.47b
58.70 ± 3.50a
35.20 ± 2.46b
59.20 ± 1.09a
Eosinophils (%)
0.70 ± 0.21a
0.50 ± 0.22a
1.40 ± 0.42a
0.90 ± 0.31b
Basophils (%)
1.60 ± 0.26a
0.40 ± 0.26b
1.20 ± 0.35a
0.30 ± 0.15b
Heterophils-toLymphocyte ratio
1.38 ± 0.11a
0.75 ± 0.12b
1.83 ± 0.20a
0.68 ± 0.21b
WBC (× 103/µL)
1
Data is express as mean ± standard error. Means within a row between treatments groups for each trial respectively with different superscripts differ P < 0.05; n = 10 chickens/group, for each trial.
a–b
Table 3. Effect of a selected lactic acid bacteria -based probiotic on Salmonella Enteritidis (SE) intestinal colonization and serum FITC-d concentration in neonatal broilers chickens. Experiment 2. CONT
PROB
Log10 CFU of SE in
Negative for salmonella
Negative for salmonella
cecal contents
upon arrival
upon arrival
b
0.30 ± 0.08b
Serum FITC-d 0.34 ± 0.09
CONT+SE
PROB+SE
3.09 ± 0.75a
1.08 ± 0.69b
a
0.76 ± 0.01b
1.59 ± 0.07
(µg/mL) a,b
Superscripts within rows indicate difference at P < 0.05; n = 12 chickens/group. Data are expressed as mean ± standard error. Ceca-cecal tonsils were cultured to enumerate Log10 CFU of SE/g of ceca content.
