Effect of Indigenous Probiotics on Gut Morphology and Intestinal Absorption Capacity in Broiler Chicken Challenged with Salmonella enteritidis

Imran Khan1, Muhammad Nawaz1,*, Aftab Ahmad Anjum1, Mansur-ud-Din Ahmad2, Adnan Mehmood1, Masood Rabbani1, Amina Mustafa1 and Muhammad Asad Ali1

1Department of Microbiology, University of Veterinary and Animal Sciences, Lahore

2Department of Epidemiology and Public Health, University of Veterinary and Animal Sciences, Lahore

ABSTRACT

Probiotics are useful in modulating and strengthening the gut microbiota. Present study was designed to determine the effect of three previously characterized probiotic lactobacilli i.e. L. fermentum IKP 23, L. fermentum IKP 111 and L. salivarius IKP 333 on histomorphology of small intestine and D-xylose absorption capacity in broiler challenged with S. enteritidis. Negative control group was not supplemented with probiotics. Positive control groups received only the challenge bacteria (S. enteritidis) ATCC 13076 at day 07. Groups (3, 4 and 5) received probiotics (IKP23, IKP111, IKP333) at day 01 to 35 and challenge bacteria at day 07 in prevention model. Groups (6, 7 and 8) started receiving probiotic at day 07 to day 35 and challenge bacteria at day 07 in treatment model. Group 09 started receiving commercial probiotic Protexin (1g/litre) at day 01 to 35 and challenge bacteria at daye 07. Group 10 started receiving antibiotic at day 01 to 10 and challenge bacteria at day 07. Birds were challenged with a single dose of 106 cfu of Salmonella enteritidis by oral gavage. D-xylose absorption capacity and gut morphometric parameters (villus height, crypt depth and villus height to crypt depth ratio) were studied at day 35. Broiler administered with IKP23, IKP111 and IKP333 significantly improved villus height and villus height to crypt depth ratio as compared to positive control. D-xylose absorption was also enhanced in groups administered with probiotics. It is concluded that IKP23, IKP111 and IKP333 may improve gut morphometric parameters and absorption capacity of broiler challenged with S. enteritidis which insinuate for their possible role in efficient broiler production.

Article Information

Received 18 May 2019

Revised 12 June 2019

Accepted 29 June 2019

Available online 18 May 2020

Authors’ Contribution

MN, MUD and MR designed the project. IK and MAA collected samples. IK and AM executed experiments. MN and AAA analysed data. AM, IK and MN prepared the manuscript.

Key words

Probiotics, S. enteritidis, Prevention model, Protexin, D-xylose.

DOI: https://dx.doi.org/10.17582/journal.pjz/20190518090547

* Corresponding author: muhammad.nawaz@uvas.edu.pk

0030-9923/2020/0005-1825 $ 9.00/0

Copyright 2020 Zoological Society of Pakistan

Introduction

Probiotics are live microorganisms which provide benefit to host upon sufficient administration (FAO-WHO, 2001). Probiotics provide a number of health benefits to poultry i.e. immune boosting, protection against gut pathogens, binding of mycotoxins, strengthening of gut function and microbiota, and increased absorption of nutrients leading to enhanced and efficient poultry production (Patterson and Burkholder, 2003; Park et al., 2016; Azeem et al., 2019). Poultry gastric tract is composed of crop, gizzard, jejunum, ileum, duodenum, and ceca. Intestinal health of poultry is directly linked with the gut function and nutrient absorption. Poultry gut contains a huge diversity of microorganisms including lactobacilli (Saleem et al., 2018). Further strengthening of gut microbiome with probiotics provides huge benefits. Probiotics increase villus height and crypt depth ratio in poultry gut intestine (Kim et al., 2012). Increased villus height and crypt depth ratio are associated with growth performance in poultry (Ali et al., 2017). Probiotics help in the improvement of gut health and can be used as growth promotor in broiler diets (Awad et al., 2009).

Probiotics play an important role in improving the nutritional approach of poultry gut function and provide potential alternatives to antimicrobial growth promoter in poultry nutrition (Mountzouris et al., 2007; Applegate et al., 2010). Salmonella enteritidis is generally present in poultry gut and its transmission to human food chain is a major threat to public safety. It is one of the major causes of food poisoning and gastro-intestinal infections in human. It negatively impacts the nutrient absorption, gut function and intestinal health of birds (Ansari et al., 2017). Use of antibiotics to control the Salmonellae in poultry is not only a risk factor for emergence of antibiotic resistance, it also leads to decreased gut microbiota and gut function (Apata, 2012). Decreased gut microbiota may lead to reduced gut function and nutrient absorption. Prophylactic use of antibiotics in poultry industry for the control and prevention of Salmonella have aggravated the problem of antibiotic resistance and antibiotic residues in animal and human food chains (Siddique et al., 2018). Thus, the use of antibiotics especially as growth promoter should be prohibited in Pakistan and alternatives i.e. probiotics should be developed.

Probiotics play an important role in improving the nutrients availability and poultry gut function (Mountzouris et al., 2007; Applegate et al., 2010). Probiotics diet increase the villus height and villus height/crypt depth ratio in duodenum of poultry (Afsharmanesh and Sadaghi, 2014). Lactobacilli significantly increase weight of small intestine (jejunum and ileum) (Olnood et al., 2015). Probiotics dietary treatments influence the histomorphological measurements in poultry and probiotics diet significantly increase the villus height/crypt depth ratio in duodenum and ileum (Awad et al., 2009). D-xylose absorption test is very useful tool to assess the intestinal function efficiency. D-xylose is well absorbed from the small intestine and has been successfully used to evaluate the mal-absorption syndrome in birds caused by bacteria and viruses (Doerfler et al., 2000). The birds with different health status show D-xylose absorption capacity (Shomali et al., 2012).

Although there are few published reports on development of indigenous probiotics from Pakistan (Asghar et al., 2016; Arif et al., 2018) , there still is insufficient data on effect of indigenously characterized probiotics on gut morphology and nutrient absorption capacity of broiler challenged with Salmonella from Pakistan. Therefore, current study was designed to analyse the effect of indigenously characterized probiotic strains on gut morphology and D-xylose absorption capacity in broiler.

 

Material and Methods

Microorganisms and growth conditions

Previously characterized potential probiotic strains Lactobacillus fermentum IKP 23 (MK350329), L. fermentum IKP 111 (MK350330) and L. salivarius IKP 333 (MK346270) were grown and maintained in MRS media. Salmonella enteritidis ATCC 13076 was procured from department of Microbiology, University of Veterinary and Animal Sciences, Lahore and grown in SS agar at 37oC.

Study design

Day old broiler chicks (n=150) were procured from the local market and reared for 35 days in experimental shed in Department of Microbiology, University of Veterinary and Animal Sciences, Lahore. Chicks were randomly distributed in 10 groups (15 chicks per groups). Negative control group was not supplemented with probiotics. Positive control groups received only the challenge bacteria (S. enteritidis ATCC 13076) at day 07. Groups (3, 4 and 5) received probiotics (IKP23, IKP111, IKP333) from day 1 to 35 and challenge bacteria at day 7 in prevention model (PM). Groups (6, 7 and 8) started receiving probiotic at day 7 to day 35 and challenge bacteria at day 7 in treatment model (TM). Group 09 started receiving commercial probiotic Protexin (Hilton Pharma) 1g/litre (107 cfu/ml) at day 1 to 35 and challenge bacteria at day 7. Group 10 started receiving antibiotic (enrofloxacin) at day 1 to 10 and challenge bacteria at day 7. Birds were challenged with a single dose of 106 cfu of Salmonella enteritidis by oral gavage, while probiotics were administered 108 cfu daily.

Intestinal morphometric parameters

On day 35, 5 birds from each group were randomly slaughtered and intestinal tissue samples (duodenum, ileum and jejunum) were collected. Samples were fixed in 10% neutral buffered formaldehyde, embedded in paraffin, cut into fine sections, and stained with haematoxylin and eosin stain as described previously (Awad et al., 2009). Slides were examined with a light microscope (Olympus CX31, USA) attached with a digital imaging system and analysed by using Labomed pixel pro software. The morphometric variable including villus height, crypt depth and villus height/crypt depth ratios were measured.

D- xylose absorption capacity test of broiler

D-xylose absorption capacity in broiler of different groups was also determined on day 35 as described previously (Mansoori et al., 2012). Briefly, five birds from each group were randomly selected for D-xylose absorption test. D-xylose (Merck, Germany) was given via oral route @ 500mg D-xylose/kg body weight to each bird. The blood samples were drawn from ulnar vein in wings at pre-inoculation and post-inoculation (30 min and 60 min) of D-xylose. D-xylose was measured from plasma using phloroglucinol reagent as described previously (Regassa et al., 2016). Briefly, 20 µl plasma was mixed with 2 ml phloroglucinol reagent, heated for 4 min at 100°C, cooled to room temperature followed by reading the absorbance at 554 nm using spectrophotometer (UV-150-02 Shimadzu Corporation, Japan) Concentration of D-xylose in plasma was measured from a D-xylose standard curve prepared from different concentrations (5-75 mg/dl) of D-xylose.

Statistical analysis

Data were expressed as Mean±S.D and means of different groups were compared by one way ANOVA followed by Tukey’s multiple comparison test at P value <0.05 by GraphPad Prism Software.

 

Table I.- Effects of probiotic on gut (duodenum) morphology in broiler chickens.

Groups	Villus height (um) (Mean± S.D)	Crypt depth (um) (Mean± S.D)	Villus height/ Crypt depth ratio
Negative control	646.67±52.51	98.33±16.07	6.58a
Positive control	709.67±74.56	115.67±8.14	6.14a
IKP 23-PM	818.53±52.47	108.67±24.24	8.03b
IKP 111-PM	741.67±66.01	91.53±10.63	8.10b
IKP 333-PM	725.43±83.71	90.33±11.67	8.07b
Protexin-PM	660.59±101.91	86.28±5.46	7.66a
Antibiotic-PM	656.67± 78.47	107.90± 5.85	6.09a
IKP 23-TM	683.18±61.74	91.71±4.77	7.45a
IKP 111-TM	596.40±28.71	96.74±14.41	6.17a
IKP 333-TM	629.24±82.19	92.42±4.72	6.81a

 

a,b, different superscript show statistically significant difference at p ≤ 0.05.

 

Table II.- Effects of probiotics on gut (jejunum) morphology in broiler chickens.

Groups	Villus height (um) (Mean± S.D)	Crypt depth (um) (Mean± S.D)	Villus height/ Crypt depth ratio
Negative control	805.73±65.76	123.52±14.87	6.52 a
Positive control	686.67±37.52	107.57±2.40	6.38a
IKP 23-PM	1231.47±45.36	103.67±10.88	11.88b
IKP 111-PM	953.47±149.53	100.00±18.02	9.53b
IKP 333-PM	1185.83±122.04	143.80±23.68	8.25b
Protexin-PM	564.28±6.89	79.95±10.52	7.06a
Antibiotic-PM	670.30±62.56	110.98±10.70	6.04a
IKP 23-TM	1015.87±8.20	138.67±54.26	7.33a
IKP 111-TM	675.99±10.53	100.33±21.70	6.74a
IKP 333-TM	1036.78±29.30	109.77±19.98	9.45b

 

a,b, different superscript show statistically significant difference at p ≤ 0.05.

 

Results

Probiotics improve intestinal histomorphological measurement of broiler

Mean villus height, crypt depth and villus height/crypt depth ratio of duodenum, jejunum and ileum of different groups are presented in Tables I, II and III, respectively. Representative intestinal histomorphology (duodenum) of different groups is shown in Figure 1. Villus height/crypt depth ratio of negative and positive control group were non-significantly different (P<0.05) in duodenum (6.58 vs 6.14), jejunum (6.52 vs 6.38) and ileum (5.28 vs 6.06), respectively. Villus height/crypt depth ratio in broiler groups (IKP 23-PM, IKP 111-PM, IKP 333-PM) administered with probiotic before challenge was significantly increased in duodenum (8.03, 8.1, and 8.07, respectively), jejunum (11.88, 9.53, and 8.25, respectively) and ileum (7.59, 8.53, 8.25, respectively) as compared with positive control group. Results also revealed that broiler groups (IKP 23-TM, IKP 111-TM, IKP 333-TM) administered with probiotic post challenge showed non-significant treatment increase in villus height/crypt depth ratio in duodenum. IKP111 and IKP333 significantly increased villus height/crypt depth ratio in jejunum and ileum in respective groups of broilers.

 

Table III.- Effects of probiotic on gut (ileum) morphology in broiler chickens.

Groups	Villus height (um) (Mean± S.D)	Crypt depth (um) (Mean± S.D)	Villus height/ Crypt depth ratio
Negative control	632.30±65.90	119.77±8.88	5.28a
Positive control	666.67±20.81	110.00±5	6.06a
IKP 23-PM	762.47±27.06	100.43±20.70	7.59b
IKP 111-PM	864.67±25.60	101.40±4.54	8.53c
IKP 333-PM	761.63±73.55	92.23±17.03	8.26c
Protexin-PM	710.47±37.21	102.29±3.93	6.94b
Antibiotic-PM	417.91±35.67	78.67±13.86	5.31a
IKP 23-TM	771.33±47.58	93.93±6.12	8.21c
IKP 111-TM	664.13±48.24	95.08±9.06	6.99b
IKP 333-TM	611.12±57.41	87.08±27.90	7.02b

 

a,b,c, different superscript show statistically significant difference at p ≤ 0.05.

 

Table IV.- D-xylose concentration in plasma of broiler chicken at 0, 30 and 60 min on day 35 of age.

Groups	D-Xylose concentration (Mean±SD, mg/dl) in plasma of broiler in different groups
	0 min	30 min	60 min
Negative control	0	14.11±2.31a	22.09±2.38a
Positive control	0	13.51±1.89a	22.30±1.95a
IKP 23-PM	0	30±5.21b	58.06±7.23b
IKP 111-PM	0	26.92±4.32b	52.75±5.64b
IKP 333-PM	0	24.16±3.74b	53.09±4.89b
Protexin-PM	0	25.05±3.23b	50.97±5.41b
Antibiotic-PM	0	16.01±2.73a	22.10±3.54a
KP 23-TM	0	20.03±3.12a	48.81±4.26b
IKP 111-TM	0	22.80±2.56a	46.71±8.23b
IKP 333-TM	0	22.41±2.43a	48.02±5.69b

 

a,b, different superscript in different rows of same column show statistically significant difference at p ≤ 0.05.

 

Probiotics enhance D-xylose absorption from intestine

Results of D-xylose absorption capacity of broiler is given in Table IV. There was no significant difference (P˂ 0.05) in the D-xylose absorption capacity of negative control group and positive control group. D-xylose concentration in plasma of negative control group and positive control group was 22.09 mg/dl vs 22.30 mg/dl, respectively at 60 min post administration. Results revealed that plasma D-xylose concentration in broiler groups administered with probiotics pre-challenge (58.06±7.23, 52.75±5.64 and 53.09±4.89 mg/dl, respectively) or post challenge of Salmonella (48.81±4.26, 46.71±8.23 and 48.02±5.69 mg/dl, respectively) was significantly higher (P˂ 0.05) compared to negative control (22.09±2.38 mg/dl), positive control (22.30±1.95 mg/dl) and antibiotic group (22.10±3.54). Commercial probiotic product protexin also significantly increased the D-xylose absorption (50.97±5.41 mg/dl) from intestine as compared to positive and negative control group.

 

Discussion

Intestinal morphology including duodenal, ileal and jejunum villus height and villus height crypt depth ratio are indicative of gut health. Increased villus height and villus height to crypt depth ratio are directly associated with an improved epithelial turn over (Fan et al., 1997; Liu et al., 2009) and longer villi are interrelated with stimulation of cell mitosis (Samanya and Yamauchi, 2002), while shortening of villi and reduced crypt depth ratio is indicative of poor nutrient absorption, increased secretions of gastrointestinal tract and reduced gut performance (Xu et al., 2003). In the present study, supplementation of L. fermentum IKP 23, L. fermentum IKP 111 and L. salivarius IKP 333 in drinking water of broiler resulted in increased villus height and villus height to crypt depth ratio in duodenum, jejunum and ileum at day 35. Result obtained in the present study are consistent with previous study reported increased villus height and villus height to crypt depth ratio in duodenum, jejunum and ileum of broiler supplemented with probiotics (Lactobacillus). Similar study reported that supplementation of Lactobacillus in broiler increased villus height and villus height to crypt depth ratio in duodenum (Awad et al., 2010; Ashraf et al., 2013; Song et al., 2014). Lactobacillus treatment caused similar changes in intestinal morphology of broiler (Awad et al., 2009; Thanh et al., 2009; Salim et al., 2013). Scientific data shows that probiotics have positive effect on physiological function in small intestine. Probiotics involved in crypt cells proliferation of small intestine increased with the probiotics as compared to control (Ahmad, 2006; Awad et al., 2009; Sohail et al., 2012). In past, researcher claimed that villus height and crypt depth ratio increased in broiler with probiotics supplementation (Awad et al., 2009; Al-Fataftah and Abdelqader, 2014; Song et al., 2014). Result obtained here in provides information that L. fermentum (IKP 23), L. fermentum (IKP 111) and L. salivarius (IKP 333) have potential of improving gastrointestinal morphology in broiler chicken as growth promotor. Present study also reported that the use of probiotics helped in an increase in villus height and crypt depth ratio in small intestine (duodenum, jejunum and ileum). Villus height and crypt depth ratio of duodenum, jejunum and ileum was measured in all experimental groups. In duodenum the highest villus height and crypt depth ratio (8.10) was observed in IKP 111 supplemented (day 01 to 35) group and lowest ratio (6.09) was in antibiotic supplemented group. Significant difference (p˂0.05) was observed in villus height and crypt depth ratio in probiotics group (IKP 23, IKP111 and IKP 333 supplemented at day 01 to 35) and other experimental groups. In jejunum the highest villus height and crypt depth ratio (11.88) was measured in IKP 23 supplemented (day 01 to 35) group and lowest ratio (6.04) was measured in antibiotic supplemented. In ileum the highest villus height crypt depth ratio (8.53) was measured in IKP111 supplemented (day 01 to 35) group while lowest (5.28) in negative control group. It was evident, that IKP 23, IKP111 and IKP 333 probiotics supplemented groups had higher villus height and crypt depth ratio in all experimental groups.

D-xylose absorption test is very effective test to assess the intestinal absorption capacity of broiler (Mansoori et al., 2012). Moreover, D-xylose absorption test has been proven a reliable display of intestinal absorptive function in poultry (Doerfler et al., 2000). D-xylose absorption test is a sensitive tool used for the evaluation of intestinal absorption capacity of chicken and birds with different nutritional demands showed different result of D-xylose absorption capacity (Mansoori et al., 2012). D-xylose absorption is a good serum selected parameter for broiler chicken (Shomali et al., 2012). The intestine of broiler absorb D-xylose practically completely, thus any change in plasma concentration of D-xylose in early hours after consumption is sign of absorption capacity of intestinal tract (Schutte et al., 1991; Doerfler et al., 2000). In present study, it was reported that differences exist in absorption function of small intestine for D-xylose in different groups of broilers which were supplemented with probiotics and without probiotics. D-xylose test was used to assess the small intestine absorption capacity of broiler groups administered with probiotics and broiler group without supplementation of probiotics in present study. Dietary supplementation of probiotics in broiler resulted in an increase in villus height and surface area of villi in small intestine (Awad et al., 2009). D-xylose is well absorbed from the small intestine as D-glucose in birds (Doerfler et al., 2000). In past, Mansoori (2010) claimed that reduced growth rate in chicken are linked with low D-xylose absorption capacity in small intestine. Groups had better D-xylose absorption capacities which were supplemented with probiotics at day 1 to 35 as compared to those groups in which probiotics were supplemented at day 7 to 35. The highest concentration of D-xylose 58.06 mg was measured in plasma of broiler group supplemented with IKP 23 at day 1 to 35 in all experimental groups. The lowest concentration 22.10 mg of D-xylose was measured in plasma of group supplemented with antibiotics. There was significant difference (p˂0.05) in D-xylose concentration in plasma of probiotics supplemented groups both in preventive and treatment model in comparison with negative control group, positive group and antibiotic supplemented group.

 

Conclusion

It is concluded that potentially probiotic L. fermentum IKP 23, L. fermentum IKP 111and L. salivarius IKP 333 may have improve gut morphology and enhance nutrient absorption in broiler gut. It also insinuate for formulation and development of probiotic poultry product containing these strains.

 

Acknowledgments

This project was partially supported by Higher Education Commission/NRPU Project No. 7069/Punjab/NRPU/R&D/HEC/2017.

 

Statement of conflict of interest

We declare that there is no conflict of interest

 

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