Research Background and Objectives

Lactic acid bacteria (LAB), as common microorganisms in foods and the intestine of human and most animals, are widely used as probiotics in humans and animals to restore the ecological balance of different mucosa. Some studies showed high effect of LAB on the economic yield properties (lay intensity, day egg weight and mean egg weight) indexes and resistance to pathogens in laying or meat chickens. The presence of some Lactobacillus in the chicken gastrointestinal tract (GIT) has been described to be of great importance for regulating the composition of the intestinal microflora, developing immunity of the intestine, and promoting the health of chickens. The results of these studies have demonstrated that some LABs of poultry origin were able to inhibit the growth of pathogens and survive through the gastrointestinal tract. So, the objective of the present study is to isolate and characterize new LAB strains with high probiotic potentials from Iranian indigenous chickens for application in the poultry nutrition industry. A megaproject containing 3 projects was designed to isolate, identify and characterize probiotic bacteria from digestive systems of native chickens.

The performed works and results

The objective of the first project was to isolate, identify and characterize lactic acid bacteria with high probiotic potential from native chickens of West Azerbaijan, Esfahan and Mazandarn provinces. About 500 lactobacillus isolates were obtained from ileum by using three screening culture media, including MRS broth (pH 5.5), MRS broth (pH 2.5) and MRS broth (pH 2.5 + 0.3% pepsine). After primary screening of the isolates (Gram staining and Catalase test), strains with high probiotic potential were identified using biochemical evaluations, including low pH and bile salts tolerance. The strains with high resistance to acidic gastric conditions (pH 2.5) and high concentrations of bile salts (0.5%) were selected.  Molecular identification was performed by amplification and sequencing of the 16s rDNA gene (by the PAR/PAF primers). The sequenced fragments were analyzed using bioinformatics software, including Chromas, NCBI Blast, BioEdit and ClustalW, and were subsequently submitted to the GenBank (NCBI database). The selected strains belonged to the species L. crispatus, L. reuteri, L.  salivarus, L.  vaginalis, L.oris, L.  agilis, L.  fermentum. Finally, in order to study microbial flora of the native chickens gut, the metagenomics studies was performed using PCR and DGGE electrophoresis techniques. Theresults showed high diversity of the microflora of digestive systems of the studied native chickens. In the second project, the selected strains were studied for their important probiotic characteristics, including auto-aggregation, temperature and salt tolerance, antibiotic resistance, antimicrobial characteristics and capability of adhesion to epithelial cells (Caco2).  The strains showed auto-aggregation after 10 to 120 minutes, and the strains ES1, OR9, OR10, M4, M9 showed the fastest aggregation (less than 15 minutes).  The majority of the strains were capable to have growth in the culture containing 15% NaCl, and also in the temperatures 4.5 to 45 C°. The results of antibiotic tests showed that the strains had different tolerance level for 16 different antibiotics used in veterinary and medicine. Totally, the majority of the strains was sensitive to ampicillin and linco-spectinomycin, and were semi-tolerant to other antibiotics. The antimicrobial activities of the strains were studied against 7 different pathogens, including Pseudomonas aeroginosa, E.coli, Streptoccus mutans, Clostridium defficile, Enterococcus hirea, Salmonella enteric,  Staphilococcus aureus. The maximum inhibitory effect was observed against E. hirea and S. enteric, whereas the minimum antimicrobial activity was observed for S. mutans and P. aeroginosa. The maximum antimicrobial activity was observed for M18, M16 and M8, whereas the lowest activity was for ES2, ES3, Es4 andEs8. Also, the strains showed adhesion activity to epithelial cells with rate 0 to 40 bacterial cells/cell Caco2. Finally based on the results of evaluations, 10 strains with maximum probiotic characteristics potentials were selected for the next farm experiments.

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The objective of the third project was to evaluate beneficial effects of the selected strains on the broiler chickens yield and health parameters. The field experiments were performed to determine the effects of the selected Lactobacillus and Bacillus subtilis strains on the yield (mortality rate, feed intake, weight, feed conversion ratio (FCR), organ weights and carcass yield percentages), hematological (serum cholesterol and triglyceride), immunological (antibody titers against Salmonella and bronchitis) parameters and also on the expression of Muc2 gene (using Real Time PCR) in the broilers which were challenged or unchallenged with Salmonella.  The experiments were performed in the format of a 5x2 Factorial design. The first factor was 5 levels of probiotic application, including A: control (without probiotic), B: Commercial probiotic, C: 6 Lactobacillus strains, D: 4 Bacillus strains, E: 6 Lactobacillus and 4 Bacillus strains. The second factor was challenging or unchallenging the broilers with salmonella (oral application of 1 m Salmonella enterica subsp. enterica serovar Paratyphi A (CFU: 5x10⁴) at day 32). Application of Lactobacillus strains enhanced reduction of feed intake and a significant increase in weight, carcass rate, and thigh and breast rate in comparison to the control and other probiotics and as result enhanced a significant reduction in FCR. Moreover, application of probiotics (especially Lactobacillus strains) could significantly decrease the quantity of serum cholesterol and triglyceride, and increase antibody titers against salmonella and bronchitis, and also increased MUC2 gene expression in both challenged and unchallenged chickens. Finally, it could be concluded that application of the native Lactobacillus strains could positively affect yield, hematological and immunological parameters of broilers.

Future program

Isolation and chractreization of new effective probiotics for poultry applications will be continued. Also, developing efficient fermentation and formulations processes at pilot and semi-industrial levels will be performed in the private firm.  

Scientific Achievments

• Technology transfer to private sector (Contract No: 3153/251(17/9/94), Technology title: Specific and efficient probiotic strains for poultry applications)
• Aazami N., Gholamreza  Salehi Jouzani, Zohreh Khodaei, Amir Meimandipour4, Mohammad Safari,  Mahdi Goudarzvand, 2014, Characterization of some potentially probiotic lactobacillus strains isolated from Iranian native chickens, The Journal of General and Applied Microbiology (IF: 1), 
• Kafili Tiva, Seied Hadi Razavi, Zara Emam Djomeh, Gholamreza Salehi Jouzani, Pablo Álvarez-Martín, and Baltasar Mayo, 2010, Antibiotic resistance-susceptibility profiles of Lactobacillus strains from Lighvan, a traditional Iranian raw milk cheese, Milchwissenschaft , Milk Science International, , 65 (1):59-64 (Impact Factor: 1).
• Mehdi Ghaderi-Joybari1, Ali Asghar Sadeghi, Gholamreza Salehi-Jouzani, Mohammad Chaman1, Mehdi Aminafshar, 2014, Immune tissue development in pathogen challenged broiler chicks fed diet supplemented with probiotic (Bacillus subtilis),  International Journal of Biosciences, Vol. 5, No. 12, p. 197-203, 2014
•  Salehi Jouzani Gh., M. Ghaderi-Joybari, N. Aazami1, AA. Sadeghi, M. Chamani, M.A. Afshar, 2015, Designing specific and efficient Lactobacillus based probiotics for broiler chickens, VI International Conference on Environmental, Industrial and Applied Microbiology - BioMicroWorld2015 (Barcelona-Spain, 28-30 October 2015),
• Akbari Vala S., Gh. Salehi Jouzani and J. M. Sinaki, 2015, Determination of an efficient and economic medium for growth of some probiotic Lactobacillus strains using response surface methodology, VI International Conference on Environmental, Industrial and Applied Microbiology - BioMicroWorld2015 (Barcelona-Spain, 28-30 October 2015)