Effect of Cellulase Genes Recombinant L. reuteri on Digestibility of Diet Crude Fiber and Adhesion in Gut of Chicken

The present research project was conducted at Northwest A and F University, Shannxi, China and Henan University of Science and Technology, Luoyang, China, after approval from the directorate of advanced studies of the University for Ethical use of birds from 2015-2017.
 
Strains and reagents
 
Recombinant L. reuteri XNY-Cel15 and XNY-Cel73 were preliminarily constructed and used as the strains in this research (Li et al., 2012).
 
Bacterial genomic DNA kits, Taq and endonucleases were purchased from the Promega Corporation (USA); Tryptone and yeast extract were obtained from the OXOID Corporation (UK), other reagents used in this research were purchased from the Sigma Corporation (USA) (Liu and Yu 2012).

Primers for the detection of target gene Cel15 and Cel73 in recombinant Lactobacillus Xny15 and Xny73 were synthesized by the Shanghai Jierui High-Tech. Co. Ltd. (China) and were same as previous work (Li et al., 2012). The amplification program was 35 cycles of 94°C for 45s, 54°C for 30s and 72°C for 90 s with initial 2 min denaturation at 94°C.
 
Animals and experimental diets
 
Seven-day-old leghorn chicks were purchased from a hatchery in Yangling District, Shaanxi Province, China, for the collection of intestinal epithelial cells (Conlon and Kita 2002).
 
Another 100 one-day-old Leghorn chicks (Fahey et al., 2007) procured from the same source and used for determination of adhesion and crude fiber digestibility evaluation. Feed was reserve laying hen feed (for chicks aged less than six weeks) purchased from the Shaanxi Shiyang Group (China). The composition and the nutrition level of the diet are showed in Table 1.
 

 
Adhesion test of recombinant L. reuteri in vitro and in vivo
 
The seven-day-old chicks were aseptically sacrificed and take 10 cm small intestine to isolate small intestinal epithelial cells. The coculture, dyeing and testing of cells and bacteria in vitro was carried out according to previous reports(Lin 2006).
 
The 100 one-day-old leghorn chicks were randomly divided into four groups; each group was replicated five times with 5 birds per replicate and distinguished in different cages. Chicks in control were orally gavaged with 200 μL of MRS broth; Chicks in C15 were orally gavaged with 200 μL of fermentation broth containing recombinant Lactobacillus reuteri XNY-Cel15 (Viable bacteria count is 8.3×10⁹ CFU/mL); Chicks in C73 were orally gavaged with 200 μL of fermentation broth containing recombinant Lactobacillus reuteri XNY-Cel73 (Viable bacteria count is 6.9×10⁹ CFU/mL); And, chicks in C1573 were orally gavaged with 100 μL broth containing XNY-Cel15 and 100 μL broth containing XNY-Cel73. During feeding, the feed and water were provided ad libitum, without immunization. At the end of the experiment (30 days), 40 chicks from each group were aseptically sacrificed to detect the recombined bacteria by PCR amplification of the target genes from microbe in the crop, intestine and cecum. The target gene amplification products were detected by agarose gel electrophoresis.
 
Apparent digestibility assay
 
Ten 30-day-old chicks of similar body weight were selected from each group and housed in 4 wire cages (40×30×38 cm) for crude fiber digestibility trial. The method of animal feeding was carried out according to previous reports (Chen et al., 2007). The samples were collected and determined according to previous reports (Soest et al., 1991). The contents and the apparent digestibility of crude fiber were then calculated (Liu et al., 2016) as follows:
   
Statistical analysis
 
All the data were analyzed via one-way ANOVA using SPSS software version 11.5 and means were deemed significantly different at P < 0.05.

Adhesion of recombinant Lactobacillus to intestinal epithelial cells
 
The recombined Lactobacillus was cultured in MRS broth for 10h (Fig 1A), cocultured with the intestinal epithelial cells which were cultured for 24 h (Fig 1B). After 3 h of co-culture, a large number of Gram-positive bacilli were found surrounding the cells (Fig 1C, Fig 1D), suggesting that recombinant L. reuteri XNY-Cel15 and XNY-Cel73 both can adhere to the surface of intestinal epithelial cells, thus demonstrating the adhesion characteristics of most Lactobacillus.
 

 
Epithelial-specific adhesion is a significant characteristic of Lactobacillus and most Lactobacillus bacteria derived from the animal gut are capable of adhering to the mucosal epithelium (Edelman et al., 2003). In the present study, we found that recombinant Lactobacillus gathered around the intestinal epithelial cells, indicating that recombinant Lactobacillus maintained the characteristics of adhesion to mucosal cells. This is consistent with the previous research results (Yu et al., 2007). In addition, Lam also found that recombinant Lactobacillus was also capable of adhering to ulcer surfaces, suggesting that Lactobacillus adheres to most mucosal epithelia (Lam et al., 2007).
 
Recombinant Lactobacillus colonization of the chicken gut
 
The 30 days old chicks were sacrificed. Lactobacilli were isolated from the contents of the crop, small intestine and cecum of chicks for PCR amplification. The amplification results are shown in Fig 2, a 1.5 kb fragment was detected in the crop, small intestine and cecum of chicks in the C15 group, corresponding to the Cel15 gene fragment; a 740 bp fragment was detected in the crop, small intestine and cecum of chicks in the C73 group, corresponding to the Cel73 gene fragment; the 740 bp fragment corresponding to the Cel73 gene but not the 1.5 kb fragment corresponding to the Cel15 gene was successfully detected in the crop, small intestine and cecum of chicks in C1573 group. No DNA fragments were detected in the control. These results suggest that recombinant Lactobacillus strains carrying the target gene still flourished in the gut of 30 days old chicks.
 

 
Previous studies regarding microbial colonization of the gut have mainly focused on heredity and classification (Tierney et al., 2004). Fluorescent labeling is commonly used to track the distribution and colonization of microorganisms in the gut (Fortineau et al., 2000). In the present study, the distribution and colonization of recombinant Lactobacillus were determined by detecting the reporter genes in different parts of the gut and at different stages. The results demonstrated that recombinant Lactobacillus was capable of colonization in all parts of the chicken gut, with a colonization time of at least 30 days. However, the amount of recombinant Lactobacillus colonization in different parts of the chicken gut and at different stages needs further investigation.
 
Effects of different treatments on feed crude fiber digestibility
 
The air-dried fecal samples collected from each experimental group and feed samples were crushed according to the requirements of analytical experiments. Statistical data of each group and the crude fiber content results of each sample are shown in Table 2. The digestibility value of feed crude fiber of C15 was higher than those of the other three groups; the digestibility values of the C15 and C1573 were significantly higher than that of the control group (P<0.01); the difference in the digestibility value between C73 and control was not significant.
 

 
Previous studies regarding Lactobacillus colonization of the chicken gut were mainly concerned with mucosal immunity against pathogenic microorganisms (Dalloul et al., 2005; Radhika et al., 2020). In the present study, the effect of recombinant Lactobacillus on feed crude fiber digestibility was analyzed. The results were consistent with most studies regarding the addition of exogenous cellulase in animal feed or cellulase expressed by microorganisms (Liu et al., 2016). The feed crude fiber digestibility was improved by recombined Lactobacilli, with significant differences observed between the 30-day-old chicks. Barley- wheat- or rye-based diets high in crude fiber and mucopolysaccharides have been used in most previous studies (Ginindza et al., 2017; Liu et al., 2007), with the results obtained in those studies showing more significant differences. The results on feed crude fiber digestibility showed that the cellulase gene Cel15 was a typical endo-β-1, 4 glucanase, acting on the crystal structure of cellulose and thus improving the digestibility of feed crude fiber. The crude fiber digestibility of the C73 group was significantly low, indicating that the expression products of the Cel73 gene showed poor capability of decomposing natural crude fiber, because the product of this gene is a xylanase which cannot decompose the crude fiber in animal diet (Li et al., 2009).

In a conclusion, the recombinant L. reuteri XNY-CEL15 and XNY-CEL73 were capable of adhering to the chicken intestinal epithelial cells co-cultured in vitro and could adhere in the crop, small intestine and cecum for 30-days. The digestibility of feed crude fiber of chicks were higher than those in the control group; and the C15 group were significantly high (P<0.01).

This study was under the auspices of Special project of the Ministry of science and technology of China (2017 YFE0129900).