Dietary Supplementation of Bacillus subtilis Probiotics Improves Growth Performance and Immune Response of Ducks-Experimentally Infected with Riemerella Anatipestifer
Received Date: Mar 15, 2019 / Accepted Date: Apr 15, 2019 / Published Date: May 25, 2019
Riemerella anatipestifer (RA) is a major bacterial disease affecting the poultry industry worldwide, particularly causing high economic losses in duck farms in Egypt. The objective of this experimental study was firstly to investigate the effects of RA-experimental infection on the morbidity and mortality rates and growth performance in Pekin ducks. Secondly the effects of RA on the transcriptomic expression of the immune related genes (interleukins (IL)1β, 6, 8, 10 and 17A) in the infected ducks were also examined using real time quantitative PCR. Thirdly, the protective effects of Bacillus subtilis (B. subtilis) as a probiotic dietary supplementation against RA infection in ducks were also investigated. The achieved results indicated that the mortality rate in RA-experimentally infected group was 80% and this percentage was reduced to 20% when birds received probiotics in their diet before challenge with RA. Furthermore, RA infection reduced the average feed intake, average body gain with poor average feed conversion ratio, while dietary supplementation of probiotics to ducks ’ feed significantly improved growth performance parameters. RA modulated the gene expression of the immune related genes, however, probiotics significantly upregulated mRNA expressions of the examined cytokines as a mechanism for the protective roles of probiotics against RA infection in ducks. Therefore, it is highly recommended to use probiotics dietary supplementation in duck farms in order to improve the growth performance and reduce the disease burden.
Keywords: Riemerella anatipestifer; Bacillus subtilis; Probiotics; Immunity; Growth performance; Ducks
Riemerella anatipestifer (RA) is a Gram’s negative bacteria that cause respiratory disease in ducks, geese, turkey and chicken . Several outbreaks of RA infection occurred worldwide, particularly in the duck [2,3]. The diseased birds suffer from respiratory and nervous symptoms and during necropsy, the dead birds had pericarditis, perihepatitis, and meningitis . RA was responsible for many outbreaks in ducks and ducklings reared in Egypt causing severe economic losses .
Antimicrobials are extensively used in poultry and duck farms in Egypt and other African countries for the purposes of prevention and control of bacterial diseases and as feed additives to improve the bird’s performance and feed-conversion ratio . However, the abuse of these antimicrobials led to development of multidrug resistant pathogens. Multidrug-resistant RA was isolated from intensively reared ducks, water fowls and migratory birds in several reports [7-9].
Probiotics are beneficial microorganisms that can be added into the bird’s ration in order to replace the gut harmful microbiota and can improve the overall performance and productivity of the birds mainly via a) activation of the immune system , b) maintain the gut microbial homeostasis , c) antimicrobial functions , d) improvement of the metabolic functions and detoxification of toxins and mutagens . However, few reports had investigated the protective effects of probiotics supplementation on RA infection in ducks.
In sight of the previous facts, this study aimed at investigating the protective and beneficial effects of probiotics supplementation on RA experimental infection in ducks via studying the feed conversion ratio, feed intake and body gain in the experimental groups. Furthermore, the effects of probiotics on RA-down regulated immune related genes were investigated using quantitative real-time PCR (qPCR).
Materials and Methods
Birds and experimental groups
Eighty (one day old) Pekin ducklings (Anas platyrhynchos domesticus) were purchased from a commercial hatchery in Egypt. Birds were grouped separately into four experimental groups; the first group received normal starter duck ration (crude protein 22%, crude fat 3.59%, crude fiber 3.84% and energy 2900 kcal/kg, Alfa Feed International, Egypt) and considered as a control group, the second group received the same ration and challenged with RA serotype 1 via intramuscular injection on their 15th day and named here and after RA group, the third group received Bacillus subtilis (probiotic) - supplemented ration and challenged with an intramuscular injection of RA on their 15th day and named RA+probiotic group, the fourth group received Bacillus subtilis -supplemented ration with no microbial challenge and named probiotic group. Bacillus subtilis (1 × 107 CFU/g, Weifang, China) was added to the commercial duck starter ration at 250 g/ton). RA serotype 1, the challenging pathogenic strain, was kindly gifted from Animal Research Institute, Cairo, Egypt and it was originally isolated from a commercial duck farm and prepared according to Fernandez et al. . All birds received free access to antibiotic free-water and feed. Constant light was provided during the experimental duration (30 days). Survival percentages and growth performance were recorded. On the age of 30th day, five birds from each group were sacrificed by cervical dislocation and liver and spleen tissues were collected and snap frozen for total RNA isolation. All animal experiments were done according to the guidelines for the animal use adopted by Faculty of Veterinary Medicine, Zagazig University, Egypt.
Re-isolation of RA from the liver and spleen of the dead birds or from collected tissue at the end of the experiment according to Fernandez et al.  with slight modifications. Briefly, one g of liver and spleen was aseptically collected and homogenized separately in 500 ml of tryptic soy broth using tissue homogenizers. A loopful from each homogenized sample was streaked on 5% sheep blood agar plates, and incubated at 37°C. The bacterial isolates were gram-negative coccobacilli and identification was confirmed biochemically and serologically.
Growth performance parameters including average feed intake (AFI), average body gain (ABG) and average feed conversion ratio (AFCR) were recorded at five-day intervals according to Abudabos et al. .
Total RNA was extracted from liver and spleen of the sacrificed ducks according to the method published before  using TRI reagent (Sigma, Sigma, St. Louis, USA) combined with homogenization (Bead Blaster 24 Tissue Homogenizer, Sayreville, NJ 08872, USA). The clear lysate was strongly mixed with chloroform for 2 min and followed by centrifugation at 13000 rpm for 20 min at 4°C. RNA was precipitated using isopropanol and centrifugation at the same conditions followed by washing with 70% ethanol and RNA was dissolved in RNAse free water. RNA concentrations and qualities were determined using a Nanodrop ND-1000 spectrophotometer (DYMO, Stamford, Conn., USA).
For cDNA synthesis, Rever Tra Ace® qPCR RT Master Mix with gDNA remover (Toyobo Co. Ltd., Osaka, Japan) was used as described in the manufacturer’s instructions. cDNA samples were stored at -20°C for further analysis.
Quantitative real time PCR (qPCR) for immune response genes
The mRNA expression levels of the immune response genes including interleukins (IL-) 1β, 6,8,10 and 17A were determined using qPCR, in Step One Plus Real-Time PCR system (Applied Biosystems, Foster, CA). The PCR mixture contained 600 ng of cDNA, Fast SYBR® Master Mix, 5 μM of each primer, with RNase-free water added to a final volume of 10 μL. The reaction cycle started with initial incubation for 20 s at 95°C, followed by 40 cycles of 3 s at 95°C (denaturation), 30 s at 60°C (annealing) and 15 s extension at 95°C. Single amplicon amplification was confirmed using melting curve analysis. The absence of primer dimers and genomic DNA amplification were confirmed by agarose gel electrophoresis. GAPDH was used for normalization by the comparative ΔΔCt method. Primer sets used were prepared according to published reports [17,18] and were displayed in Table 1.
|Target||Nucleotide sequence||Product size (bp)||Accession number|
Table 1: Primer sequences used in the present study.
Statistical significances among experimental groups were analyzed using Tukey’s Kramer HSD test (JMP program, SAS Institute, Cary, NC, USA) with P<0.05 considered as significant.
Results and Discussion
Ducks experimentally infected with RA in the present study suffered from loss of appetite, poor growth nervous manifestations, respiratory symptoms, and diarrhea. The morbidity rate in RA-experimentally infected group was 100%. While this percentage was reduced to 70% in the RA-experimentally infected group that received probiotics-supplemented ration (Table 2). Similar symptoms were reported in ducks naturally infected with RA . The mortality rate in RA-experimentally infected group was 80% and this percentage was reduced to 20% when birds received probiotics in their diet before challenge with RA. Likely, Subramaniam et al.  demonstrated that RA causes mortality rates as high as 75% in ducks. The re-isolation percentages were 100% and 70% in both RA and RA +probiotics group (Table 2).
Table 2: Effects of probiotic supplementation on the survival rates of ducks experimentally infected with RA.
At necropsy, the most prominent findings were fibrinous perihepatitis, pericarditis, and airsacculitis in RA group which agrees with Sandhu . Relatively higher mortality rate (16%) in RA-naturally infected ducks was reported in Japan during 2014. The diseased birds were 15-21 days old, suffering from ataxia, leg paddling, and dorsal decumbency; while at postmortem inspection, diseased birds showed meningitis, air sacculitis, pericarditis and perihepatitis . Furthermore, high prevalence rate (65.6%) for RA in naturally infected waterfowls was reported in Taiwan . Concerning the growth performance of the ducks experimentally infected with RA, the achieved data were reported in Table 3.
|Group||AFI (g/day)||ABG (g/day)||AFCR|
|Control||50.22 ± 5.15a||105.22 ± 9.15a||27.14 ± 4.22a||45.22 ± 3.32a||1.85||2.33|
|RA||55.17 ± 4.12a||72.17 ± 6.12b||29.22 ± 5.25a||21.22 ± 2.11b||1.88||3.4|
|RA+Probiotic||81.11 ± 7.14b||100.11 ± 5.14a||53.61 ± 8.15b||43.51 ± 5.17a||1.51||2.3|
|Probiotic||85.14 ± 7.21b||152.14 ± 9.21c||55.18 ± 5.17b||80.28 ± 9.77c||1.54||1.89|
Table 3: Effect of probiotic supplementation on the growth performance of ducks experimentally-infected with Riemerella anatipestifer. RA: Riemerella anatipestifer; AFI: average feed intake; ABG: average body gain; AFCR: average feed conversion ratio. Values in the same column carrying different super script letter are significantly different with each other at P<0.05.
It was clear that AFI (g/day) was significantly reduced in RA infected group (72.17 ± 6.12) compared with the control (105.22 ± 9.15) and RA+Probiotic (100.11 ± 5.14). Similarly, the ABG (g/day) was the lowest in RA group (21.22 ± 2.11) in a comparison with the control group (45.22 ± 3.32) and RA+Probiotic (43.51 ± 5.17). When birds received only probiotics-supplemented ration, their AFI (152.14 ± 9.21 g/day) and ABG (80.28 ± 9.77 g/day) were the highest among the tested group. Likely, AFCR value was the best in ducks receiving probiotics in their diet, while RA raised AFCR (3.4) reflecting the poor growth performance in the diseased birds, which much improved in the ducks infected with RA while receiving probiotics (2.3) in their diet (Table 3). Similar poor growth performance was reported in RA-naturally infected dusks in Egypt , Japan  and Taiwan .
Interleukins have emerged as critical molecules in host protective immunity and inflammatory diseases [22,23]. Therefore, screening of the transcriptomic changes of immune related interleukins expressed in both liver and spleen was investigated in order to explain the possible reasons for the high morbidity and mortality rates in RA-experimentally infected group. This screening was conducted using qPCR analysis as clear in Figures 1 and 2. RA mainly targets both liver and spleen tissues as reported before . The achieved results in Figure 1 indicated significant down regulation (fold relative to control) of IL-8 (0.33 ± 0.04) and IL-10 (0.39 ± 0.09) in the liver tissue of RA-experimentally infected ducks. In the spleen, the expression levels of these interleukins were in the same trend as the liver as clear in Figure 2. The reduction (fold relative to control) levels were as follows: IL-8 (0.44 ± 0.04) and IL-10 (0.47 ± 0.15). Interestingly, IL-1β (1.35 ± 0.03 and 1.83 ± 0.15), IL-6 (2.61 ± 0.05 and 1.54 ± 0.02) and IL-17A (2.11 ± 0.07 and 3.35 ± 0.11) were drastically up regulated as a response for RA infection in the liver and spleen, respectively. Supplementation of probiotics to ducks significantly up regulated the examined cytokines in the challenged group with RA as clear in Figures 1 and 2. This might explain the protective roles of the probiotics against RA infection and the significant reduction in the morbidity and mortality rates.
The achieved results indicate different roles for each interleukin. In agreement with the achieved results, Zhoe et al.  reported that RA infection in ducks modulated the transcriptomic expression of several genes in the affected ducks. For instances, immune response genes such as beta-defensins were downregulated, however IL-6 was upregulated as it plays an important role in the inflammatory response of the birds. In addition, Fernandez et al.  had reported significant decreases in the gene expression of IL-2 and IL-4 and increase in the expression of IL-1β, IL-6 and IL17A as a response to RA infection.
It notes worthy to mention that the supplementation of the duck ration with B. subtilis probiotics had led to a significant elevation in the immune response. For instances, the expression levels (fold relative to control) of IL-1β (4.13 ± 0.09), IL-6 (3.18 ± 0.09), IL-8 (8.32 ± 1.08), IL-10 (9.70 ± 1.07) and IL-17A (5.99 ± 0.49) in the liver. Similarly, the expression levels (fold relative to control) of the tested interleukins were significantly up regulated, IL-1β (6.92 ± 0.19), IL-6 (2.75 ± 0.09), IL-8 (8.71 ± 0.39), IL-10 (15.88 ± 1.21) and IL-17A (7.17 ± 0.35). The protective effects of B. subtilis probiotics were reported in ducks challenged with E. coli and novel duck reovirus, as B. subtilis improved innate immune response, growth performance and resistance to pathogens . The possible mechanisms for the protective effects of the dietary probiotics were suggested to include improving gut barrier function, antimicrobial activity, improving the resistance against pathogens, competition for nutrients, degradation of toxins and secretion of inhibitory substances .
This experimental study demonstrated the protective roles of B. subtilis as a probiotic supplemented to duck ration against RA experimental infection. This probiotic improved the growth performance survival rates and reduced the morbidity of the disease. The protection mechanism is suggested to involve up regulation of the immune and inflammation related genes in the liver and spleen of ducks. Therefore, it is highly recommended to use this probiotic type in duck farms as a preventive measure against RA infection.
We would like to thank stuff members of Avian and Rabbit Medicine Department and Educational Veterinary Hospital, Faculty of Veterinary Medicine, Zagazig University, Egypt for their kind technical support.
Conflict of Interest
- Rubbenstroth D, Ryll M, Behr KP, Rautenschlein S (2009) Pathogenesis of Riemerella anatipestifer in turkeys after experimental mono-infection via respiratory routes or dual infection together with the avian metapneumovirus. Avian Pathol 38: 497-507.
- Hinz KH, Ryll M, Kohler B, Glunder G (1998) Phenotypic characteristics of Riemerella anatipestifer and similar micro-organisms from various hosts. Avian Pathol 27: 33-42.
- Li JX, Tang Y, Gao JY, Huang CH, Ding MJ (2011) Riemerella anatipestifer infection in chickens. Pak Vet J 31: 65-69.
- Sandhu TS (2008) Riemerella anatipestifer infection. In: Diseases of poultry. 12th edn. In: Saif YM, Barnes HJ, Fadly AM, Glisson JR, McDougald LR, et al. (eds.). Blackwell Publishing Ltd., Oxford, UK, pp: 758-764.
- Deif H, Samir A, Mohamed KF, El-Jakee J (2015) Identification of duck septicemia in Egypt. Global Veterinaria 15: 397-400.
- Darwish WS, Eldaly EA, El-bbasy MT, Ikenaka Y, Nakayama S, et al. (2013) Antibiotic residues in food: the African scenario. Jpn J Vet Res 61: S13-S22.
- Cha S, Seo H, Wei B, Kang M, Roh J, et al. (2015) Surveillance and characterization of Riemerella anatipestifer from wild birds in South Korea. J Wildlife Dis 51: 341-347.
- Rubbenstroth D, Ryll M, Hotzel H, Christensen H, Knobloch JK, et al. (2013) Description of Riemerella columbipharyngis sp. nov., isolated from the pharynx of healthy domestic pigeons (Columba livia f. domestica), and emended description of the genus Riemerella, Riemerella anatipestifer and Riemerella columbina. Int J Syst Evol Microbiol 63: 280-287.
- Sun N, Liu JH, Yang F, Lin DC, Li GH, et al. (2012) Molecular characterization of the antimicrobial resistance of Riemerella anatipestifer isolated from ducks. Vet Microbiol 158: 376-383.
- Hyland NP, Quigley EM, Brint E (2014) Microbiota-host interactions in irritable bowel syndrome: Epithelial barrier, immune regulation and brain-gut interactions. World J Gastroenterol 20: 8859-8866.
- Lin CS, Chang CJ, Lu CC, Martel J, Ojcius DM, et al. (2014) Impact of the gut microbiota, prebiotics, and probiotics on human health and disease. Biomed J 37: 259-268.
- Arauz LJ, Jozalaa AF, Mazzolab PG, Vessoni Pennaa TC (2009) Nisin biotechnological production and application: a review. Trends Food Sci Technol 20: 146-154.
- Kondepudi KK, Ambalam P, Karagin PH, Nilsson I, Wadström T, et al. (2014) A novel multi-strain probiotic and synbiotic supplement for prevention of Clostridium difficile infection in a murine model. Microbiol Immunol 58: 252-258.
- Fernandez CP, Kim WH, Diaz JA, Jeong J, Afrin F, et al. (2016) Upregulation of duck interleukin 17A during Riemerella anatipestifer infection. Dev Comp Immunol 63: 36-46.
- Abudabos AM, Alyemni AH, Dafallah YM, Khan RU (2016) The effect of phytogenic feed additives to substitute in-feed antibiotics on growth traits and blood biochemical parameters in broiler chicks challenged with Salmonella typhimurium. Environ Sci Poll Res 23: 24151-24157.
- Darwish WS, Ikenaka Y, El-Ghareeb WR, Ishizuka M (2010) High expression of the mRNA of cytochrome P450 and phase II enzymes in the lung and kidney tissues of cattle. Animal 4: 2023-2029.
- Wei L, Jiao P, Song Y, Cao L, Yuan R, et al. (2013) Host immune responses of ducks infected with H5N1 highly pathogenic avian influenza viruses of different pathogenicities. Vet Microbiol 166: 386-393.
- Li N, Wang Y, Li R, Liu J, Zhang J, et al. (2014) Immune responses of ducks infected with duck Tembusu virus. Front Microbiol 6: 425.
- Subramaniam S, Huang B, Loh H, Kwang J, Tan HM, et al. (2000) Characterization of a predominant immunogenic outer membrane protein of Riemerella anatipestifer. Clin Diagn Lab Immunol 7: 168-174.
- Chikuba T, Uehara H, Fumikura S, Takahashi K, Suzuki Y, et al. (2016) Riemerella anatipestifer infection in domestic ducks in Japan. J Vet Med Sci 78: 1635-1638.
- Chang FF, Chen CC, Wang SH, Chen CL (2019) Epidemiology and Antibiogram of Riemerella Anatipestifer Isolated from Waterfowl Slaughterhouses in Taiwan. J Vet Res 63: 79-86.
- Bettelli E, Korn T, Oukka M, Kuchroo VK (2008) Induction and effector functions of TH17 cells. Nature 453: 1051-1057.
- Pappu R, Ramirez-Carrozzi V, Sambandam A (2011) The interleukin-17 cytokine family: critical players in host defence and inflammatory diseases. Immunology 134: 8-16.
- Zhou Z, Li X, Xiao Y, Wang X, Tian W, et al. (2013) Gene expression responses to Riemerella anatipestifer infection in the liver of ducks. Avian Pathol 42: 129-136.
- Guo M, Hao G, Wang B, Li N, Li R, et al. (2016) Dietary administration of Bacillus subtilis enhances growth performance, immune response and disease resistance in Cherry Valley ducks. Front Microbiol 7: 1975.
- Bajaj BK, Claes IJJ, Lebeer S (2015) Functional mechanisms of probiotics. J Microbiol Biotechnol Food Sci.
Citation: Ibrahim WFSE, Sameaa LA, Darwish WS (2019) Dietary Supplementation of Bacillus subtilis Probiotics Improves Growth Performance and Immune Response of Ducks-Experimentally Infected with Riemerella Anatipestifer. J Vet Sci Technol 10:578.
Copyright: © 2019 Ibrahim WFSE, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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