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ISOLATION, CHARACTERIZATION AND EVALUATION OF ANTI-SALMONELLA PROBIOTIC POTENTIAL OF INDIGENOUS

LACTOBACILLI IN POULTRY

IMRAN KHAN 2013-VA-02

A THESIS SUBMITTED IN THE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE

OF

DOCTOR OF PHILOSOPHY

IN

MICROBIOLOGY

UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES, LAHORE

2019

To,

The Controller of Examinations,

University of Veterinary and Animal Sciences,

Lahore.

We, the supervisory committee, certify that the contents and form of the thesis, submitted by Imran Khan, Regd. No. 2013-VA-02 have been found satisfactory and recommend that it to be processed for the evaluation by the External Examiner(s) for award of the degree.

Supervisor ________________________________________

Dr. Muhammad Nawaz

Member: __________________________________________

Prof. Dr. Aftab Anjum

Member: __________________________________________

Prof. Dr. Mansur-ud-Din Ahmed

DEDICATION

Dedicated to

i

ACKNOWLEDGEMENTS

First of all, I bow my head to the most gracious and ALMIGHTY ALLAH (AZAWAJAL) who gave me the strength and prospect to complete this work. I humbly pay my respect to HOLY PROPHET MUHAMMAD (PEACE BE UPON HIM) who is ever an ember of guidance and knowledge for humanity and whose life is an ultimate source of guidance for mankind.

I feel great honor to place on the record my sincere thanks to my supervisor Dr. Muhammad Nawaz, Department of Microbiology, University of Veterinary and Animal Sciences, Lahore. He supervised my research light heartedly and proficiently made the dispatch of intimidating work load possible by persistent guidance and scholarly criticism communicated to me during the course of this study and execution of this manuscript. The co-operation extended by the members of my supervisory committee Prof. Dr. Aftab Ahmad Anjum, Department of Microbiology, Prof. Dr. Mansur-ud-Din Ahmad Department of Epidemiology and Public Health, and Prof. Dr. Tahir Yaqub, Chairman, Department of Microbiology, University of Veterinary and Animal Sciences, Lahore is very sincerely appreciated for skillful suggestions during the whole span of this study.

I am also thankful to the all of my Lab fellows and Laboratory staff of Department of Microbiology, University of Veterinary and Animal Sciences Lahore.

Last but not least, I must acknowledge my thanks to my loving parents, sisters and my elder brother (Zakir khan) and whole family for the motivation to take up this program of studies and their hands in prayers for my success throughout the fairly long period of training at this institution.

Finally, as is customary, the errors that remain are mine alone.

Thanks a lot to everyone!

IMRAN KHAN

ii

CONTENTS

DEDICATION .................................................................................................................................. (i)

ACKNOWLEDGEMENTS ............................................................................................................ (ii)

LIST OF TABLES .......................................................................................................................... (iv)

LIST OF FIGURES........................................................................................................................ (vi)

LIST OF ANNEXURES............................................................................................................... (viii)

ABBREVIATIONS......................................................................................................................... (ix)

ABSTRACT...................................................................................................................................... (xi)

SR.NO. CHAPTERS PAGE NO.

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 3

3 MATERIALS AND METHODS 11

4 RESULTS 18

5 DISCUSSION 74

6 SUMMARY 78

779

LITERATURE CITED

iii

LIST OF TABLES

TABLE NO. TITLE PAGE NO.

2.1 Major microbes used as probiotics in poultry 05

2.2 Some commercial probiotic products 06

2.3 Studies reporting anti-Salmonella probiotics 07

3.6.1 Primer used in Genus specific PCR of lactobacilli 13

3.6.2 Recipe of reaction mixture for Lactobacillus genus specific PCR 13

3.7.1 Primer used for amplification of 16S rDNA 14

3.7.2 Recipe of reaction mixture for amplification of 16S rDNA 14

4.1 Isolation of lactobacilli from droppings, cecum and ileum of poultry 19

4.2 Antibacterial activity of selected lactobacilli isolates against Salmonella Enteritidis 25

4.3Growth of selected lactobacilli isolates in MRS broth supplemented with different concentrations of pH represented as mean O.D after 24 hours

28

4.4Growth of selected lactobacilli isolates in MRS broth supplemented with different concentrations of bile salts represented as mean O.D after 24 hours

33

4.5 Percentage auto aggregation of lactobacilli isolates at different time intervals 36

4.6 Percentage co-aggregation of lactobacilli isolates against Salmonella Enteritidis at different time intervals 37

4.7 Antibiotic susceptibility pattern of lactobacilli isolates 38

4.8 Antibiotic resistance profile of lactobacilli 40

4.9 Selection of potentially probiotic isolates 40

4.10 Inhibition of Salmonella Enteritidis by lactobacilli in broth culture at different time intervals 40

4.11 Identification of selected lactobacilli to specie level 43

4.12 Experimental design for in vivo evaluation of probiotics 46

4.13 Effect of selected lactobacilli strains on gut lactobacilli count in broiler 48

iv

4.14 Effect of selected lactobacilli on Salmonella count in broiler gut 50

4.15 Effect of selected lactobacilli on Coliform count in broiler gut 52

4.16 Geometric mean antibody titer in broiler chickens fed with different lactobacilli strains against NDV vaccine 54

4.17 Antibody titer in broiler chickens fed with different lactobacilli strains against AIV H9 vaccine 55

4.18 Effect of selected lactobacilli strains on broiler weight gain 56

4.19 Effects of selected lactobacilli on gut (duodenum) morphology in broiler chickens 63

4.20 Effects of selected lactobacilli on gut (jejunum) morphology in broiler chickens 66

4.21 Effects of selected lactobacilli on gut (ileum) morphology in broiler chickens 68

4.22 D-xylose concentration in plasma of broiler chickens 71

v

LIST OF FIGURES

FIGURE

NO.TITLE PAGE

NO.

4.1 Representative dropping sample (A); Sample transport box (B); Processing of Poultry Intestinal sample (C) 18

4.2Representative anaerobic growth conditions (A); Growth of lactobacilli on MRS agar (B); Purification of lactobacilli on MRS agar (C); Gram staining characteristics of lactobacilli (D)

24

4.3 Zone of inhibition of selected lactobacilli isolates against Salmonella Enteritidis 26

4.4 Activity of CFS (cell free supernatant) at pH 6.5 of selected lactobacilli isolates 26

4.5 Tolerance of selected lactobacilli at pH 7 29




4.9 Tolerance of selected lactobacilli at 0.30% bile salt conc. 34

4.10 Tolerance of selected lactobacilli at 1 % bile salt conc. 35

4.11 Tolerance of selected lactobacilli at 1.8 % bile salt conc. 36

4.12 Lactobacillus genus specific amplification of 16S rDNA-23S rDNA inter-spacer region 41

4.13 Amplification of 16S rDNA by universal Primers 41

4.14 Sequence chromatogram of IKP 333 43

4.15 Genetic relationship of 16SrRNA aligned sequence of Lactobacillus fermentum IKP 23 44

4.16 Genetic relationship of 16SrRNA aligned sequence of Lactobacillus salivarius IKP 333 45

4.17 Birds in different groups in 1st week 47

4.18 Birds in different groups in 4th week 47

vi

4.19 Poultry dropping sample collection 47

4.20 Gut lactobacilli count in different experimental groups at day 35 49

4.21 Salmonella count in different experimental groups at day 35 51

4.22 Coliform count in different experimental groups at day 35 53

4.23.1 Mean weight (g) of chickens at day 01 57






4.24.1 Villus length/crypt depth ratio comparisons in chicken (duodenum) between different experimental groups after use of probiotics 64

4.24.2 Effect of probiotics on gut morphology of broiler challenged with Salmonella Enteritidis 65

4.24.3 Villus length/crypt depth ratio comparisons in chicken (jejunum) between different experimental groups after use of probiotics 67

4.24.4 Villus length/crypt depth ratio comparisons in chicken (ileum) between different experimental groups after use of probiotics 69

4.25 D-xylose absorption in different experimental groups at 30 and 60 min. 70

4.26 D-xylose absorption capacity in broiler chicken between different experimental groups after use of probiotics 72

vii

LIST OF ANNEXURES

ANNEX. NO. TITLE PAGE NO.

1. Poultry dropping Samples collected from different areas of Punjab 89

2. Poultry Cecum Samples collected from different areas of Punjab 91

3. Poultry Ileum Samples collected from different areas of Punjab 93

viii

ABBREVATIONS

AIV Avian influenza virus

ANOVA Analysis of variance

ATCC American type culture collection

BLAST Basic local alignment search tool

BWG BWG

CFU Colony forming Unit

CFS Cell Free Supernatant

DNA Deoxyribonucleic acid

FAO Food and Agriculture Organization

FASTA FAST-ALL

FCR Feed Conversion Ratio

FDA Food and Drug Authority

FI Feed Intake

g Gram

GIT GIT

EFI Expected Feed Intake

GALT Gut Associated Lymphoid Tissue

GMT Geometric Mean Titer

GRAS Generally Recommended as Safe

HA Haemagglutination

HI Haemagglutination Inhibition

ITS Internal transcribed spacer

LAB Lactic acid bacteria

LSM Lab Susceptibility Medium

MIC Minimum Inhibitory Concentration

MRS De Man, Rogosa and Sharpe

ix

NCBI National Centre for Biotechnology Information

NDV New castle Disease Virus

NE Necrotic Enteritis

O.D Optical Density

OIE World Organization for animal

PBS Phosphate buffer saline

PCR Polymerase chain reaction

PM Preventive model

RBC Red blood cell

RNA Ribonucleic acid

SD Standard Deviation

SPSS Statistical package for Social Sciences

TM Treatment model

µm micrometer

UVAS University of Veterinary and Animal Sciences

VRB Violet red bile

XLD Xylose Lysine Deoxycholate

ZOI Zone of inhibition

x

ABSTRACT Lactobacilli (n=84) were isolated from the droppings, ileum and caecum of back-yard poultry birds on De Man Rogosa and Sharpe medium. Lactobacilli isolates were screened and selected on the basis of their antimicrobial activity (6.33±0.57-20.33±1.15 mm) against Salmonella Enteritidis by well diffusion assay. In vitro characterization it was revealed that IKP23, IKP 111 and IKP 333 had pH tolerance, survival and growth in bile salts, no acquired antibiotic resistance, good auto-aggregation and co-aggregation capacity. Therefore, these three isolates were selected as potential probiotics. IKP23, IKP 111 and IKP 333 were identified as L. fermentum, L. fermentum and L. salivarius, respectively on the basis of homology of their 16S rRNA sequences. Selected isolates (IKP23, IKP 111 and IKP 333) were evaluated in vivo. For in vivo characterization, Day old broiler chicks (n=150) were randomly divided ten different groups. Group one was negative control group. Group 2 was positive control which received only the challenge bacteria (Salmonella Enteritidis) ATCC 13076 at day 07. Groups (3, 4, 5) received probiotics at day 01 to 35 and challenge bacteria at day 07 in preventive model (PM). Groups (6, 7, 8) started receiving probiotic at day 07 to day 35 and challenge bacteria at day 07 in treatment model (TM). Group 09 started receiving commercial probiotic Protexin (1g/liter) at day 01 to 35 and challenge bacteria at day 07. Group 10 started receiving antibiotic at day 01 to 05 and challenge bacteria at day 07. Birds were challenged with a single dose of ~106 CFU of Salmonella Enteritidis by oral gavage, while probiotics were administered with ~108CFU/ml daily. Weight of birds was recorded on weakly basis. Enumeration of microbes (lactobacilli, total coliforms and Salmonella) and antibodies against NDV and AIV H9 was done at different days. D-xylose absorption capacity and gut morphometric parameters (villus height, crypt depth and villus height to crypt depth ratio) were studied at day 35. Results revealed that Salmonella count (log10 CFU) was significantly increased (P˂ 0.05) in positive control group (4.88±0.29) as compared negative control (3.66±0.23). Salmonella counts were significantly lower in groups administered with IKP 23, IKP 111 and IKP 333 before Salmonella challenge (2.92±0.04, 3.05±0.10, 2.99±0.25) or after Salmonella challenge (3.37±0.12, 3.49±0.50, 3.55±0.45, respectively ) as compared to positive control group (4.88±0.29). Weight of broiler at day 35 was significantly higher (P˂ 0.05) in groups administrated with IKP 23, IKP 111 and IKP 333 prior to Salmonella challenge (1640±48.1, 1608±59.7 and 1590±49.0 gm respectively) and post Salmonella challenge (1569±45.1, 1515±47.8, and 1530±51.7 gm respectively) as compared to negative control (1466±49.6 gm), positive control group (1320±44.8 gm). Immunomodulatry effects of probiotics were higher in preventive model as compared to treatment model. Broilers 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 have probiotic potential for poultry and these strains may prevent or competitively exclude Salmonella from poultry gut. These strains may provide additional benefit of better weight gain, improvement in gut morphometric parameters and absorption capacity in broiler challenged with Salmonella Enteritidis which insinuate for their possible role in efficient broiler production.

Keywords: Lactobacilli, Poultry, Salmonella Enteritidis, in vitro, in vivo, bile salts, treatment model, preventive model,

xi

CHAPTER 1INTRODUCTION

Poultry is as the most vital sector of agriculture in Pakistan and it contributes 1.3% of total GDP of Pakistan. In Pakistan, commercial poultry production was established in 1960s and now providing a significant protein portion in diet on daily basis (Hussain et al. 2015). Although, poultry sector is growing fast, it still faces many challenges such as infections due to Escherichia coli, Salmonella spp., Clostridium perfringens, Clostridium botulinum and viral diseases as well. Apart from bacterial and viral diseases, poultry industry also faces problems of mycotoxicosis resulting in decreased feed conversion ratio and increased disease burden. Salmonella is one of the major pathogens of poultry which not only affect poultry and its production; it also enters into food chain and cause problems in human. Salmonella cause economic loss of billions every year (Wales and Davies 2011). Salmonella cause severe problems of poultry sector and is one of the biggest obstacles in growth of poultry sector in Pakistan. Salmonella is a motile, Gram negative bacillus from Enterobacteriaceae. It is facultative anaerobe having more than 2600 serotypes.

Food safety is much more concerned about human diet which is associated with poultry related products. In many recent reports it is mentioned that Salmonella is frequent cause in human food borne bacterial diseases in poultry (Resanovic et al. 2008). Salmonella Enteritidis is one of the major issue in commercial egg production and poultry production (Foley et al. 2011). To solve the Salmonella problem, prophylactic use and mis-use of antibiotics is very common in poultry industry. Large scale use of antibiotics in poultry industry results in increased antibiotic resistance and antibiotic residues transfer in human through food chain. The purpose of antibiotics uses to promote the growth rate and prevent the enteric pathogens in bird is the reason to development of antibiotic resistance and drug residues in human. Probiotics are the alternative source of antibiotics use and to decrease antibiotics and antibiotics residues, scientists now a day’s prefer to improve the health (Kogut and Swaggerty 2012). To control and prevention of Salmonella by prophylactic use of antibiotics have only aggravated the other problems such as increased antibiotic resistance and antibiotic residues in animal and human food chain. So, to control Salmonella, prophylactic use of antibiotics, solve one problem and cause many others which adversely affect human beings in turn as well (Mathur and Singh 2005).

Probiotic is an alternative approach to control Salmonella issue in poultry. In the recent studies, probiotics are constantly used because of their inhibitory effects on pathogenic bacteria. Probiotics definition according to FAO is ‘‘live microorganisms which give benefit to host after administration of adequate amount’’. The word “pro” meaning “for life” has been derived from Greek (Gibson and Fuller 2000). Probiotic were also defined as “ live microorganisms which promotes the intestinal balance (Fuller 1989). One health benefit of probiotics is their capability to prevent the growth of pathogenic bacteria like Salmonella spp. and Escherichia coli in broiler or poultry gut (Jin et al. 1996). Mechanism of action of probiotics, although is not fully elucidated, is dependent on properties of the probiotic strains. Probiotics control enteric pathogens by reducing the gut pH in microenvironment and secrete antimicrobial substances like bacteriocins (Abbas et al. 2010) and strengthen the normal flora (Wang and Gu 2010). Probiotics are also involved in boosting mucosal and overall immune response (Brisbin et al. 2008; Tsai et al. 2012). Probiotics act as immune boosters and increased feed conversion ratio of birds by producing digestive enzymes. Probiotics are commonly used in supplementation of human and animals because of their beneficial use (Shokryazdan et al. 2017). The growth factor is an important part of poultry production and main reason to replace antibiotics with probiotics use as a growth promoter in poultry for researchers. Several countries have banned the use of antibiotic as growth promotor in broiler production. The major probiotics have beneficial effects on poultry performance and gut health. Growth performance is a straight indicator in poultry because it is involved in feed conversion and has general impact in poultry production (Ajuwon 2015). Probiotics enhanced the immunity of broiler when administered through orally. Efficiency of probiotics when compared with control group (without probiotic) then it was settled that antibody forming cells, contents of immunoglobulin and thymocytes were increased in probiotic group. The length of cecal tonsils and density of microvilli were also improved. Probiotics enhanced the intestinal mucosal immunity at early age of bird (Yang et al. 2007; Shao et al. 2010). Probiotics also have antihypertensive, hypo-cholesterolemic and immuno-modulatory effects. Probiotics increased the growth performance of chickens, egg quality, egg production and feed intake in broiler (Getachew 2016). Normally Probiotics are non-infectious pathogens supplemented to both human and poultry, but it may be infectious in

1

Introductionimmuno-compromised patients (Peret Filho et al. 1998). The main function of the GIT is to absorbed and digest the feed and to maintain the gut microbiota. The GIT of broiler is the main barrier to protect the body from harmful pathogens (Peret Filho et al. 1998).

Competitive exclusion is a common mechanism in which probiotics protect the colonization of pathogenic bacteria like Salmonella in gut. Probiotics exclude pathogen replication site in gut. Competitive exclusion by probiotics include competition through pathogenic bacteria for attachment sites, production of antagonistic substances and boosting of immunity (Callaway et al. 2008).

Use of lactic acid bacteria (LAB) as probiotic is very common (Wali and Beal 2011). In 1965, many scientist and researcher suggested LAB named as probiotic or bio therapeutic agent. LAB also have antibiotic characteristics. The lactobacilli strains are very effective in modulating the immune system in broiler and lying hens. Immunoprobiotic lactobacilli are used in enhancing the both cellular and humoral immune response in broiler (Koenen et al. 2004). Antibacterial actions of local lactic acid producing bacteria and commercial probiotics were compared against Salmonella spp., Staphylococcus aureus and Escherichia coli. It was concluded that local LAB had significant higher antibacterial activity as compared to commercial probiotics (Darabi et al. 2014).

Probiotics are very efficient in the development of immune system of poultry. Probiotics have good interaction with gut associated lymphoid tissue (GALT) which activate B-1 cells of GALT and activation of these cells enhance the antibodies production (Haghighi et al. 2006). Probiotics uses in poultry are also associated with increased intestinal morphological parameters i.e. height of villi and ratio of villi to crypt depth. These two parameters are highly linked with growth performance of broiler. Probiotics use as growth promotor in broiler diet have good effect on gut (Awad et al. 2009). Nowadays researchers are focused to see the probiotics effect on broiler health, performance and total bacteria count (Fuller 1989; Patterson and Burkholder 2003). Probiotics play an enormous role in nutrition and gut function of poultry hence considered as good alternative to antibiotics in poultry (Mountzouris et al. 2007; Applegate et al. 2010). D-xylose absorption test is very effective tool to assess the intestinal capability of broiler (Rutgers 2005). This test was used to assess the mal-absorption disorder caused by bacteria and viruses in birds (Doerfler et al. 2000). Birds with different intestinal health show different ability to absorb D-xylose (Shomali et al. 2012).

The rapid emergence of antibiotic resistant pathogens and antibiotics residues as environmental contaminants is a concern worldwide. There is a need to develop new and effective substitutes to antibiotics which are also efficient in production of poultry (Suresh et al. 2018). The administration of probiotics may improve health status of host, help in fighting against diseases, improve growth rate and gut function (Merrifield et al. 2010). There is dire need of system-based approach for well understanding of physiological mechanisms of action of probiotics. This may require the use of advance molecular technique and computational techniques. These techniques can be used for better predictability of probiotics effect in poultry industry (Ajuwon 2015).

2

CHAPTER 2REVIEW OF LITERATURE

2.1. Poultry Industry of Pakistan and its challengesPoultry sector is one of the most vital part and major contributor of livestock sector in Pakistan. It

provides jobs (directly /indirectly) to more than 1.5 million people. Eggs, poultry meat and other products are cheaper protein source for consumption. Poultry sector is vital in keeping the prices of mutton and beef in check. Investment in poultry sector is estimated as more than Rs.700 billion in 2018. During 2017-2018, poultry contributed 32.7% (1.39 million tons) in total meat production in Pakistan. During 2017-2018, growth of commercial layer, breeders, broiler and rural poultry was 7.0%, 5.0% , 10% and 1.5% respectively (Anonymous 2018). Although, poultry sector is showing good growth, it still encounters many challenges i.e. disease outbreaks, sensational variations in prices, feed containing high level of mycotoxins, lack of specific pathogen free chicken, poor implementation of biosecurity plans and sensational propaganda by media. All these challenges threaten the profitability and still hinder the growth of poultry sector. Poultry sector in Pakistan still has potential for huge growth (Hussain et al. 2015). Among all these challenges, infectious diseases are the biggest threat to poultry industry. Some of the bacterial diseases of poultry include spirochetosis, avian tuberculosis, ulcerative enteritis, erysipelas, fowl typhoid and salmonellosis (Porter 1998). These bacterial diseases are controlled by either using vaccines or by antibiotics. There also are many bacterial agents i.e. Salmonella Enteritidis which inhabit poultry gut and their transmission to human food chain is a threat to public safety (Eng et al. 2015).

2.2. Salmonella Salmonellae are gram negative bacilli. It is motile, facultative anaerobic member of Enterobacteriaceae. One the basis of 16S rRNA sequences, genus Salmonella has been divided into two species including S. enteric and S. bongori. The S. enterica is classified into S. enteric subsp. enterica; S. enteric subsp. salamae, S.enterica subsp. arizonae, S.enterica subsp. diarizonae, S.enterica subsp. houtenae, and S. enterica subsp. indica on the basis of their biochemical characteritics and genomic relatedness. On the basis of three antigens i.e. somatic (O), capsular (K) and flagellar (H) each sub-speice is further divided into different serotypes by Kauffmann (Brenner et al. 2000). There have been more 2600 serotypes of Salmonella identified. Most of the serotypes belong to S. enterica subsp. enterica (Brenner et al. 2000). Some of the important serovars of Salmonella include S. Typhi, S. Paratyphi, S. Pullorum, S. Gallinarum, S. Typhimurium, and Salmonella Enteritidis. S. Typhi, S. Paratyphi are host specific serovars and cause typhoid fever in human. S. Pullorum, S. Gallinarum are also host specific serovars and cause white diarrhea and fowl typhoid respectively in chicken. S. Typhimurium and Salmonella Enteritidis are non-host specific inhabitants of poultry gut. Many of the serotypes of S. enterica are classified as typhoid serovars and non-typhoid serovars (Eng et al. 2015). Non typhoid serovars (S. Typhimurium and Salmonella Enteritidis ) are ubiquitous in nature and present in soil, feces, water and GIT of different animals including chicken (Murray 1991). Salmonellosis caused by Salmonellae enterica non-typhoid serovars is one of the leading causes of food poisoning and food poisoning associated mortality in human throughout the world. Although a large number of S. enterica non-typhoid serovars are associated with food borne infections, Salmonella Enteritidis is the dominant serovar isolated from food borne associated infections from human (Andino and Hanning 2015). 2.2.1. Salmonella enterica subsp. enterica serovar Enteritidis

Salmonella Enteritidis cause infection of reproductive tract in chicken and can be transmitted vertically to chicks through egg (Suzuki 1994). It can colonize the egg before the addition of shell to egg (Gantois et al. 2009). Bird may secrete it with showing symptoms as well. Once few birds in a flock are positive for Salmonella Enteritidis whole flock is expected to contract it. Broilers infected with Salmonella Enteritidis generally remain asymptomatic because of its physiology and possibly higher body temperature (Troxell et al. 2015). Its transmission from poultry through egg, meat and chicken associated products to human is a safety concern (Girmay et al. 2015). It causes salmonellosis in human which is a GIT infection. It generally colonizes and reproduces in mucosa of GIT and then spread to spleen, liver, and various other organs and tissues (Eng et al. 2015). It appears 6-48 hours after the ingestion of contaminated food. It is characterized by nausea, vomiting, abdominal cramps, diarrhea, abdominal pain and fever. Infants, elderly and immuno-compromised people are more susceptible to the symptoms. Infectious doze also vary

3

Review of Literaturedepending on physiology and immune status of the infected person. Generally a large number of cells (10 5-106) are required to cause the disease but it can be as low as a single cell in immune-compromised individuals. Therefore, Salmonella is not generally enumerated from ready to eat foods rather it is detected after enrichment. It should be absent from ready to eat foods. Salmonella Enteritidis can survive on surfaces (egg, fruits, vegetable), dried foods (spices etc.) and sewage for relatively longer time. Another prominent characteristics of Salmonella Enteritidis is its higher D value (D60 = 2-6 min). Following a heat shock its resistance to temperature further enhances. Freezing generally causes disruption of Salmonella cells but it can survive in frozen meat. All these characteristics make it harder to control Salmonella from food and increase the chances of contracting salmonellosis from contaminated foods (Kang et al. 2006; Sakha and Fujikawa 2012; Andino and Hanning 2015).

2.2.2. Strategies for control of Salmonella Enteritidis in ChickenSalmonella Enteritidis has frequently been detected, isolated and characterized from different sources

including poultry, poultry egg, poultry meat, spices, milk and dairy products, sewage and environment from throughout the world (Barnhart et al. 1991; Poppe et al. 1991; Waltman et al. 1992; Schutze et al. 1996; Schaar et al. 1997; Suresh et al. 2006; Rincon-Reyes and Figueroa 2008; Arnold et al. 2010; Betancor et al. 2010; Kozoderovic et al. 2012; Thung et al. 2016; Campioni et al. 2017) as well as Pakistan (Shaheen et al. 2004; Sajid and Schwarz 2009; Hassan Ali et al. 2010; Kitadai et al. 2010; Asif et al. 2017; Wajid et al. 2019). Salmonella have been isolated from all animals and can be the reason of mortality and morbidity in cattle, sheep, swine and poultry (Callaway et al. 2008). Therefore, with an expansion of poultry industry an increase in food borne infections has also elevated dramatically in developing countries. Thus, bacterial control is one of vital management strategies for the success in production of poultry and human health. Control of Salmonella is important not only for poultry and human health in a country, Salmonella free food production is also required for compliance with export quality food. Salmonella is controlled in food production systems by use of appropriate integrated management systems, strict compliance to current good manufacturing practices and by implementing hazard analysis critical control points. Since poultry is the biggest reservoir of Salmonella Enteritidis, it is imperative to control Salmonella from its source. Antibiotic are generally used to treat Salmonella and other bacterial infections in poultry. Antibiotics are also used as prophylactic measure to control the bacterial pathogens and to enhance the overall growth of the birds. Overuse and misuse of antibiotics as growth promoters has led to the emergence of antibiotic resistant Salmonella, which when transmitted to human is recalcitrant to common antibiotic treatments. There are many recent reports throughout the world including Pakistan on the prevalence of multiple drug resistant or extremely drug resistant Salmonella in poultry or human food chain (Cizek and Kovarik 1994; Tassios et al. 1997; Rankin and Coyne 1998; Rouahi et al. 2000; Busani et al. 2004; Mathur and Singh 2005; Velge et al. 2005; Suresh et al. 2006; Mirmomeni et al. 2007; Betancor et al. 2010; Kozoderovic et al. 2012; Thung et al. 2016; Campioni et al. 2017; Zhou et al. 2019). A report from Hyderabad, Pakistan revealed 38% prevalence of Salmonella in meat samples (Soomro et al. 2011). In another report from Faisalabad, Pakistan, 75.24% prevalence of Salmonella was recorded. All Salmonella Enteritidis isolates were resistant to bacitracin, erythromycin and novobiocin. These result showed that poultry meat and poultry products are the foundation of multidrug resistant Salmonella Enteritidis (Shahzad et al. 2012). A research was conducted in Shaanxi province of China in 2007-2008 to check the Salmonella prevalence. Fifty four percent (276) samples of chicken were positive of Salmonella and Salmonella Enteritidis was found in 31.5% samples. In total, 80% of isolates were resistant to as a minimum of one antimicrobial and 53% of the isolates were resistant to in excess of three antimicrobials. Resistance was most likely to tetracycline, sulfamethoxazole, and trimethoprim/sulfamethoxazole. Most of the isolates were also resistant to quinolones and flouroquinolones (Yang et al. 2010). In a comprehensive study from china, it was revealed that Salmonella of poultry origin had high level of antibiotic resistance with more than 60% Salmonellae carrying multiple drug resistance (Cui et al. 2016). Use of antibiotics also results in presence of antibiotic residues in meat and egg which may have deleterious effects (Mathur and Singh 2005; Jing et al. 2011; Moscoso et al. 2015). Problem of antibiotic resistance and strict regulations on use of the antibiotic as growth promoters have compelled scientists to explore alternatives of antibiotic to control and treat bacterial infections including Salmonella in Poultry. Alternatives of antibiotics such as nutraceuticals, bacteriophages, nanoparticles antimicrobial peptides and probiotics have been a focus of study recently (Cho et al. 2005; Chambers and Gong 2011; Diaz-Sanchez et al. 2015; Gadde et al. 2017; Islam and Yang 2017; Abudabos et al. 2019). Probiotics is an

4

Review of Literatureimportant alternative of antibiotics as probiotics may also provide many other health benefits along with control of bacterial pathogens including Salmonella. Salmonella control is an important prerequisite for production of safe food. Control of Salmonella is difficult because of multiple sources of Salmonella in food and absence of clinical symptoms in broiler. Intestinal microbiota and its modulation have considerable success in controlling the Salmonella in poultry gut. Probiotics, prebiotics, postbiotics and synbiotics are generally used to modulate and strengthen the intestinal microbiota in chicken. Furthermore, increased consumer demand of organic poultry insinuate for exploration of natural Salmonella control measures (Chambers and Gong 2011).

2. 3. Probiotics Probiotic are live microorganisms which provide health benefits to host upon adequate administration (FAO-WHO 2001). There are many different microbes i.e. lactobacilli, peidiococci, enterococci, streptococci, bacilli, lactococci, Escherichia, saccharomyces yeast etc. which are used as probiotics. However, most of the characterized and commercial probiotics belong to genera Lactobacillus and Bifidobacteria. Table 2.1 summarizes some of the probiotics.

Table 2.1 Major microbes used as probiotics in Poultry

Probiotic generaRepresentative species/strain

1. LactobacillusLactobacillus acidophilus; Lactobacillus casei; Lactobacillus rhamnosus; Lactobacillus salivarius; Lactobacillus fermentum; Lactobacillus plantarum

2. Bifidobacterium Bifidobacterium lactis; Bifidobacterium longum; Bifidobacterium bifidus

3. Peidiococcus Peidiococcus pentosaceua

4. Saccharomyces Saccharomyces cerevisiaeSaccharomyces boulardii

5. Bacillus

Bacillus subtilisBacillus clausiiBacillus pumilus

6. Escherichia E. coli strain Nissle 1917

There are certain pre-requisites, a potential strain should fulfill before being claimed as probiotics. These prerequisites are given below

i) Candidate strain should be of host origin. A probiotic strain developed for poultry should come from poultry gut or its environment. A strain isolated from human should not be used as probiotics in poultry and vice versa. If a strain isolated from human is used in poultry, it may adopt poorly, may not survive and may also interfere with the normal microbiota of the host (Girmay et al. 2015).

ii) Probiotics should be identified to at least its specie level. Identification to specie and strain level is an important criterion for probiotic strains to fulfill as probiotic effects are specie and strain specific. For identification to specie level, a polyphasic taxonomic approach is applied which include the culture characters, microscopic characters of the strain, biochemical characters and genetic characters. Amplification of 16S rRNA is generally employed for the identification of potential probiotic strains to specie level, if 16S rRNA sequencing end up with poor resolution, sequencing of 23S rRNA or 16S rRNA-23S rRNA interspace region is employed for better resolution of the candidate strain to specie level. In case of yeast candidates 18S rRNA or ITS region proves good for specie identification. Restriction fragment length polymorphism, pulse field gel electrophoresis or other appropriate techniques are also employed for strain differentiation and characterization. Now a day, with the availability of commercial and cheaper

5

Review of Literaturesequencing technologies, whole genome sequencing of the candidate strains is also beneficial (Girmay et al. 2015).

iii) Potential probiotic candidate should survive/tolerate or grown in the physicochemical barriers of the host. Physicochemical barriers of poultry include the digestive enzymes, low pH and bile salts. Probiotics should tolerate and survive in low pH (3-4) for at least 90 min so that it can further travel to chicken intestine to exert its beneficial effect. Resistance to digestive enzymes and low pH can be determined by exposing candidate strains to simulated conditions followed by determination of percent survival. Intestine of the chicken contain bile salts which can inhibit the growth of microbes, so it is important to determine in vitro that potential candidate not only tolerate the bile salts (at least 0.3%) but also grow in its presence (Suzuki 1994).

iv) Potential probiotic candidate should be safe for the host. Potential probiotic should have established non-pathogenicity for the host. Lactobacilli have acquired “Generally Recognized As Safe (GRAS) status so there is no need to establish their non-pathogenicity. If a Bacillus spp. or other genera is used as probiotic, appropriate procedures must be applied to determine the deleterious effect and possible pathogenesis of the candidate strain on host. Another safety concern is the presence of acquired antibiotic resistance in probiotics. Since acquired antibiotic resistance can be transferred horizontally to other bacteria, probiotic should not have it. Presence of acquired antibiotic resistance in probiotics or any other strain intentionally added to human food chain or administered in poultry is strictly prohibited as it will act as antibiotic resistance reservoir. It is important that antibiogram of the candidate strain be determined before being declared as probiotics. Antibiotic susceptibility pattern should be determined by standard protocols provided by Clinical Laboratory Standards Institute (CLSI) wherever possible. If standards protocols and microbiological breakpoints are not available in CLSI then guidelines provided by other international regulatory bodies’ i.e. European Food Safety Authority, WHO, FAO or OIE may be used (Suzuki 1994).

v) Potential probiotic candidate should have ability to adhere and colonize the intestinal epithelial cells. Colonization of potential probiotics to intestinal mucosa indicates their retention and survival in host gut. Since probiotics exert their effect on the host by virtue of their presence and growth, a strain with ability to colonize and attach with intestinal epithelial cells indicate that the strain can retain itself in host gut for longer time and exert better beneficial effect (Girmay et al. 2015).

vi) A probiotic after fulfilling all above pre-requisites should also exert at least one health benefit on the host. Some of benefits of probiotics in chicken include enhanced gut function, better feed conversion ratio, binding of mycotoxins, mitigation of bacterial pathogens, enhancement of immune system, and enhanced digestion and availability of nutrients (Campioni et al. 2017). Table 2.2 presents some of the commercial poultry probiotic products.

Table 2.2 Some commercial poultry probiotic products

Sr. No

Commercial Probiotic product

Company Probiotic Strains

1. PoultryStar® Biomin, USA E. faecium, P. acidilactici, B. animalis, L. salivarius, L. reuteri

2. Lavipan JHJ, Poland

Lactococcus lactis B/00039; Carnobacterium divergens KKP2012pLactobacillus casei B/0008;Lactobacillus plantarum B/00081 Saccharomyces cerevisiae KKP 2059p

3. Protexin Soluble Protexin Lactobacillus plantarum; Lactobacillus bulgaricus,

Lactobacillus acidophils, Lactobacillus rhamnosus4. Max-Grow Max Biotech Lactobacillus acidophilus, Lactobacillus rhamnosus5. CBT XL Vee Excel Drugs

and Lactobacillus acidophilus

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Review of LiteraturePharmaceutical

6. Max Econo Vital Provital Lactobacillus acidophilus

7. SiloSolve F.C Chr. Hansen Lactobacillus buchnaeri, Lactobacillus lactis

8. Bovamine Daily Chr. Hansen Lactobacillus animalus

9. Product 11C33 Pioneer Seeds Lactobacillus buchnaeri, Lactobacillus plantarum

10. BioStabil Biomin, USA Lactobacillus plantarum

2.3.1. Anti-Salmonella ProbioticKeeping in mind the threat to public safety, Salmonellae should be controlled from entering into food

chain and best way to do that is its control at the farm level (Vandeplas et al. 2010; Desin et al. 2013). Besides the antibiotic treatment, alternative approach for controlling Salmonella in poultry is it competitive exclusion by probiotics (Chambers and Gong 2011). Salmonella is inhibited by probiotic by the increase in the founding of normal intestinal microbiota in newly born chickens (Mead 2000). The oral dosage of lactobacilli or other probiotics that stop the growth of Salmonella would decrease the burden of Salmonella in the intestine and ultimately reduced its infection (ovarian or other). Probiotics i.e. lactobacilli can colonization of reproductive tracts of birds and stop Salmonella infection (Van Coillie et al. 2007). Salmonella spp. cause major problems in poultry and human worldwide. They are responsible for the GIT infection in human. The use of Probiotics often improves the intestinal activity against Salmonella spp. (Casey et al. 2007). Lactic acid producing bacteria have a better activity against the Salmonella spp. These bacteria produce lactic acid and to reduce the pH in the intestine (Hotel and Cordoba 2001). There are different criteria set for the microorganism to be run as effective Probiotics. Actions include the attachment to the host cells in the GIT, also some other properties may include like exclusion of pathogenic microbes from the GIT, ability to persist in the GIT pH and to stimulate the immunity of the host against pathogen (Fernández et al. 2003). There are several mechanisms against Salmonella but some major mechanism includes production of hydrogen peroxide (H2O2). They destroy the cell wall of bacteria because free radicals produce. Also, these bacteria produce bacteriocins. Bacteriocins are the chemicals which destroy the Salmonella spp. in the intestine of the poultry (Bakken 2014). Some Probiotics produce lactic acid due to which it reduces the pH of the intestine so most of the bacteria cannot multiply at such a low pH (Khodadad et al. 2013). There is a lot of literature which report anti-Salmonella activity of probiotics in laboratory experiments, in eggs, in cell culture studies as well as in vivo studies (Lehto and Salminen 1997; Audisio et al. 1999; Van Coillie et al. 2007; Ruby and Ingham 2009; Chen et al. 2012; Lim and Ahn 2012; Tellez et al. 2012; Thirabunyanon and Thongwittaya 2012; Yamazaki et al. 2012; Menconi et al. 2013; Yamawaki et al. 2013; Penha Filho et al. 2015; Feng et al. 2016; Ferrari Ida et al. 2016; Carter et al. 2017; Ceugniez et al. 2017; Prado-Rebolledo et al. 2017; Adetoye et al. 2018; Okamoto et al. 2018; Burkholder et al. 2019; Mohanty et al. 2019; Smialek et al. 2019). All such studies have been summarized in table 2.3.

Table 2.3 Studies reporting anti-Salmonella probiotics

Sr. No

Probiotic product/Probiotic strains Study

Inhibition activity against

SalmonellaReference

1. Lactobacillus plantarum DM 69

In vitro and in Caco-2 cells

S. enterica ATCC 35640 (Mohanty et al. 2019)

2.Lavipan (JHJ, Poland)Lactobacillus spp.Saccharomyces cervisae

Poultry Salmonella Enteritidis (Smialek et al. 2019)

3.Lactobacillus acidophilusLactobacillus rhamnosusLactobacillus casei

Intestinal epithelium Salmonella enterica Javiana

(Burkholder et al. 2019)

4. Lactobacillus spp In vitro and in Poultry S. Heidelberg (Okamoto et al.

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Review of Literature2018)

5.

Lactobacillus amylovorus C94;Lactobacillus salivarius C86

In vitro co-culture Salmonella Enteritidis (Adetoye et al. 2018)

6.

Lactobacillus salivarius 59;Enterococcus faecium PXN-33

In vitro and in Poultry Salmonella Enteritidis (Carter et al. 2017)

7.Kluyveromyces marxianus; Kluyveromyces lactis

In vitro and in Poultry S. typhimurium (Ceugniez et al. 2017)

8.FloraMax-B11(R)(probiotic containing multiple strains)

In Poultry Salmonella Enteritidis

(Prado-Rebolledo et al. 2017)

9. Lactobacillus spp. In vitro and in cheese S. typhimurium (Ferrari Ida et al. 2016)

10.Lactobacillus plantarum; Lactobacillus salivarius;Pediococcus acidilactici

In vitro and in Poultry

Salmonella Enteritidis ATCC 13076S. Typhimurium ATCC 14082

(Feng et al. 2016)

11.

Lactobacillus acidophilusLactobacillus fermentumLactobacillus reuteri Lactobacillus salivarius

Poultry Salmonella Enteritidis

(Penha Filho et al. 2015)

12. Lactobacillus spp EggsSalmonella Enteritidis(no inhibition)

(Yamawaki et al. 2013)

13. Bacillus spp Broiler Salmonella Enteritidis (Menconi et al. 2013)

14. Lactobacillus plantarum GK81 In vitro S. Typhimurium (Lim and Ahn 2012)

15. Lactobacillus spp In vitroSalmonella EnteritidisS. typhimurium

(Yamazaki et al. 2012)

16. Bacillus subtilis NC11 Intestinal Epithelial cells

Salmonella Enteritidis

(Thirabunyanon and Thongwittaya 2012)

17. Lactobacillus casei CRL 431 BalbC mice S. typhimurium

18.

Lactobacillus acidophilus (LASW)Lactobacillus fermentum (LF33)Lactobacillus plantarum (LPL05)Enterococcus faecium (TM39)

Chicken epithelial cells

S. Typhimurium ST70 (Chen et al. 2012)

19. Bacillus sppLactobacillus spp In vitro and in vivo Salmonella

Enteritidis (Tellez et al. 2012)

20. Lactobacillus reuteri; Lactobacillus johnsonii In vitro and in Poultry Salmonella

Enteritidis(Van Coillie et al. 2007b)

21. Enterococcus faecium J96 In vitro and in Poultry S. Gallinarum (Audisio et al. 1999)

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Review of LiteratureS. PullorumSalmonella EnteritidisS. typhimurium

22. Lactobacillus sakei 10-EGR-a In vitro S. newport

S. typhimurium)(Ruby and Ingham 2009)

23. Lactobacillus rhamnosus GG Caco-2

S. typhimurium(Inhibtion of adhesion of Salmonella)

(Lehto and Salminen 1997)

2.3.2. Lactobacilli-as a probiotic Lactobacilli are gram positive rods which are non-spore former and present in the GIT, mouth and in

female genitourinary tracts of human. They are also present in the intestine of poultry as normal flora (Cannon et al. 2005). Lactobacilli are facultative anaerobes, microaerophiles, aerotolerant or obligate anaerobes (Summanen et al. 1993). Lactobacilli belong to LAB group because of their ability to form lactic acid from reduction of pyruvic acid at the end of glycolytic pathways. Terminal electron accepter in energy generation pathway of lactobacilli is the pyruvic acid. Lactobacilli are non-spore forming, non-motile, catalase negative rods. GC content in genome of lactobacilli is generally below 50% (Ennahar et al. 2003). There is a huge diversity in Lactobacillus genus. More than 140 species of lactobacilli have been identified and many more are expected to be explored. There also are many uncultivable lactobacilli (Satokari et al. 2003). Lactobacilli are categorized as homo-fermentative or hetro-fermentative. Homo-fermentative lactobacilli produce lactic acid as a major end product while hetro-fermentative produce lactic acid, ethanol and acetic acid as well as end products of energy production pathways. Lactobacilli require high carbohydrate and amino acids for growth making them dominant microbiota of GIT, spoiled foods, fermented foods and surface of fruits and vegetables (Ennahar et al. 2003). Lactobacilli are dominant microbiota of GIT of human, animal and birds where they play a key role on maintain the proper health and physiology of host as a component of so called organ of host-microbiome (Dec et al. 2014). Other than GIT, lactobacilli are also frequently isolated from vagina of human and cloaca of laying hens where they play enormous role in antagonizing the other pathogens and maintenance of overall health of host (Miyamoto et al. 2000).

2.4. Status of probiotics development in PakistanAlthough multiple probiotic products are available in Pakistan for poultry industry, there still is

insufficiency of local probiotic development for both human and poultry. There are only limited reports on development of probiotics for human and poultry. Nawaz et al. (2011) isolated and characterized probiotic strains from stool of breast fed healthy babies and further evaluated their anti-allergic potential in allergic airway inflammation model of BalbC mice. L. fermentum NWS29 was concluded as a potential anti-allergic probiotics (Nawaz et al. 2015). In another study, Asghar et al. (2016) developed and evaluate probiotic L. casei and L. crispatus for poultry with ability to increase body weight of broilers. Arif et al. (2018) showed that lactobacilli isolated from local poultry have phosphorous solubilization activity in vitro and may be used as probiotic candidates for phytase solubilization in poultry gut. Anti-campylobacter probiotics have also been evaluated in vitro as well as in vivo (Khan et al. 2019). Azeem et al. (2019) determined anti-aflatoxigenic effect and aflatoxin binding capacity of selected probiotic strains. There also are few other studies on strains isolated from Pakistan having probiotic properties (Anwar et al. 2012; Mahmood et al. 2013; Anwar and Rahman 2016; Patrone et al. 2016; Amer et al. 2018; Manzoor and Tayyeb 2019).

2.5. Regulation of probioticsDifferent countries have different set of regulations for the use of probiotics in poultry production.

European Food Safety Authority has more strict rules for the registration of probiotics as feed additive and it takes three to four years. In United States, the registration process is less complicated as compare to Europe. If the bacterial species is already in feed ingredient list then registration is not compulsory on the federal

9

Review of Literaturelevel but product label is must be registered. Different regulatory bodies across the globe have different regulations for probiotics depending upon on their intended use (Arora et al. 2013).

2.6. Statement of ProblemAlthough many probiotics have been developed in different parts of world, there are only few reports

from Pakistan (Asghar et al. 2016; Arif et al. 2018; Azeem et al. 2019). Furthermore there are no anti-Salmonella targeted probiotics developed indigenously. Keeping in mind insufficiency of local anti-Salmonella probiotics and importance of Salmonella to public safety, present study is designed with following objectives

To isolate, and screen lactobacilli of poultry origin for anti-salmonella activity. 16 S rRNA gene sequencing and phylogenetic analysis of selected lactobacilli. In vitro evaluation of probiotic potential of selected lactobacilli. In vivo competitive exclusion of Salmonella in broiler by selected lactobacilli.

10

CHAPTER 3MATERIALS AND METHODS

3.1. Sampling MethodA total of 150 samples were collected from indigenous chicken for the isolation of lactobacilli.

Samples included caeca (n=50), ileum (n=50) and droppings. Samples were collected from different areas in Punjab as given in Annexture 01-03. All samples were collected in clean plastic bags/containers. Samples were transported in a transport container having temperature of at 4°C to the Department of Microbiology, University of Veterinary and Animal Sciences (UVAS), Lahore. Samples were processed immediately on arrival to Laboratory or stored at -20°C until processing for isolation of lactobacilli.

3.2. Isolation of lactobacilli (Preliminary identification)All samples were subjected to 10 fold serial dilution on in normal saline/phosphate buffered saline.

All dilution (100 µl) were plated on De Man, Rogosa and Sharpe media (MRS Agar Merck, Germany) supplemented with nystatin (100 µg /100 ml) for the isolation of lactobacilli. Nystatin was added to inhibit the fungal growth on MRS media. All media plates were incubated at 37°C for 24-48 hours in anaerobic/microaerophilic conditions. Anaerobic or microaerophilic conditions were developed by lighting a candle in glass desiccator or by using anaerogen sachet 3.5 L (Oxoide, UK) followed by sealing with paraffin or wax. Following incubation, MRS plates were observed for growth and plates showing appropriately separated colonies were selected. Morphology of distinguished colonies was noted and distinguished colonies were designated as ‘isolate’. All isolated marked from primary culture plates were further streaked on MRS media for purification. After purification, isolates were stored in MRS broth (Merck, Germany) supplemented with 15% glycerol. All isolates were subjected to Gram’s stain procedure and catalase test for preliminary identification.

3.2.1. Preparation of Normal salineWith the help of weighing balance (OHAUS, USA), 8.5 gm of NaCl (Merck, Germany) was weighed

and added into 800 ml distilled water in a beaker. Mixing of NaCl was achieved by a stirrer until complete dissolution. After that final volume was made upto 1000mL. Normal saline solution was sterilized by autoclaving (121°C, 15 psi for 15 minutes).

3.2.2. Gram staining ProcedureFor Gram staining, clean glass slides were de-waxed and an appropriate smear was prepared. To

make smear, a drop of normal saline was taken on slide. Pure culture of isolates from MRS agar was picked with help of sterilized loop and mixed with normal saline to prepare a uniform smear/film of bacteria. For staining, crystal violet was applied for 60 seconds. After that slides were rinsed with tap water. In next step, Gram’s iodine was applied for 60 seconds followed by rinsing it tap water. In next step, Ethyl alcohol (95%) was used as decolorizer for 30 seconds. In last step, counter stain safranin was applied on slide for 01 minute followed by washing with tap water again. After that, slides were dried in air. Slides were later observed under light microscope (CX 23 Olympus, Japan) at 100 X lens and microscopic characters were noted. Characters noted were shape of bacterial cell, Gram’s reaction and arrangement of cells. Lactobacilli were supposed to be Gram positive rods.

3.2.3. Catalase test ProcedureLactobacilli are catalase negative bacteria. To determine catalase reaction, few drops of hydrogen

peroxide (3%) were taken on a glass slide. Fresh growth of isolates was picked from MRS media with the help of sterilized match stick. Growth was mixed with hydrogen peroxide previously placed on slide. Gas bubbles were observed over as positive reaction.

3.3. Screening of lactobacilli for anti-Salmonella activityLactobacilli isolates were screened for their activity against Salmonella of poultry origin using well

diffusion assay as described elsewhere well (Bao et al. 2010). Salmonella Enteritidis of poultry origin and ATCC (13076) culture of Salmonella Enteritidis was procured from the Department of Microbiology (UVAS), Lahore. Cell free supernatants (CFS) were obtained from overnight cultures of lactobacilli by centrifugation at 10000 rpm for 5 minutes. Supernatant collected in a syringe and sterilized by filtration. For

11

Materials and Methodsfilter sterilization a filter of appropriate pore size (0.45 µm) was used. Salmonella Enteritidis (0.5 McFarland) was inoculated on Muller Hinton agar (Oxoid, UK) plates by a swab to obtain confluent growth. Wells were made in Muller Hinton Agar plates and sealed with molten agar. CFS (80 -100 µl) of each isolate were added in respective wells. Plates were incubated aerobically for 24 hours at 37°C. After incubation plates were read for the growth of Salmonella and inhibition of growth of Salmonella around the well. Diameter of zone of inhibition (mm) was recorded as well.

3.4. In vitro characterization of lactobacilli for their probiotic properties

3.4.1. Resistance to low pH To be probiotic, lactobacilli must have ability to tolerate low pH encountered in poultry gut.

Tolerance to low pH was analyzed through method as described by determined as described by Delgado et al. (2007). Isolates were grown in MRS broth tubes for 24-48 hrs. Broth was centrifuged and growth was collected as pellet. Growth was washed twice with PBS to remove traces of media. After that, growth was re-suspended in PBS and its optical density (O.D) was adjusted to equivalent of 1 Macfarland (~3×108

CFU/ml). Re-suspended growth was added in PBS tubes having different pH (pH 2, 3, 4,7) at the rate of 100 µL/mL and incubated at 37°C for 90 min. After 90 minutes, 100 µl from each tube was inoculated in MRS broth tubes (10 ml) for 24 hours at 37°C. After incubation, growth was assessed by taking O.D at 600 nm in a spectrophotometer (UV-150-02 Shimadzu Corporation, Japan).

3.4.2. Preparation of PBSAll salts [NaCl (8 g/l), KCL (0.2 g/l), Na2HPO4 (1.44 g/l), KH2PO4 (0.24 g/l)] were mixed in 800

ml distilled water and pH was adjusted as 7.4. Final value was made to 1000 ml.

3.4.3. Resistance to bile saltsProbiotic should tolerate and grow in presence bile salts. Tolerance and growth of lactobacilli in bile

salts was determined following the method developed by Bao et al. (2010). Isolates were grown in MRS broth for 24 hours. MRS broth tubes having different concentrations (0.3%, 1% and 1.8%) of bile salts (Oxoid, UK) were prepared. MRS broth tubes (10 ml) containing bile salts were inoculated with 100 µl of fresh growth of each isolate in MRS broth. Tubes were incubated for 24 hours. Growth was analyzed by taking O.D at 600 nm through spectrophotometer (UV-150-02 Shimadzu Corporation, Japan).

3.5. Antibiotic resistance profileProbiotic should not have transferable antibiotic resistance. Antibiogram of selected lactobacilli was

determined by disc diffusion method as described by Velasco et al. (2005). Lactobacilli were grown on MRS media plates. Loopful of growth was re-suspended in PBS and O.D was adjusted to equivalent of 1 McFarland (~3 x 108 CFU) to prepare the inoculum. Inoculum was swabbed on MRS media plates and antibiotic disc of different antibiotic i.e penicillin G, erythromycin, ampicillin, polymyxin B, bacitracin, tetracycline, fusidic acid, chloramphenicol, kanamycin, ciprofloxacin, imipenem and vancomycin were placed on MRS plates. MRS plates were incubated at 37°C for 24 hours anaerobically. Following incubation, diameter of zone of inhibition (mm) surrounding the antibiotic disc was measured. Isolates were designated as sensitive, resistant or intermediate according to breakpoints adopted from Clinical and Laboratory Standards Institute (CLSI) or European Food Safety Authority

3.6. Auto-aggregation of selected lactobacilli isolates and co-aggregationAuto-aggregation of lactobacilli was determined following the previously established protocol by

Reniero et al. (1992). Freshly grown cultures were centrifuged, pellets were washed twice in PBS, re-suspended in PBS and O.D of suspension was measured at 600nm. Lactobacilli suspensions were incubated at 37°C for measuring O.D at 600 nm by spectrophotometer (UV-150-02 Shimadzu Corporation, Japan) after 1 hour and 2 hours. Auto-aggregation was expressed as % auto-aggregation. Auto-aggregation was considered positive when suspended cells were settled down in bottom of tube and left a clear supernatant (Heravi et al. 2011).Auto-aggregation % = (OD1 -OD2) /(OD1) ×100)OD1=O.D of (strain1) lactobacilli at 1 hour

12

Materials and MethodsOD2=O.D of (strain1) lactobacilli at 2 hours

3.7. Co-aggregation of selected lactobacilli isolates with Salmonella Enteritidis Co-aggregation of lactobacilli with Salmonella Enteritidis was also determined as described by Jin et

al. (1996). Overnight cultures of lactobacilli and Salmonella Enteritidis were centrifuged at 10000×g for 15 minute and washes twice in PBS. Cells were re-suspended in PBS and an equal quantity (2 mL) of each lactobacilli isolate was mixed gently with Salmonella Enteritidis suspension. After 2 hours of incubation at 37°C, O.D of each mixture was determined at 600 nm by spectrophotometer. Co-aggregation was expressed as % co-aggregation. Co-aggregation indicates the ability of lactobacilli to possibly inhibit indicator organism. Co-aggregation results from formation of the aggregates of cells settled in tubes leaving a supernatant fluid on top within 2 hour having reduced O.D (Bujñáková et al. 2004).Co-aggregation % = 100× (OD1 +OD2) - 2(OD3)/(OD1+OD2)OD1=O.D of (strain1) lactobacilliOD2=O.D of (strain 2) Salmonella EnteritidisOD3=O.D of strain 1and strain 2 after 2 hours

3.8. Inhibition of Salmonella in broth cultureEqual amounts of freshly grown Salmonella (100 µl) and selected lactobacilli (100 µl) were mixed in

10 ml nutrient broth (Himedia, India). Tubes were incubated at 37°C. The Salmonella were enumerated from the incubated tubes at different time intervals to determine the effect of lactobacilli on growth of Salmonella as described previously elsewhere (Todoriki et al. 2001).

3.9. Molecular level Identification of selected isolates For molecular identification of selected isolates to genus level Lactobacillus genera specific PCR

was used. For specie identification amplification and sequencing of 16S RNA gene was employed.

3.9.1. DNA extraction DNAs of lactobacilli were extracted by a commercial Kit (Gene All, Korea) following the

manufacturer’s instructions. Loopful of freshly grown isolates on MRS agar was transferred to lysis buffer containing 20 mg lysozyme and lysis was done for 30 min. After that, 20 µl (20mg/ml) of Proteinase K was added. The 200 µl of a buffer from the kit (BL buffer) was added into the tube. Mixing was done by gentle vortex and incubated at 56°C for ten minutes. After incubation, 200 µl of absolute ethanol was added in tubes. Tubed were vortexed followed by transfer of whole mixture in SV column. SV columns containg the previous mixture were subjected to centrifuge at 6000 × g for one minute. Following that SV columns were put in new collection tubes and 600 µl of BW buffer of the kit was added in SV columns and columns were again subjected to centrifugation at 6000 × g for one minute, after centrifugation collection tube were again replaced. After that, 700 µl of TW buffer was added followed by centrifuged at 6000 ×g for one minute and replacement of collection tube. After that, SV columns in collection tubes were again subjected to high speed centrifugation (13000 ×g for one minute) to remove the remaining buffer residues. At last, SV columns were placed in 1.5 ml DNAse/RNAse free sterile microcentrifuge tube for collection of DNA. DNA was eluted from columns by addition of 200 µl of AE buffer followed by one minute incubation at room temperature and centrifugation at high speed (13000 ×g for one minute). At this final step, filtrate was collected and SV columns were discarded. Filtrate contained extracted DNA. DNA was run on agarose gel electrophoresis for confirmation of its extraction.

3.9.2. Genus specific PCR of lactobacilli Genus specific PCR using forward primer and reverse primers was used for the confirmation of

lactobacilli using XB5 and LbLMA-1 primers as given in table 3.6.2 (Nawaz et al. 2011). A total of 25 µl reaction mixture contained following: 15 pmoles of each primer and 2µl (~20 ng) DNA. Complete composition of reaction mixture is given in table 3.6.2. Reaction mixture was placed for amplification of target DNA in thermocycler. DNA amplification was carried out in Thermocycler (BIO-RAD, USA) by utilizing following program. Initial denaturation at 95 oC for five minutes; followed by thirty five cycles of denaturation at 95 oC for sixty seconds, annealing at 55 oC for sixty seconds and extension at 72 oC for sixty seconds; and final extension at 72 oC for seven minutes followed by storage of the amplicons at 4 oC.

13

Materials and MethodsLactobacillus spp. specific amplification was confirmed by subjecting the amplicons (∼250-bp) to agarose gel electrophoresis. Agarose gel 2 % (W/V) was prepared in TAE buffer with 0.5 µg per ml of ethidium bromide. In each well 2 µl amplicon (~250 bp) was loaded and molecular weight of amplicons was assessed by running 100 bp DNA markers. Amplicons of specific bands were observed using gel documentation system (UVITEC, UK).

Table 3.6.1: Primers used in Genus specific PCR of lactobacilliPrimers Sequence (5’------------3’) Amplicon sizeXB5 (forward) GCCTTGTACACACCGCCCGT ∼250-bpLbLMA1(reverse) CTCAAAACTAAACAAAGT

Table 3.6.2: Recipe of reaction mixture for Lactobacillus genus specific PCR

Ingredients Quantity (µl)2X Master Mix 12.510 µM Forward primers [XB5] 1.510 µM Reverse primers [LbLMA1] 1.5Water 7.5Template DNA 2Total Mixture 25

3.9.3. Amplification of 16S rRNA of lactobacilliThe 16 rDNA amplification and sequencing is one of the gold standards in identification of bacteria

to their specie level. The 16 S rDNA of selected lactobacilli was amplified using universal primers (8FLP and XB4 as given in table 3.7.1) as described previously (Nawaz et al. 2011a). A total of 25 µl reaction mixture contained following: 15 pmoles of each primer and 2µl (~20 ng) DNA. Complete composition of reaction mixture is given in table 3.7.2. Reaction mixtures were placed for amplification of target DNA in thermocycler. DNA amplification was carried out in Thermocycler (BIO-RAD, USA) by utilizing following program. Initial denaturation at 95 oC for five minutes; followed by thirty five cycles of denaturation at 95 oC for sixty seconds, annealing at 55 oC for sixty seconds and extension at 72 oC for sixty seconds; and final extension at 72 oC for seven minutes followed by storage of the amplicons at 4 oC. The 16S rDNA specific amplification was confirmed by subjecting the amplicons (∼1400 bp) to agarose gel electrophoresis. Agarose gel 1.5% (W/V) was prepared in TAE buffer having 0.5 µg per ml of ethidium bromide. In each well 2 µl amplicon was loaded and band size was assessed by running 100 bp DNA markers. Amplicons of specific bands were observed using gel documentation system (UVITEC, UK).

Table 3.7.1: Primer used for amplification of 16S rDNA Universal Primers Sequence (5’------------3’) Amplicon size8FLP (forward) AGTTTGATCCTGGCTCAG ∼1400-bp XB4 (reverse) AGTTTGATCCTGGCTCAG

Table 3.7.2: Recipe of reaction mixture for amplification of 16S rDNA

Ingredients Quantity (µl)2X Master Mix 12.510 µM Forward primer 1.510 µM Reverse primer 1.5Water 7.5Template DNA 2Total Reaction Mixture 25

3.10. Sequencing and Phylogenetic analysisPCR products of genus specific amplification (~250 bp) and 16S rRNA amplification (~1400 bp)

were sent for sequencing to a commercial company 1st BASE, Korea. Sequences were viewed by Chromas

14

Materials and Methods2.0, analyzed by BioEdit software and FASTA files were prepared. Retrieved sequences were subjected to BLAST analysis for specie identification. Sequences were submitted to a public databank (NCBI GenBank) and their GenBank accession numbers were obtained. Relatedness of the isolates of current study with other lactobacilli was revealed through constructing a phylogenetic treewith mega 7 software.

3.11. In vivo evaluation of selected lactobacilli Probiotics Lactobacilli fulfilling all prerequisites to be a probiotic in vitro were subjected to In vivo evaluation in broiler. In in vivo evaluation following parameters were studied: (i) effect of lactobacilli on mitigation of Salmonella in poultry gut, (ii) weight gain in broiler, (iii) intestinal morphometric parameters, (iv) D-xylose absorption from gut and (v) effect on antibody response against NDV and AIV H9 vaccine.

For in vivo evaluation, a total of 150 day old broiler birds were obtained from market and housed in experimental shed for 35 day. Broilers were divided intro ten different groups. Each group had 15 birds. Randomized complete block design was used. At a proper age, fully susceptible broilers were divided into ten (10) different groups. Negative control group was not supplemented with probiotics. Positive control groups received only the challenge bacteria (Salmonella Enteritidis) ATCC 13076 at day 07. Groups (3, 4, 5) received probiotics at day 01 to 35 and challenge bacteria at day 07 in prevention model (PM). Groups (6, 7, 8) started receiving probiotic at day 07 to day 35 and challenge bacteria at day 07 in treatment model (TM). Group 09 started receiving commercial probiotic Protexin (1g/liter) at day 01 to 35 and challenge bacteria at day 07. Group 10 started receiving antibiotic at day 01 to 05 and challenge bacteria at day 07. Birds were challenged with a single dose of ~106 CFU of Salmonella Enteritidis by oral gavage (Wolfenden et al. 2007), while probiotics were administered with ~108CFU/ml daily (Kizerwetter-Swida and Binek 2009). Droppings of birds in each group were collected daily and evaluated for the bacterial shedding of probiotics and challenged bacteria by enumeration of bacteria on selective culture media.

3.12. Dropping or intestinal content samples collection Fresh dropping samples were collected in sterilized cotton swab from on different days.

3.13. Enumeration of bacteria in poultry dropping samplePoultry droppings/intestinal contents were collected from different groups at different days for the

enumeration of salmonellae, total coliforms and lactobacilli. Conventional microbiological techniques were used for the enumeration of bacteria. Samples were homogenized and serially decimal dilution (10 1 to 107) was prepared in PBS. Each dilution was spread on plate with L shape glass spreader on selective media for lactobacilli, Coliform and Salmonella. For the enumeration of lactobacilli, Coliform and Salmonella, MRS agar (Merck, Germany), VRB (Violet Red Bile) agar (Merck, Germany) and XLD (Xylose Lysine Deoxycholate) agar (Himedia, India), respectively were used. Results were expressed as Mean±S.D log 10

CFU/g of dropping/intestinal contents.

3.14. Body weight gain (BWG)

Weight of birds was determined weekly (Day 1, 7,14,21,28 and 35).

3.15. Immuno-modulatory effects on NDV (Newcastle disease virus) Vaccine and (AIV) avian influenza virus H9

Blood was collected from at least 5 birds from each group at different days (14, 21, 28 and 35 days) to determine serum antibody titer against NDV and AIV H9. Serum was separated from blood samples and stored at the -20 °C. Antibody titer against NDV and AIV H9 was determined using haem-agglutination inhibition assay (HI) as described previously by Allan et al. (1978). Commercially available live vaccine of NDV Lasota (Solvay, USA) and AIV H9 killed vaccine GPVAC flu-9 (Grand pharma, Pakistan) were used as an antigen source for the test. HI titer of serum sample was presented as reciprocal of serum dilution. Geometric mean titer (GMT) of different groups was calculated using the ‘tube number and table/modified log 2’ method.

15

Materials and Methods3.15.1. Haem-agglutination Inhibition (HI) assay Procedure

The Blood (3ml) was collected from wing vein of chicken and centrifuged the blood at 3000 rpm for 3 minutes. Discarded the supernatant, added 5 ml of normal saline and re-suspended. Repeated this step until supernatant was clear solution. Finally, added 2ml of this solution to 98 ml of normal saline to prepared 2% of erythrocyte solution. For haem-agglutination (HA) Test with the help of multichannel pipette 50 ul of normal saline was added from the 1 to 12 wells of immunoassay plates. Virus (50 µl) was added in first well, with the help of pipette the content in the first well was mixed well, transferred to 2 nd well and process was continued through well no.11. Washed RBC (50ul) was mixed in 1st well and transferred to each well through pipette. The plate was incubated at room temperature for 25 minutes and results were recorded. For HI test 4 HA diluted virus was used. With the help of multichannel pipette 50 ul of normal saline was added from the 1 to 12 wells of immunoassay plates. Serum sample 50 ul of chicken was placed in the first well and mixed through 10th well with pipette. The virus antigen (50ul of 4HA) was added from 1 to 11 well. The plate was incubated at room temperature for 30 minutes. Chicken RBC (50 µl of 2% suspension) was added from 1 to 12 well. The plate was moved backward and forward from side to make uniform suspension of the erythrocyte in the mixture. The plate was kept at room temperature until a clear pattern of HA or HI (button formation) was seen. The highest dilution of serum sample causing inhibition of HA was the end point.

Calculation of GMT titer (5 birds/group) Log was taken of each reading Summation of log Then took mean log Finally took the antilog of mean log GMT = (anti Σ log /n)

3.16. Intestinal morphometric parameters analysis At the age of 35 days, 05 birds from each group were randomly selected and slaughter. The intestine of broiler chicken was separated into three parts, duodenum, jejunum and ileum. Two-centimeter portion was cut at the midpoint with the help of scissor from each part of small intestine, flushed with saline and open longitudinally. The intestine segments were fixed in 10% neutral buffered formaldehyde, then tissues were processed embedded in paraffin, sections were cut and stained with hematoxylin and eosin (Awad et al. 2009). Slides were examined with a light microscope (Olympus CX31, USA) installed with digital imaging system and analyzed by using LABOMED pixel pro software. The morphometric parameters were villus height, crypt depth and villus height/crypt depth ratio were measured as described by De Los Santos et al. (2007).

3.17. D- xylose absorption capacity in broiler chicken Food and water were withdrawn from each group of the bird 12 hour before the first collection of

blood. Five birds from each group were randomly selected for sampling. The birds were weighed individually. D-xylose solution was prepared by dissolving 50 mg D-xylose powder into 1ml distilled water and solution was given via oral gavage at a concentration of 500mg D-xylose/kg body weight to each bird. The blood samples were drawn from ulnar vein in wings at 0 minutes, 30 minutes and 60 minutes of post inoculation of D-xylose using sterile syringes. The blood was collected in commercially available heparinized micro hematocrit capillary tube. The tubes were centrifuged at 6000 rpm for 15 minutes for plasma separation. To each 20ul plasma sample, 2ml phloroglucinol reagent was added and heated for 4 minutes at 100 ◦C. The samples were cooled to room temperature and absorbance of each sample was taken at 554 nm using spectrophotometer (Regassa et al. 2016). The phloroglucinol color reagent was prepared by adding 0.5g of phloroglucinol in 100 ml of glacial acetic acid and 10ml of conc. HCl in amber colored bottle. D-xylose standard solution was prepared by dissolving D-xylose in deionized water to make 0, 10, 20, 30, 40, 50, 60, 70, 80 and 90mg/2ml concentrations. Color reagents were also added in these standard concentrations and were processed similarly. The 20ul of each standard solution was added to appendroff tubes and 2ml phloroglucinol color reagent was added. The solution was heated for 4 minutes at 100 ◦C and after cooling absorbance was taken at 554nm.The standard curve was prepared using O.D values of samples

16

Materials and Methodsand D-xylose standards. The O.D values were used to determine D-xylose concentration in blood by comparing with standard absorbance value (Mansoori et al. 2007).

3.19. Statistical AnalysisO.D data was expressed as Mean±S.D and means in different groups were compared by one-way

ANOVA followed by Tukey’s multiple comparison test. Enumeration data was expressed as Mean±S.D log10 CFU per gram or ml and compared by one-way ANOVA followed by Tukey’s method at significance level P value <0.05 by using SPSS.

17

CHAPTER 4RESULTS

The aim of the research was to collect the indigenous poultry samples from all Punjab (Pakistan) cities to get the lactobacilli potential of probiotics. Total of 150 samples of indigenous poultry including contents from poultry droppings (n=50), caeca (n=50) and ileum (n=50) of backyard poultry (n=50) were collected. Samples were collected and transported in sample box with controlled temperature at 4°C to Department of Microbiology, UVAS, Lahore, Pakistan. Samples were stored at -20°C until for further analysis. In primary identification lactobacilli were isolated by platting on MRS agar followed by Gram staining and catalase test. According to Berges Manual of determinative bacteriology all lactobacilli are Gram +Ve rods and catalase -Ve. In further identification DNA of lactobacilli was extracted, confirmation was done by using genus specific PCR and species specific PCR using universal primers. In vitro characterization, lactobacilli were screened for their ability to survive in low pH, different bile salt concentrations, auto aggregation activity, co aggregation activity and inhibition of Salmonella Enteritidis in broth culture. In vivo characterization, lactobacilli were screened for reduction of Salmonella count in broiler, increased weight gain in broiler, better intestinal morphological parameters (villi height and Villus/crypt depth ratio) and improved D -xylose absorption capacity of broiler.

4.1. Isolation and preliminary identification of lactobacilliTotal of 150 samples of indigenous poultry including contents from poultry droppings (n=50), caeca

(n=50) and ileum (n=50) of backyard poultry (n=50) were collected. Representative dropping sample, sample transport box and processing of intestinal samples is shown in figure 4.1. Figure 4.2 shows Representative anaerobic growth conditions (A); growth on lactobacilli MRS agar (B); Purification of lactobacilli on MRS agar (C); Gram staining characteristics of lactobacilli (D). From 150 samples of poultry origin, a total of 403 isolates were recovered on the basis of distinguished colony morphology on MRS plate. Table 4.1 represents isolates recovered from each sample. Out of 403 isolates, 84 were identified as lactobacilli on the basis of colony morphology, Gram staining and catalase test.

18

Results

Figure 4.1: Representative dropping sample (A); Sample transport box (B); Processing of Poultry Intestinal sample (C)

19

ResultsTable 4.1: Isolation of lactobacilli from dropping, cecum and ileum of poultry

Sr. No Samples Isolates recovered

1. PDSO1 IKP01, IKP02, IKP03, IKP04, KP05, IKP06, IKP07, IKP08, IKP09

2. PDSO2 IKP10, IKP11, IKP12, IKP13, IKP14, IKP15

3. PDSO3 IKP16, IKP17, IKP18, IKP19, IKP20, IKP21

4. PDSO4 IKP22, IKP23

5. PDSO5 IKP24, IKP25, IKP26, IKP27

6. PDSO6 IKP28

7. PDSO7 IKP29, IKP30, IKP31

8. PDSO8 IKP32, IKP33, IKP34

9. PDSO9 IKP35, IKP36, IKP37, IKP38, IKP39, IKP40, IKP41

10. PDS10 IKP42, IKP43, IKP44

11. PDS11 IKP45, IKP46, IKP47, IKP48


13. PDS13 IKP52, IKP5, IKP54, IKP55, IKP56, IKP57

14. PDS14 IKP58


16. PDS16 IKP62, IKP63, IKP64, IKP65, IKP66

17. PDS17 IKP67

18. PDS18 IKP68, IKP69

19. PDS19 IKP70



22. PDS22 IKP078

23. PDS23 IKP079, IKP80

24. PDS24 IKP81, IKP82, IKP83, IKP84, IKP85, IKP86, IKP87


26. PDS26 IKP92

27. PDS27 IKP93, IKP94, IKP95, IKP96, IKP97


29. PDS29 IKP102, IKP103, IKP104, IKP105,

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Results

30. PDS30 IKP106, IKP107

31. PDS31 IKP108, IKP09

32. PDS32 IKP110, IKP111, IKP112

33. PDS33 IKP113, IKP114, IKP115,

34. PDS34 IKP116, IKP117

35. PDS35 IKP0118

36. PDS36 IKP119, IKP120, IKP121,

37. PDS37 IKP122, IKP123, IKP124,

38. PDS38 IKP125, IKP126, IKP127

39. PDS39 IKP128, IKP129,

40. PDS40 IKP130, IKP131, IKP132

41. PDS41 IKP133, IKP134

42. PDS42 IKP0135,

43. PDS43 IKP136, IKP137, IKP138, IKP139, IKP140, IKP141, IKP142

44. PDS44 IKP143, IKP144, IKP145, IKP146, IKP147, IKP148, IKP149, IKP 150, IKP151

45. PDS45 IKP152, IKP153, IKP154, IKP155, IKP156,

46. PDS46 IKP157, IKP158

47. PDS47 IKP159, IKP160, IKP161

48. PDS48 IKP162

49. PDS49 IKP163, IKP164

50. PDS50 IKP165, IKP166, IKP167,

51. PCSO1 IKP168, IKP169, IKP170, IKP171

52. PCSO2 IKP172, IKP173,

53. PCSO3 IKP174, IKP175, IKP176

54. PCSO4 IKP177






60. PCS10 IKP195

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Results

61. PCS11 IKP196, IKP197, IKP198, IKP199, IKP200, IKP201

62. PCS12 IKP202, IKP203, IKP204

63. PCS13 IKP205, IKP206, IKP207

64. PCS14 IKP208

65. PCS15 IKP209, IKP210

66. PCS16 IKP211,

67. PCS17 IKP212, IKP213, IKP214

68. PCS18 IKP215, IKP216, IKP217

69. PCS19 IKP218

70. PCS20 IKP219, IKP220

71. PCS21 IKP22, IKP222, IKP223

72. PCS22 IKP224, IKP225, IKP226, IKP227

73. PCS23 -

74. PCS24 IKP228, IKP229, IKP230

75. PCS25 IKP231, IKP232, IKP233


77. PCS27 IKP228

78. PCS28 IKP229, IKP0231

79. PCS29 IKP232,

80. PCS30 IKP233, IKP234, IKP235

81. PCS31 IKP236, IKP237

82. PCS32 IKP238

83. PCS33 -

84. PCS34 IKP239, IKP240, IKP241, IKP242, IKP243

85. PCS35 IKP244

86. PCS36 IKP245, IKP246, IKP247


88. PCS38 IKP252, IKP253, IKP254, IKP255, IKP256

89. PCS39 -

90. PCS40 IKP257

91. PCS41 IKP258, IKP0259

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Results

92. PCS42 IKP260, IKP261, IKP262


94. PCS44 IKP267, IKP268

95. PCS45 IKP269

96. PCS46 IKP270, IKP271, IKP272


98. PCS48 IKP277, IKP278,279.280, IKP281

99. PCS49 -

100. PCS50 IKP282, IKP283

101. PISO1 IKP284

102. PISO2 IKP285, IKP286, IKP287, IKP288, IKP289, IKP290

103. PISO3 IKP291, IKP292

104. PISO4 IKP293, IKP294, IKP295

105. PISO5 -

106. PISO6 -

107. PISO7 IKP296, IKP297.IKP298, IKP299

108. PISO8 IKP300, IKP301

109. PISO9 IKP302, IKP303, IKP304, IKP305

110. PIS10 IKP306, IKP307

111. PIS11 IKP308

112. PIS12 IKP309, IKP310, IKP311

113. PIS13 -

114. PIS14 IKP312, IKP313, IKP314

115. PIS15 IKP315

116. PIS16 IKP316, IKP317

117. PIS17 IKP318, IKP319, IKP320, IKP321

118. PIS18 IKP322, IKP323, IKP324, IKP325, IKP326, IKP327

119. PIS19 IKP328

120. PIS20 IKP329, IKP330

121. PIS21 IKP331, IKP332, IKP333, IKP334, IKP335, IKP336, IKP337


23

Results

123. PIS23 IKP342, IKP343, IKP343

124. PIS24 IKP344

125. PIS25 IKP345, IKP346, IKP347

126. PIS26 IKP348, IKP349

127. PIS27 -

128. PIS28 IKP350, IKP351, IKP352

129. PIS29 IKP353, IKP354,

130. PIS30 IKP354, IKP355, IKP356

131. PIS30 IKP357, IKP358

132. PIS32 IKP359, IKP360

133. PIS33 IKP361

134. PIS34 IKP362, IKP363,

135. PIS35 IKP364, IKP365

136. PIS36 IKP366, IKP367, IKP368, IKP369, IKP370

137. PIS37 IKP371, IKP372, IKP373

138. PIS38 -

139. PIS39 IKP374, IKP375

140. PIS40 IKP376, IKP377, IKP378, IKP379, IKP380

141. PIS41 IKP381


143. PIS43 IKP386

144. PIS44 IKP387, IKP388, IKP389

145. PIS45 IKP390, IKP391

146. PIS46 -

147. PIS47 IKP392

148. PIS48 IKP393, IKP394, IKP396, IKP,397, IKP398,

149. PIS49 IKP399, IKP400, IKP401

150. PIS50 IKP402, IKP403

24

Results

Figure 4.2: Representative anaerobic growth conditions (A); Growth of lactobacilli MRS agar (B); Purification of lactobacilli on MRS agar (C); Gram staining characteristics of lactobacilli (D)

25

Results4.2. In vitro characterization of lactobacilli for their probiotic properties

4.2.1. Anti-Salmonella activity of selected isolatesOnly 15 lactobacilli isolates showed antimicrobial activity against Salmonella Enteritidis out of 84

isolates and these isolates were selected for further analysis. In statistical analysis as indicated in table 4.2, IKP 111, IKP 192, IKP 333 and IKP 402 showed highest antimicrobial activity (20.33±0.57, 20.33±0.57, 19.66±1.15, 20.33±0.57) respectively against Salmonella Enteritidis and significantly different (P˂ 0.05) from other lactobacilli isolates. Lowest anti-Salmonella activity was shown by IKP07 (6.33±0.57). Figure 4.3 and 4.4 indicate representative anti-Salmonella activity of CFSs of lactobacilli on nutrient agar plate.

Table 4.2: Antibacterial activity of selected lactobacilli isolates against Salmonella Enteritidis

Isolate Isolate source Antimicrobial activity (mm) expressed as Mean ± S. D

IKP07 Poultry dropping 6.33±0.57a

IKP23 Poultry dropping 17.33±0.57b

IKP41 Poultry dropping 16.33±1.52b

IKP76 Poultry dropping 18±01b

IKP94 Poultry dropping 11.66±0.57c

IKP111 Poultry dropping 20.33±0.57d

IKP138 Poultry dropping 14.66±1.15e

IKP162 Poultry dropping 9.33±0.57f

IKP183 Poultry cecum 13.33±0.57e

IKP192 Poultry cecum 20.33±0.57d

IKP229 Poultry cecum 8.66±0.57f

IKP271 Poultry cecum 12±1c

IKP333 Poultry ileum 19.66±1.15d

IKP387 Poultry ileum 17±1b

IKP402 Poultry ileum 20.33±1.15d

a,b,c,d,e,fDifferent superscripts in different rows of same columns show statistically significant difference at p ≤ 0.05

26

Results

Figure 4.3: Zone of inhibition of selected lactobacilli isolates against Salmonella Enteritidis

Figure 4.4: Activity of CFS (cell free supernatant) at pH 6.5 of selected lactobacilli isolates

a,b,c,d,e,f,gDifferent superscripts indicate statistical difference (p<0.05)

27

Results4.2.2. Resistance to low pH of selected lactobacilli isolates

IKP 111 showed better tolerance (0.777±0.02) at pH 7 among all isolates and significantly different (P˂ 0.05) from 15 lactobacilli isolates as indicated in table 4.3 IKP 138 showed lowest tolerance (0.297±0.06) at pH 7. IKP 111 showed better tolerance (0.683±0.02) at pH 4 among all isolates and significantly different (P˂ 0.05) from 15 lactobacilli isolates. IKP 07 and IKP 76 showed lowest tolerance (0.150±0.01) and (0.180±0.05) respectively at pH 4. IKP 111 showed highest tolerance (0.543±0.03) at pH 3 and IKP 07 showed lowest tolerance (0.120±0.05) at pH 3 among all isolates. All the fifteen isolates showed varying degree of tolerance at pH 2. IKP271 showed highest tolerance (0.522±0.02) and IKP 76 showed lowest tolerance (0.116±0.02) at pH 2 (Figure 4.5, 4.6, 4.7, 4.8).

28

ResultsTable 4.3: Growth of selected lactobacilli isolates in MRS broth supplemented with different

concentrations of pH represented as mean O.D after 24 hours

LactobacilliO.D (Mean± Standard Deviation)

pH 7 pH4 pH3 pH2

IKP07 0.474±0.01a 0.15±0.01b 0.12±0.05b 0.135±0.03b

IKP23 0.584±0.04a 0.56±0.05a 0.494±0.07a 0.336±0.02b

IKP41 0.334±0.03a 0.321±0.03a 0.261±0.04b 0.232±0.05bb

IKP76 0.574±0.04 a 0.18±0.05b 0.170±0.07b 0.116±0.02b

IKP94 0.592±0.01a 0.54±0.09 a 0.486±0.07b 0.386±0.02c

IKP111 0.777±0.02 a 0.683±0.0b 0.543±0.03c 0.418±0.07d

IKP138 0.297±0.06a 0.290±0.05a 0.178±0.01b 0.188±0.01b

IKP162 0.333±0.04a 0.315±0.03a 0.214±0.07b 0.166±0.05b

IKP183 0.567±0.03a 0.522±0.04a 0.517±0.01a 0.445±0.02b

IKP192 0.498±0.04a 0.476±0.05a 0.414±0.04a 0.368±0.06b

IKP229 0.444±0.01a 0.456±0.07a 0.398±0.04b 0.354±0.01b

IKP271 0.614±0.04a 0.598±0.05a 0.534±0.07b 0.522±0.02b

IKP333 0.587±0.01a 0.566±0.03a 0.497±0.06b 0.479±0.03b

IKP387 0.602±0.05a 0.555±0.01a 0.423±0.01b 0.405±0.03b

IKP402 0.543±0.05a 0.476±0.04b 0.434±0.02b 0.398±0.01c

a,b,c,dDifferent superscripts in different columns of same row show statistically significant difference at P≤0.05.

29

Results

Figure: 4.5: Tolerance of selected lactobacilli at pH 7

a,b,cDifferent superscripts indicate statistical difference

30

Results

Figure 4.6: Tolerance of selected lactobacilli at pH 4

a,b,c,dDifferent superscripts indicate statistical difference

31

Results


a,b,c,dDifferent superscripts indicate statistical difference

32

Results


a,b,cDifferent superscripts indicate statistical difference (p<0.05)

33

Results4.2.3. Resistance to bile salt of selected lactobacilli isolates

Bile salt tolerances of selected isolates were measured at different concentrations 0.30%, 1.0% and 1.8%. As indicated in table 4.4 and figures 4.9,4.10 and 4.11, IKP 162 showed better tolerance (1.341±0.04) at 0.30% concentration of bile salt and significantly different (P˂ 0.05) from other lactobacilli isolates. IKP 23 showed lowest tolerance (0.121±0.05) at 0.30% concentration of bile salt. IKP 271 and IKP 23 showed better tolerance (0.777±0.06), (0.714±0.09) respectively at 1% concentration of bile salt and significantly different (P˂ 0.05) from other lactobacilli isolates. IKP 07 and IKP 41 showed lowest tolerance (0.134±0.03), (0.165±0.10) respectively at 1.0% bile salt concentration and significantly different (P˂ 0.05) from other lactobacilli isolates. As indicated in table 4.7, at 1.8% bile salt concentration IKP183 showed highest tolerance (0.567±0.03) and IKP 07 showed lowest tolerance (0.121±0.09) at 1.8% bile salt concentration and significantly different (P˂ 0.05) from other lactobacilli isolates.

Table 4.4: Growth of selected lactobacilli isolates in MRS broth supplemented with different concentrations of bile salts represented as mean O.D after 24 hours

LactobacilliO.D (Mean± Standard Deviation)

MRS broth 0.30% 1.0% 1.8%

IKP07 1.23±0.02a 0.365±0.01b 0.134±0.03c 0.121±0.09c

IKP23 1.40±0.08a 1.210±0.05a 0.714±0.09b 0.432±0.05c

IKP41 1.11±0.04a 0.411±0.01b 0.165±0.10c 0.174±0.07c

IKP76 1.276±0.02a 1.052±0.08b 0.654±0.06c 0.444±0.01d

IKP94 0.987±0.03a 0.744±0.05b 0.543±0.09c 0.298±0.05d

IKP111 1.31±0.02a 0.823±0.10 b 0.462±0.04c 0.171±0.08c

IKP138 1.163±0.07a 0.567±0.05b 0.397±0.09c 0.243±0.05d

IKP162 1.534±0.02a 1.341±0.04b 0.598±0.06c 0.519±0.01d

IKP183 1.007±0.06a 0.698±0.01bb 0.619±0.02b 0.567±0.03c

IKP192 1.345±0.01a 0.897±0.01b 0.555±0.03c 0.256±0.08d

IKP229 0.925±0.10a 0.845±0.09a 0.390±0.03b 0.314±0.02b

IKP271 1.211±0.02a 0.921±0.08b 0.777±0.06c 0.476±0.06d

IKP333 1.083±0.11a 0.611±0.04b 0.582±0.07b 0.531±0.05b

IKP387 1.29±0.04a 0.883±0.05b 0.546±0.02c 0.478±0.02c

IKP402 1.170±0.07a 0.609±0.10b 0.455±0.06c 0.286±0.07d

a,b,c,dDifferent superscripts in different columns of same row show statistically significant difference at P≤0.05.

34

Results

Figure 4.9: Tolerance of selected lactobacilli at 0.30% bile salt conc.

a,b,c,d,e,f Different superscripts indicate statistical difference (p<0.05)

35

Results

Figure 4.10: Tolerance of selected lactobacilli at 1 % bile salt conc.

a,b,c,d,eDifferent superscripts indicate statistical difference (p<0.05)

36

Results

Figure 4.11: Tolerance of selected lactobacilli at 1.8 % bile salt conc.

a,b,c,dDifferent superscripts indicate statistical difference (p<0.05)

4.2.4. Auto aggregation activity of selected isolatesO.D values were recorded at different time intervals (1 hour and 2 hour) in auto aggregation

experiments as mentioned in table 4.5. IKP 138 and IKP 111 showed highest auto aggregation activity (65.87±3.12), (60.70±2.44) respectively after 2 hours time interval and showed significantly difference (P˂ 0.05) from other lactobacilli isolates. IKP 192 showed poor auto aggregation activity (25.40±1.28) after 2 hours time interval.

Table 4.5: Percentage auto aggregation of lactobacilli isolates at different time intervals

Lactobacilli Isolates 1hour 2hour

IKP07 16.22±0.1a 27.05±0.72a

IKP23 28.09±1.31b 51.23±0.93b

IKP41 34.25±0.65c 45.70±0.99c

IKP76 14.50±0.55a 33.60±1.20d

IKP94 29.32±0.47b 46.53±0.32c

37

Results

IKP111 41.10±0.55d 60.70±2.44e

IKP138 50.10±0.35e 65.87±3.12f

IKP162 24.20±0.47f 37.90±1.40g

IKP183 25.30±0.1.44f 42.13±0.39c

IKP192 14.20±0.14a 25.40±1.28a

IKP229 37.15±0.89g 46.40±0.98c

IKP271 33.30±0.59c 44.60±0.77c

IKP333 27.88±0.20b 41.40±0.82h

IKP387 15.10±0.10a 37.40±0.40g

IKP402 28.09±0.80b 51.23±1.50b

a,b,c,d,e,f,g,h Different superscripts in different rows of same column show statistically significant difference at P≤0.05.

4.2.5. Co-aggregation activity of selected isolates

O.D values were taken in co-aggregation experiment after 1 hour and 2 hours time interval as mentioned in table 4.6. IKP 111 showed highest coaggregation activity (55.70±1.32) after 2 hours time interval and significantly different (P˂ 0.05) from other lactobacilli isolates. IKP 387 showed poor coaggregation activity 6.33±0.11 after 2 hours time interval and significantly different (P˂ 0.05) from other isolates.

Table 4.6: Percentage co-aggregation of lactobacilli isolates against Salmonella Enteritidis at different time intervals

Lactobacilli Isolates 1hour 2hours

IKP07 17.10±0.30a 19.11±0.26a

IKP23 23.33±0.82b 26.20±0.75b

IKP41 27.44±0.10c 25.80±0.46b

IKP76 29.05±0.77c 24.30±0.40b

IKP94 21.10±0.94b 33.40±0.10c

IKP111 40.10±0.34d 55.70±1.32d

IKP138 14.50±0.22e 20.01±0.68a

IKP162 18.57±0.18a 24.80±0.90b

IKP183 22.19±0.96b 25.16±0.87b

IKP192 08.34±0.43f 07.14±0.09e

IKP229 17.28±0.41a 17.90±0.08a

IKP271 19.25±0.74a 23.45±0.88b

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Results

IKP333 17.88±0.19a 26.40±0.34b

IKP387 10.39±0.52f 6.33±0.11e

IKP402 16.49±0.45a 19.87±0.21a

a,b,c,d,e,f,g,h Different superscripts in different rows of same column show statistically significant difference at P≤0.05.

4.2.6. Antibiotic susceptibility patternAntibiotic susceptibility profiles of selected isolates are presented in table 4.7 IKP 23, IKP 111 and

IKP 333 were resistant to vancomycin only and sensitive to all other antibiotics. IKP 76, IKP 183, IKP 229 and 271 were resistant to Penicillin. IKP 138, IKP 162, IKP 271 and IKP 387 were resistant to tetracycline as indicated in table 4.7 and 4.8.

Table 4.7: Antibiotic susceptibility pattern of lactobacilli Isolates

Isolates PEN ERY AMP POL BA

C FUS CHL KAN CIP IMP TET VAN

IKP07 S S S S R S S S S S S R

IKP23 S S S S S S S S S S S R

IKP41 S S S S S S S S S S S S

IKP76 R R S S R R S R S R S R

IKP94 S R R S S S S S S S S R


IKP138 S S R R R S S R I S R R

IKP162 S S S S S S S S S S R R

IKP183 R S S S R S S R S S S R

IKP192 S R S S S S S S S S S R

IKP229 R S R R S S S S S R S R

IKP271 R S S R R S S S S S R R


IKP387 S S S S S S R S S S R R

IKP402 I R R R S S R S R R S R

ZOI: Zone of inhibition, n: Number of isolates; R: Resistant, I: Intermediate, S: Sensitive; PENː penicillin, AMPː ampicillin, IMPː imipenem, VANː vancomycin, BACː bacitracin, POLː polymyxin B, ERYː erythromycin, GENː gentamicin, KANː kanamycin, CHLː chloramphenicol, TETː tetracycline, CIPː ciprofloxacin, FUSː fusidic acid

39

Results

Table 4.8: Antibiotic resistance profile of lactobacilliSelected Isolates Resistance Profile

IKP07 BACR, VANR

IKP23 VANR

IKP41 -

IKP76 PENR, ERYR, BACR, FUSR, KANR, IMPR, VANR

IKP94 AMPR, ERYR, VANR

IKP111 VANR

IKP138 AMPR, POLR, BACR, CIPR, TETR, VANR

IKP162 TETR, VANR

IKP183 PENR, BACR, KANR, VANR

IKP192 ERYR, VANR

IKP229 PENR, AMPR, POLR, IMPR, VANR

IKP271 PENR, POLR, BACR, TETR, VANR

IKP333 VANR

IKP387 CHLR, TETR, VANR

IKP402 ERYR, POLR, AMPR, CHLR, CIPR, IMPR, VANR

4.3. Selection of isolates for further analysisIn table 4.9, it was mentioned that 12 isolates were rejected on the basis of in vitro characterization of

selected lactobacilli. Only 03 isolates (IKP 23, IKP 111 and IKP 333) out of 15 were selected for further study on the basis of anti-Salmonella activity, tolerance to pH, tolerance to bile salt, auto-aggregation activity and co-aggregation activity.

40

ResultsTable 4.9: Selection of potentially probiotic isolates

Isolates rejected for further study

Sr. No. Isolates Reason

01 IKP07 Absence of tolerance at different pH


03 IKP76 Resistance to different antibiotics





08 IKP192 No aggregation potential



11 IKP387 No aggregation potential


Isolates Selected for further study

01 IKP 23 Presence of anti-Salmonella properties and fulfilled prerequisites of probiotics

02 IKP 111 Presence of anti-Salmonella properties and fulfilled pre-requisites of probiotics

03 IKP 333 Presence of anti-Salmonella properties and fulfilled prerequisites of probiotics

4.4. Inhibition of Salmonella in broth culture

As indicated in table 4.10, IKP111 was recorded highest inhibition activity against Salmonella Enteritidis as compared to IKP 23 and IKP 333. IKP 111 was significantly different (P˂ 0.05) from IKP 23 and IKP 333.

Table 4.10: Inhibition of Salmonella Enteritidis by lactobacilli in broth culture at different time intervals

IsolatesLog10 CFU/ml (Mean ± S.D) of Salmonella co-cultured with lactobacilli

0 min 6 hours 24 hours Mean Log Reduction % reduction

IKP23 5.21±0.46 5.09±0.10 a 4.47±0.55a 0.74a81a

IKP111 6.11±0.18 5.66±0.47b 3.95±0.11b 2.16b99.3b

IKP333 5.78±0.40 5.23±0.15c 4.62±0.45b 1.16c93c

41

Results a,b,cDifferent superscripts in different rows of same column show statistically significant difference at p ≤ 0.05

4.5. Molecular characterization of selected lactobacilliGenus specific PCR amplification of ~250 bp amplicons confirmed that IKP 23, IKP 111 and IKP 333

are lactobacilli as indicated in figure 4.12. Universal primers were used in species identification PCR of IKP 23, IKP 111 and IKP 333 by using 16S rRNA gene (~1400bp) as indicated in figure 4.13. 16 S RNA gene sequences of IKP 23, IKP 111 and IKP 333 were also obtained.

+

Figure 4.12: Lactobacillus genus specific amplification of 16S rDNA-23S rDNA inter-spacer region

Figure 4.13: Amplification of 16S rDNA by universal Primers

42

Results16s RNA gene sequence of IKP 23 (lactobacilli isolate)

>TCTCAAAACTAAACAAAGTTTCGACAATGTGTAGGTTTCCGATTAATTCCTTAGAAAGGAGGTGATCCAGCCGCAGGTTCTCCTACGGCTACCTTGTTACGACTTCACCCTAATCATCTGTCCCACCTTAGGCGGCTGGCTCCTAAAAGGTTACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCA

16s RNA gene sequence of IKP 111 (lactobacilli isolate)

>TTGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAGCCAGCCGCCTAAGGTGGGACAGATGATTAGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTTTCTAAGGAATTAATCGGAAACCTACACATTGTCGAAACTTTGTTTAGTTTTGAGAA

16s RNA gene sequence of IKP 333 (lactobacilli isolate)

>TATACATGCAAGTCGAACGAAACTTTCTTACACCGAATGCTTGCATTCACCGTAAGAAGTTGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTAAAAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATATCTCTAAGGATCGCATGATCCTTAGATGAAAGATGGTTCTGCTATCGCTTTTAGATGGACCCGCGGCGTATTAACTAGTTGGTGGGGTAACGGCCTACCAAGGTGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACACGAGTGAGAGTAACTGTTCATTCGATGACGGTATCTAACCAGCAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGGGAACGCAGGCGGTCTTTTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGTAGTGCATTGGAAACTGGAAGACTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGTTCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCAATAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACCTAAGAGATTAGGCTTTCCCTTCGGGGACAAAGTGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTGTCAGTTGCCAGCA

43

Results

Figure 4.14: Sequence Chromatogram of IKP333

4.6. Species identification of lactobacilliAmplicons (~1400 bp) were sequenced, submitted to NCBI GenBank and accession numbers of

IKP23, IKP111 and IKP 333 were obtained as indicated in table 4.11.

Table 4.11: Identification of selected lactobacilli to specie level

Sr. No. Selected Lactobacilli isolates ID Source Species Identification Gene bankAccession no.

1. IKP 23 Poultry dropping Lactobacillus fermentum MK350329

2. IKP 111 Poultry dropping Lactobacillus fermentum MK350330

3. IKP 333 Poultry ileum Lactobacillussalivarius MK346270

4.7. Phylogenetic analysis

44

ResultsPCR product of 16S rRNA amplification was sequenced and sequences were analyzed by Chromas

2.0. The analyzed sequences were compared with NCBI data base by Blast. After that used alignment of sequence with previously reported sequence through Bioedit software. Phylogenetic tree was built by mega 7 software. After phylogenetic analysis it was revealed that IKP 23 had (Lactobacillus fermentum) 99% similarity to China Lactobacillus fermentum strain NSW29 as indicated in figure 4.15. After phylogenetic analysis it was revealed that IKP 333 (Lactobacillus salivarius) had 99% similarity to Lactobacillus salivarius strain L6 and Lactobacillus salivarius strain L8 as indicated in figure 4.16.

Figure 4.15: Genetic relationships of 16S rRNA aligned sequences of Lactobacillus fermentum IKP 23

45

Results

Figure 4.16: Genetic relationship of 16S rRNA aligned sequences of Lactobacillus salivarius IKP 333

46

Results4.8. In vivo characterization of lactobacilli for their probiotic properties

Day old broiler chicks (n=150) were divided in 10 groups. Randomized complete block design was used. Negative control (NC) group was not supplemented with probiotics. Positive control (CC) group received only the challenge bacteria (Salmonella Enteritidis ) ATCC 13076 at day 07. Groups (3, 4, 5) received probiotics (IKP23-PM, IKP111-PM, IKP333-PM) at day 01 to 35 and challenge bacteria at day 07 in prevention model. Groups (6, 7, 8) started receiving probiotic (IKP23-TM, IKP111-TM, IKP333-TM) at day 07 to day 35 and challenge bacteria at day 07 in TM. Group 09 started receiving commercial probiotic Protexin (1g/liter) at day 01 to 35 and challenge bacteria at day 07. Group 10 started receiving antibiotic at day 01 to 05 and challenge bacteria at day 07. Droppings of birds in each group were analyzed for lactobacilli count, Salmonella count and Coliform count on selective culture media. Mean body weight of each group was calculated on weekly basis, immunomodulatory effects (GMT) of probiotics against NDV and AIV H9 were also calculated at day 14, 21, 38 and 35. Villus height and villus height to crypt ratio of broiler intestine (duodenum, jejunum and ileum) were measured and D-xylose absorption test was carried out to assess broiler absorption capacity.

Table 4.12: Experimental design for in vivo evaluation of probiotics

Groups Plan

Negative control (NC) No treatment

Positive Control (CC) Salmonella Enteritidis challenge at day 07

PM

IKP 23-PM IKP 23+challenge after day 07(IKP 23 probiotic supplemented day 01 to 35)



Protexin-PM Protexin+ challenge at day 07(Protexin probiotic supplemented day 01 to 35)

Antibiotic-PM Antibiotics+ challenge at day 07Antibiotics supplemented day 01 to 05

TM

IKP 23-TM Challenge at day 07+ then started IKP 23(IKP 23 probiotic supplemented day 07 to 35)



IKP 23= Lactobacillus fermentum, IKP 111 = Lactobacillus fermentum, IKP 333= Lactobacillus salivarius

47

Results

Figure 4.17: Birds in different groups in 1st week

Figure 4.18: Birds in different groups in 4th week

Figure: 4.19 Poultry dropping samples collection

48

Results4.8.1. Effect of selected probiotic strain on lactobacilli count in droppings of broiler

Effect of selected probiotic strain (IKP23, IKP111 and IKP333) on lactobacilli count in dropping of broiler was determined by enumeration of lactobacilli on MRS agar at different days (Table 4.13, Figure 4.20). Results revealed that lactobacilli count of broiler challenged with Salmonella (positive control group) was non-significantly lower (P˂0.05) as compared to negative control group (4.42 ±0.39 log10 vs 4.65±0.14 log10). Result also showed that lactobacilli count was significantly increased in all groups given probiotics (selected strains or Protexin) as compared to negative control, positive control and antibiotic group. Lactobacilli counts were significantly higher (P˂0.05) in groups administered with IKP-PM 23, IKP 111-PM and IKP 333-PM (6.19±0.11,,6.83±0.22, 6.30±0.41 respectively) prior to Salmonella challenge (PM) as compared to groups administered with IKP-TM 23, IKP 111-TM and IKP 333-TM (5.63±0.12, 5.49±0.31, 5.65±0.45 respectively) post Salmonella challenge (TM).

Table 4.13: Effect of selected lactobacilli strains on gut lactobacilli count in broiler

GroupsLactobacilli count (Mean log10 CFU/g ±S. D) of broiler at different days

Day 07 Day 08 Day 09 Day 10 Day 14 Day 21 Day 28 Day 35

Negative control

4.62±0.10a 4.81±0.08a 4.75±0.27a 4.35±0.11a 4.44±0.08a 4.57±0.21a 4.65±0.16a 4.65±0.14a

Positive control

4.53±0.03a 4.47±0.08a 4.39±0.04a 4.45±0.09a 4.22±0.17a 4.40±0.22a 4.40±0.16a 4.42±0.39a

IKP 23-PM

5.32=±0.27b 5.01±0.09b 5.17±0.32b 5.29±0.10b 5.54±0.17b 5.65±0.23b 5.88±0.15b 6.19±0.11b

IKP 111-PM

5.73±0.23b 5.94±0.14b 5.88±0.18b 5.27±0.22b 6.43±0.19b 6.32±0.26c 6.71±0.22c 6.83±0.22b

IKP 333-PM

5.80±0.04b 5.78±0.33b 5.98±0.19b 5.93±0.10b 6.15±0.39b 6.07±0.10c 6.24±0.12c 6.30±0.41b

Protexin-PM

4.44±0.09a 4.37±0.13a 4.40±0.10a 5.34±0.13b 5.47±0.27b 6.49±0.21c 6.54±0.33c 6.61±0.42b

Antibiotic-PM

4.19±0.05a 4.20±0.14a 4.17±0.11a 4.14±0.08a 4.10±0.12a 4.11±0.07a 4.62±0.22a 4.84±0.06a

IKP 23-TM

4.61±0.13a 4.55±0.33a 4.27±0.08a 5.45±0.12b 5.51±0.25b 5.46±0.16b 5.47±0.44b 5.63±0.12c

IKP 111-TM

4.52±0.34a 4.61±0.16a 4.69±0.52a 4.82±0.20a 5.68±0.11b 5.65±0.13b 5.52±0.24b 5.49±0.31c

IKP 333-TM

4.59±0.20a 4.59±0.11a 4.42±0.29a 4.23±0.26a 4.45±0.19a 5.52±0.18b 5.50±0.12b 5.65±0.45c

a, b, c Different superscript in different column of same row show statistically significant difference at p ≤ 0.05aData are expressed as Means ±SD of log10 bacterial counts per gram weight of dropping/intestinal content

49

Results

Figure 4.20: Gut lactobacilli count in different experimental groups at day 35a,b,cDifferent superscripts indicate statistical difference (p<0.05)

50

Results4.8.2. Effect of selected probiotic strain on Salmonella count in droppings of broiler

Effect of selected probiotic strain (IKP23, IKP111 and IKP333) on Salmonella count in dropping of broiler was determined by enumeration of Salmonella on XLD agar at different days (Table 4.14, Figure 4.21). Result revealed that Salmonella count of broiler challenged with Salmonella (positive control group) was significantly higher as compared to negative control group (4.88±0.29 log10 vs 3.66±0.23 log10). Result also showed that Salmonella count was significantly increased (P˂ 0.05) in positive control group as compared to groups administered with IKP 23-PM, IKP 111-PM and IKP 333 (PM and TM), Protexin and antibiotic supplemented group.

Table 4.14: Effect of selected lactobacilli on Salmonella count in broiler gut Groups Day 07 Day 08 Day 09 Day 10 Day 14 Day 21 Day 28 Day35

Negative control

2.48±0.09a 2.47±0.34a 3.48±0.33a 3.51±0.18a 3.46±0.10a 3.44±0.28a 3.52±0.50a 3.66±0.23a

Positive control

2.54±0.12a 3.60±0.07b 3.71±0.20a 4.89±0.70b 4.97±0.30b 5.05±0.11b 5.01±0.14b 4.88±0.29b

IKP 23-PM

2.14±0.15a 3.06±0.44b 3.72±0.70a 3.21±0.22a 3.14±0.06a 3.07±0.30a 3.16±0.11a 2.92±0.04a

IKP 111-PM

2.21±0.11a 3.21±0.05b 3.28±0.10a 3.19±0.15a 3.18±0.19a 3.13±0.02a 3.77±0.24a 3.05±0.10a

IKP 333-PM

2.45±0.40a 3.19±0.40b 3.31±0.26a 3.29±0.08a 3.27±0.13a 3.21±0.05a 3.12±0.10a 2.99±0.25a

Protexin-PM

2.32±0.14a 3.41±0.50b 3.47±0.10a 3.41±0.10a 3.35±0.30a 3.33±0.24a 3.31±0.33a 3.44±0.42a

Antibiotic-PM

2.04±0.05a 3.11±0.60b 3.13±0.11a 3.19±0.08a 3.26±0.19a 3.60±0.15a 3.63±0.20a 3.75±0.70a

IKP 23-TM

2.39±0.13a 3.47±0.33b 3.42±0.08a 3.40±0.12a 3.32±0.07a 3.23±0.05a 3.82±0.44a 3.37±0.12a

IKP 111-TM

2.34±0.34a 3.39±0.26b 3.40±0.52a 3.37±0.04a 3.35±0.09a 3.31±0.13a 3.75±0.40a 3.49±0.50a

IKP 333-TM

2.38±0.02a 2.44±0.11a 3.42±0.29a 3.55±0.26a 3.47±0.19a 3.35±0.09a 3.38±0.12a 3.55±0.45a

a, b Different superscript in different column of same row show statistically significant difference at p ≤ 0.05aData are expressed as Means ±SD of log10 bacterial counts per gram weight of dropping or intestinal

content

51

Results

Figure 4.21: Salmonella count in different experimental groups at day 35a,bDifferent superscripts indicate statistical difference (p<0.05)

52

Results4.8.3. Effect of selected probiotic strain on Coliform count in droppings of broiler

Effect of selected probiotic strain (IKP23, IKP111 and IKP333) on Coliform count in dropping of broiler was determined by enumeration of Coliform on VRB agar at different days (Table 4.15, Figure 4.22). Result revealed that Coliform count of broiler challenged with Salmonella (positive control group) was non-significantly different (P˂0.05) as compared to negative control group (4.79±0.66 log10 vs 4.92±0.20 log10). Result also showed that Coliform count was significantly low (P˂0.05) in groups given probiotics (selected strains or Protexin) prior to Salmonella challenge as compared to negative control, positive control and antibiotic group. Coliform counts were significantly low (P˂ 0.05) in groups administered with IKP 23-PM, IKP 111-PM and IKP 333-PM (3.09±0.63, 3.21±0.90, 3.18±0.41 respectively) prior to Salmonella challenge (PM) as compared to groups administered with IKP 23-TM, IKP 111-TM and IKP 333-TM (4.20±0.88, 4.17±0.55, 4.11±0.45 respectively) post Salmonella challenge (TM).

Table 4.15: Effect of selected lactobacilli on Coliform count in broiler gutGroups Day 07 Day 08 Day 09 Day 10 Day 14 Day 21 Day 28 Day 35

Negative control 4.52±0.35a 4.49±0.15a 4.45±0.18a 4.39±0.44a 4.22±0.27a 4.48±0.10a 4.47±0.36a 4.92±0.20a

Positive control 4.63±0.19a 4.58±044a 4.58±0.80a 4.59±0.56a 4.61±0.08a 4.62±0.10a 4.58±0.23a 4.79±0.66a

IKP 23-PM 4.11±0.46a 4.20±0.38a 4.17±0.32a 4.12±0.77a 4.10±0.11a 3.98±0.40a 3.60±0.15b 3.09±0.63b

IKP 111-PM 4.27±0.80a 4.42±0.46a 4.28±0.73a 4.21±0.37a 4.13±0.08a 4.02±0.02a 4.01±0.60a 3.21±0.90b

IKP 333-PM 4.19±0.16a 4.34±0.33a 430±0.19a 4.21±0.10a 4.13±0.39a 4.06±0.05a 4.06±0.03a 3.18±0.41b

Protexin-PM 4.47±0.04a 4.53±0.13a 4.42±0.07a 4.38±0.13a 4.39±0.27a 4.32±0.04a 4.27±0.33a 3.80±0.42a

Antibiotic-PM 4.22±0.30a 4.25±0.14a 4.30±0.58a 4.26±0.90a 4.28±0.77a 4.25±0.07a 4.31±0.22a 4.36±0.37a

IKP 23-TM 4.45±0.19a 4.42±0.33a 4.40±0.28a 4.35±0.18a 4.29±0.43a 4.27±0.50a 4.21±0.49a 4.20±0.88a

IKP 111-TM 4.50±0.67a 4.56±0.52a 4.44±0.25a 4.48±0.66a 4.51±0.09a 4.39±0.13a 4.27±0.24a 4.17±0.55a

IKP 333-TM 4.35±0.28a 4.35±0.11a 4.32±0.49a 4.27±0.20a 4.18±0.19a 4.15±0.09a 4.13±0.12a 4.11±0.45a

a, b Different superscript in different column of same row show statistically significant difference at p ≤ 0.05 aData are expressed as Means ± SD of log10 bacterial counts per gram weight of dropping or intestinal content

53

Results

Figure 4.22: Coliform count in different experimental groups at day 35a,b,c,dDifferent superscripts indicate statistical difference (p<0.05)

4.8.4. Effect of selected probiotic strain on broiler GMT titer against NDV vaccineImmuno-modulatory effects of probiotics against NDV vaccine in different poultry groups are

presented in table 4.16. Poultry groups which were supplemented (day 01 to 35) with probiotics IKP23-PM, IKP111-PM, IKP333-PM, Protexin had higher antibody titers 112, 108, 98.5, 96 respectively against NDV vaccine at day 35 as compared to negative control group, positive control group and antibiotic supplemented group. Immuno-modulatory effects in poultry groups were higher which were administered with probiotics before challenge bacteria.

Table 4.16: Geometric mean antibody titer in broiler chickens fed with different lactobacilli strains against NDV vaccine

54

Results

GroupsGMT of birds against NDV at different days

14 21 28 35

Negative control 47.8 36.8 95.5 83.2

Positive control 55 58.2 72.5 68

IKP 23-PM 63.1 68 125.9 112

IKP 111-PM 55 63.1 114.4 108

IKP 333-PM 63.1 47.8 110.5 98.5

Protexin-PM 63.1 74.5 108 96

Antibiotic-PM 55 55 90 82.6

IKP 23-TM 63.1 74.5 98.3 86

IKP 111-TM 63.1 68 95.8 88

IKP 333-TM 63.1 72.3 101 84

55

Results4.8.5. Effect of selected probiotic strain on broiler GMT titer against AIV H9 vaccine

Immunomodulatory effects of probiotics against AIV H9 vaccine in different poultry groups are presented in table 4.17. Poultry groups which were supplemented (day 01 to 35) with probiotics IKP23-PM, IKP111-PM, IKP333-PM, Protexin had higher antibody titers 110, 122, 103, 102 respectively against AIV H9 vaccine at day 35 as compared to negative control group, positive control group and antibiotic supplemented group. Immunomodulatory effects of probiotics were higher in PM as compared to TM.

Table 4.17: Geometric mean antibody titer in broiler chickens fed with different lactobacilli strains against AIV H9 vaccine

GroupsGMT of birds against H9 at different days

14 21 28 35

Negative control 60 55.5 83 103.4

Positive control 55.5 66 75.2 96.1

IKP 23-PM 72.4 78 118 110

IKP 111-PM 55 63.1 105 122

IKP 333-PM 63.1 47.8 88 103

Protexin-PM 63.1 74.5 109 102

Antibiotic-PM 55 55 94 84.2

IKP 23-TM 63.1 74.5 93.2 101.5

IKP 111-TM 63.1 68 87 94.2

IKP 333-TM 63.1 72.3 105 98

56

Results4.8.6. Effect of selected probiotic strains on broiler weight gain in vivo trial

There was no significant difference (P˂ 0.05) in mean body weight (g) between experimental groups at day 01 as indicated in table 4.18. There was no significant difference (P˂ 0.05) in mean body weight (g) between experimental groups at day 07 and day 14 as indicated in (table 4.18, figure 4.23.2 and figure 4.23.3). At day 21, the highest mean body weight (892±18.0) g was recorded by group IKP 333-TM among all probiotic supplemented groups (selected strain in preventive and TM, Protexin) and significantly different (P˂ 0.05) from negative group, positive group and antibiotic supplemented group of broiler as indicated in (table 4.18, figure 4.23.4). At day 28 the highest mean body weight (1225±30.3), (1216±23.5) g was recorded by IKP 23-PM and IKP 111-PM supplemented at day 01 to 35 respectively and significantly different (P˂ 0.05) from all other experimental groups. At day 35 as indicated in (table 4.18, figure 4.23.6) weight gain of negative control group was significantly higher (P˂ 0.05) as compared to positive control group (1466±49.6 g vs 1320±44.8 g). Result also showed that weight gain of broiler challenged with Salmonella significantly increased in all groups given probiotics (selected strains or Protexin) as compared to negative control, positive control and antibiotic group. Weight gain was significantly higher (P˂ 0.05) in groups administrated with IKP 23-PM, IKP 111-PM and IKP 333-PM (1640±48.1, 1608±59.7, 1590±49.0 respectively) prior to Salmonella challenge (PM) as compared to groups administered with IKP 23-TM, IKP 111-TM and IKP 333-TM (1569±45.1, 1515±47.8, 1530±51.7 respectively) post Salmonella challenge (TM).

Table 4.18: Effect of selected lactobacilli strains on broiler weight gain

GroupsWeight (g) (Mean ±SD) of Broiler at different age (day)

1 7 14 21 28 35Negative control 41.5±0.9a 160.2±4.2a 388±8.5a 787±13.5a 1140±10.8a 1466±49.6a

Positive control 40.8±1.1a 159.5±6.7a 397±8.8a 815±13.6a 1172±18.9a 1320±44.8b

IKP 23-PM 41.7±0.8a 162±5.3a 418±12.2a 855±16.8b 1225±30.3b 1640±48.1c

IKP 111-PM 40.4±1.0a 161±5.5a 399±6.8a 838±17.5b 1216±23.5b 1608±59.7c

IKP 333-PM 41.1±1.3a 158±8.0a 411±7.8a 815±10.9a 1170±31.7a 1590±49.0c

Protexin-PM 41.7± 0.9a 160.7±6.1a 401±14.9a 835±16.4b 1154±22.6a 1522±60.4c

Antibiotic-PM 40.8±1.4a 156±3.9a 390±15.5a 677±19.0c 1175±26.8a 1345±35.4b

IKP 23-TM 41.0 ±1.1a 164±4.5a 401±7.4a 790±10.2a 1185±30.2a 1569±45.1c

IKP 111-TM 41.3±1.4a 159±3.7a 396±9.6a 833±17.7b 1163±12.5a 1515±47.8c

IKP 333-TM 41.5± 1.2a 162±7.0a 405±12.3a 892±18.0b 1175±24.1a 1530±51.7c

a, b, c Different superscript in different column show statistically significant difference at p ≤ 0.05

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Results

Figure 4.23.1: Mean body weight (g) of chickens at day 01

58

Results


59

Results


60

Results

Figure 4.23.4: Mean body weight (g) of chickens at day 21 a,b,cDifferent superscripts indicate statistical difference (p<0.05)

61

Results

Figure 4.23.5: Mean body weight (g) of chickens at day 28a,bDifferent superscripts indicate statistical difference (p<0.05)

.

62

Results

Figure 4.23.6: Mean body weight (g) of chickens at day 35a,b,cDifferent superscripts indicate statistical difference (p<0.05)

63

Results4.8.7. Effect of selected probiotic strain on duodenum villus height crypt depth ratio As indicated in (table 4.19, figure 4.24.1, 4.24.2) in duodenum there was no significantly difference (P˂ 0.05) in the villus height crypt depth ratio of negative control group and positive control group (6.58 um vs 6.14 um). Results also showed that villus height crypt depth ratio of broiler challenged with Salmonella significantly increased (P˂ 0.05) only in PM of selected probiotics (IKP 23-PM, IKP 111-PM, IKP 333-PM) groups as compared to negative control, positive control, Protexin group, TM of selected probiotics (IKP 23-TM, IKP 111-TM, IKP 333-TM) and antibiotic supplemented group. Villus height crypt depth ratio was significantly higher (P˂ 0.05) in groups administrated with IKP 23-PM, IKP 111-PM and IKP 333-PM (8.03 um, 8.10 um, 8.07, respectively) prior to Salmonella challenge (PM) as compared to groups administrated with IKP 23-TM, IKP 111-TM and IKP 333-TM (7.45 um, 6.17 um, 6.81 um, respectively) post Salmonella challenge (TM).

Table 4.19: Effects of selected lactobacilli on gut (duodenum) morphology in broiler chickens

Groups Villus length (um)(Mean ± S.D)

Crypt depth (um)(Mean ± S.D)

Villus length/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-T;M 629.24±82.19 92.42±4.72 6.81a

a, b Different superscript in different column show statistically significant difference at p ≤ 0.05

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Results

Figure 4.24.1: Villus length/Crypt depth ratio in chicken (duodenum) between different experimental groups after use of probiotics

a,b,c,dDifferent superscripts indicate statistical difference (p<0.05)

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Results

Figure 4.24.2: Effect of Probiotics on gut morphology of broiler challenged with Salmonella Enteritidis: A: Positive control, B: Negative control, C: Probiotic 23(treatment), D: Probiotic IKP Probiotic (treatment), E: Probiotic 333 (treatment), F: Probiotic 23(preventive), G: Probiotic (preventive) IKP 23, H: Probiotic 333 (preventive), I: Protexin, J: Antibiotic

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Results4.8.8. Effect of selected probiotic strain on jejunum villus height crypt depth ratio As indicated in (table 4.20, figure 4.24.3), in jejunum there was no significant difference (P˂ 0.05) in the villus height crypt depth ratio of negative control group and positive control group (6.52 um vs 6.38 um). Result also showed that villus height crypt depth ratio of broiler challenged with Salmonella significantly increased (P˂ 0.05) in selected probiotics IKP 23-PM, IKP 111-PM groups (PM) and IKP 333-TM (TM) group as compared to all experimental groups. Increased villus height crypt depth ratio was obtained in IKP 23-PM, IKP 111-PM and IKP 333-TM (11.88 um, 9.53 um, 9.45um, respectively).

Table 4.20: Effects of selected lactobacilli on gut (jejunum) morphology in broiler chickens




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 8.25a

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 in different rows in same column show statistically significant difference at p ≤ 0.05

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Results

Figure 4.24.3: Villus length/crypt depth ratio in chicken (jejunum) between different experimental groups after use of probiotics

a,bDifferent superscripts indicate statistical difference (p<0.05)

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Results4.8.9. Effect of selected probiotic strain on ileum villus height crypt depth ratio As indicated in (table 4.21, figure 4.24.4) in ileum there was no significant difference (P˂ 0.05) in the villus height crypt depth ratio of negative control group and positive control group (5.28 um vs 6.06 um). Result also showed that villus height crypt depth ratio of broiler challenged with Salmonella significantly increased (P˂ 0.05) in selected probiotics IKP 111-PM, IKP 333-PM groups (PM) and IKP 23-TM (TM) group as compared to all experimental groups. Increased villus height crypt depth ratio was obtained in IKP 111-PM, IKP 333-PM and IKP 23-TM (8.53 um, 8.26 um, 8.21, respectively).

Table 4.21: Effects of selected lactobacilli on gut (ileum) morphology in broiler chickens




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, cDifferent superscripts in different rows of same column show statistically significant difference at p ≤ 0.05

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Results

Figure 4.24.4: Villus length/crypt depth ratio in chicken (ileum) between different experimental groups after use of probiotics


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Results4.8.10. Effect of selected probiotic strain on D-xylose absorption capacity of broiler

As indicated in (table 4.23), 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 vs 22.30 mg). Result revealed that D- xylose concentration of broiler challenged with Salmonella significantly increased (P˂ 0.05) in all groups given probiotics (selected strains or Protexin) as compared to negative control, positive control and antibiotic group. D-xylose concentration was higher but non significantly different (P˂ 0.05) in groups administered with IKP 23-PM, IKP 111-PM and IKP 333-PM (58.06 mg, 52.75 mg, 53.09 mg, respectively) prior to Salmonella challenge (PM) as compared to groups administrated with IKP 23-TM, IKP 111-TM and IKP 333-TM (48.81 mg, 46.71 mg, 48.02, respectively) post Salmonella challenge (TM).

Figure 4.25: D-Xylose absorptions in different experimental groups at 30 and 60 min. X-axis: Different groups, Y axis: Concentration (mg/dl) of D-Xylose

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ResultsTable 4.22: D-xylose concentration in plasma of broiler chickens

GroupsXylose concentration (Mean± S.D, mg/dl) in plasma of broilers 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

IKP 23-TM 0 20.03±3.12a 48.81±4.26a

IKP 111-TM 0 22.80±2.56a 46.71±4.08b

IKP 333-TM 0 22.41±2.43a 48.02±5.69b

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

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Results

Figure 4:26: D-Xylose absorption capacity (mg/dl) in broiler chickens between different experimental groups after use of probiotics


73

CHAPTER 5DISCUSSION

Salmonella is major pathogen of human and poultry. It can transmit horizontally as well as vertically in poultry and eventually to humans (Gole et al. 2014). The prophylactic use of antibiotics to control Salmonella infections in poultry may cause alterations in GIT flora and promote emergence of antibiotic resistant strains. It is the dire need of time to search for alternative approaches like probiotics. As defined by FAO, Probiotics are the live microbes which confers health benefits to host when ingested in adequate amounts. There are many microorganisms which can be used as probiotics but LAB are more appropriate as probiotics because they have GRAS status. WHO and FAO have already recommended the use of LAB strains as a probiotic in animals and humans (Amara and Shibl 2015).

Present study attempted to search out and characterize new indigenous probiotic lactobacilli against Salmonella Enteritidis. Present study isolated total of 84 lactobacilli from indigenous poultry droppings, ileum and ceca. Various different researches have also isolated lactobacilli from poultry and fermented food products (Asghar et al. 2016; Arif et al. 2018; Saleem et al. 2018). Lactobacilli can prevent the growth of pathogenic bacteria, such as Salmonella spp. and Escherichia coli in broiler or poultry gut (Jin et al. 1996). Lactobacilli can kill or reduce pathogen by reduction in gut pH due to lactic acid production, secretion of antimicrobial bacteriocins and H2O2, competitive exclusion of pathogen and strengthening normal flora (Wang and Gu 2010). Present study screened 15 anti-Salmonella Enteritidis lactobacilli using cell free supernatants in well diffusion assay, similar results have been also declared previously employing the same strategy (Asghar et al. 2016). Whereas other researchers have declared anti-Salmonella potential of lactobacilli using different strategies like spot test (Garriga et al. 1998), inhibition of Salmonella invasion using HT29 human intestinal cell line (Casey et al. 2004) and competitive exclusion of Salmonella in gut (La Ragione et al. 2004). Present study also observed Salmonella Enteritidis inhibition in broth cultures by three probiotic lactobacilli as reported previously (Makras et al. 2006). A previous study have also reported Salmonella Enteritidis inhibition on spinach leaf surface by LAB (Cálix-Lara et al. 2014).

Selection of probiotics is based upon tolerance to gastric pH and bile salts, adherence to epithelial cells, auto-aggregation, co-aggregation, antibiotic resistance profile, antimicrobial and antagonistic effect against potentially pathogenic bacteria (Shokryazdan et al. 2017). Probiotics should be tolerant to gut pH and bile salts. In present study, probiotic potential of anti-Salmonella Enteritidis lactobacilli was characterized by examining tolerance to different pH values (2,3,4,7) and bile salt concentrations (0.3%,1% and 1.8%), auto-aggregation, co-aggregation and antibiotic susceptibility pattern. All lactobacilli tolerated all tested pH values and bile salt concentrations for 90 minutes, but highest tolerance was observed at pH 4 and 0.3% bile salts. Various previous studies also indicated lactobacilli survival at acidic pH and bile salts (Asghar et al. 2016; Arif et al. 2018).

Acquired antibiotic resistance in lactobacilli poses a significant threat to public health. Probiotic lactobacilli should lack resistance against antibiotics so that they may not transfer it to pathogens. Lactobacilli obtained in present study were sensitive to all the tested antibiotics except tetracycline against 46.6% resistance is observed. Antibiotic resistance against tetracycline have been also previously molecularly characterized in lactobacilli (Saleem et al. 2018).

Auto aggregation and co-aggregation are important pre-requisites for the selection of probiotics. Auto-aggregation is necessary for their successful adhesion to epithelial cells and the benefit of co-aggregation is required to avoid colonization of pathogens in host gut systems. Lactobacillus strains showed non-significant auto-aggregation whereas significant auto-aggregation capability of lactobacilli have been witnessed in various previous studies (Bao et al. 2010; Asghar et al. 2016). Co-aggregation of probiotic with pathogenic bacteria is an antimicrobial characteristic of lactobacillus bacteria. IKP 76 and IKP 387 carried good capability to co-aggregate Salmonella Enteritidis. Similar co-aggregation pattern against Salmonella have been also determined previously (Asghar et al. 2016).

On the basis of better tolerance to gut conditions and other probiotic properties, IKP 23, IKP 111 and IKP 333, were selected for co-culturing experiments with Salmonella Enteritidis. All the three isolates successfully inhibited Salmonella Enteritidis, but highest inhibition was observed in case of IKP 111. Such inhibition have been also declared previously (Makras et al. 2006). Thus, these isolates can be employed as

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Discussionpotential probiotics against Salmonella Enteritidis in poultry after in vivo evaluations. Various in vivo experiments have been conducted previously (Garriga et al. 1998). During in vivo evaluations, effect of lactobacilli on other parameters like body weight, feed conversion ratio and enhanced immune response can also be examined as performed previously (Asghar et al. 2016).PCR product of 16S rRNA amplification was sequenced and sequences were analyzed by Chromas 2.0. Similarity of 16S rDNA sequences was used for specie identification and deveopment of phylogenetic tree as well. Use of 16S rDNA for specie identification is a gold standard and frequently used (Harris and Hartley 2003; Miller et al. 2016). Figure 4.15 indicated our isolate Lactobacillus fermentum showed 99% similarity to the isolate of China Lactobacillus fermentum strain NWS 29 (Wang et al. 2010) and our isolate showed less similarity to the isolates of China Lactobacillus rhamnosus strain which were isolated from human milk (Nikoskelainen et al. 2001). Phylogenetic analysis of Lactobacillus salivarius revealed that it had 99% homology to the isolates of India, China and Iran..

Present study attempted to characterize new indigenous probiotic lactobacilli for their in vivo activities against Salmonella Enteritidis. Various researches have also characterized lactobacilli for their in vivo activities against Salmonella Enteritidis (Van Coillie et al. 2007; Higgins et al. 2008; Asghar et al. 2016a; Arif et al. 2018; Saleem et al. 2018). The probiotics bacteria improved both the clinical and microbiological outcome of Salmonella infection (Casey et al. 2007).

Oral administration of LAB as probiotics reduce the colonization of Salmonella Enteritidis in chicken (Pascual et al. 1999; Higgins et al. 2007). Probiotics are involved in the promotion of the growth and health maintenance of chicken (Khan et al. 2007). Administration of probiotics daily in broiler, helped in increasing the count of beneficial microbes (lactobacilli) in gut (Jung et al. 2008; Zhang and Kim 2014; Hussain et al. 2015). In present study lactobacilli count was high in those groups which were fed with probiotics on daily basis. Lactobacilli counts were high in IKP 23-PM, IKP 111-PM, IKP 333-PM, Protexin supplemented group (day 01 to 35) as compared to treatments groups (TM) in which probiotics were supplemented at day 07 to 35 and lactobacilli counts were (6.19±0.11), (6.83±0.22), (6.30±0.41), (6.61±0.42) respectively. These groups had significantly different (P˂ 0.05) lactobacilli count from other experimental groups. The lowest lactobacilli count (4.42±0.39) was obtained in positive group because this group was only supplemented with Salmonella Enteritidis.

Competitive exclusion is a common mechanism in which probiotics protect the colonization of pathogenic bacteria like Salmonella and Coliform in gut. In present study probiotic efficiently excluded Salmonella in the broiler gut. Salmonella count 4.88±0.29 log10 CFU was high in positive group because this group was only received Salmonella challenge at day 07. Salmonella count was reduced in IKP 23, IKP 111 and IKP 333 probiotics supplemented groups both in PM and TM. The lowest count 2.92±0.04 log10 CFU of Salmonella was obtained in IKP 23-PM. There was significant difference (p˂0.05) in Salmonella count between positive control group and IKP 23-PM. Probiotics supplementation also competitively excluded and reduced the Coliform population in broiler gut as compared to control group (Jin et al. 1998; Gunal et al. 2006; Liu et al. 2007). In present study Coliform count was significantly reduced in those experimental groups in which probiotics were supplemented both in PM and TM. Coliform count 4.92±0.20 log10 CFU was high in negative control group while the Coliform count 3.09±0.63 log10 CFU was low in IKP 23-PM in all experimental groups. There was significant difference (p˂0.05) in Coliform count between negative control group and IKP 23-PM.

Mean body weights of the experimental groups were monitored on weekly basis after probiotics feeding. There was no significant difference (P˂0.05) in mean body weight between groups in 1 st and 2nd

week of broiler age. The differences were established in 3rd week of broiler age and maintained until the end of trial. Similar results have been also claimed previously (Jin et al. 1998; Perić et al. 2010; Asghar et al. 2016). At 3rd week of broiler age, the highest mean body weight 892±18.0g was recorded by IKP 333-TM among all the experimental groups and significantly higher (P˂ 0.05) from negative group, positive group and antibiotic supplemented group. At 4th week of broiler age, the highest mean body weight (1225±30.3), (1216±23.5) g was recorded by IKP 23-PM, IKP 111-PM respectively and significantly different (P˂ 0.05) among all experimental groups. Groups which were supplemented with IKP 23-PM, IKP 111-PM and IKP 333-PM gained highest mean body weight (1640±48.1), (1608±59.7.), (1590±49.0) g respectively. These groups had significantly higher (P˂ 0.05) mean body weight (g) in all experimental groups. PM of probiotics

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Discussionwas more successful as compared to TM in order to achieved maximum weight gain in broiler at the end of trial. Improvement in weight gain of broiler was observed with probiotics as compared with negative control group (Awad et al. 2009; Asghar et al. 2016). Similar result was reported in past by (Bai et al. 2013) that Lactobacillus fermentum was very effective supplementation in increasing weight gain of broiler. Many evidence has been reported that probiotics promote the growth of broiler chicken (Mountzouris et al. 2010; Yang et al. 2012; Ghahri et al. 2013)

Probiotics play a vital role in the development of immune response in poultry intestine. Probiotics are involved in the production of natural antibodies (Haghighi et al. 2006). Probiotics have positive result on humoral and cellular mediated immunity in chickens (Koenen et al. 2004). Probiotics result in an enhancement of broiler immune response (Koenen et al. 2004; Kabir 2009; Asghar et al. 2016). Lactobacillus salivarius is the member of chicken intestinal microflora and induce cytokine profiles in vitro mononuclear cells (Brisbin et al. 2011). Lactobacillus fermentum is very effective in developing immune status of broiler (Bai et al. 2013). In present study immune response of broiler was assessed with elevated antibody titer against NDV and AIV H9 virus vaccine. Probiotics have good effect on immune response of broiler. Probiotics treated groups had higher Avian influenza HI antibody titer as compared to untreated group. Antibody response to NDV was improved in probiotic treated groups in comparison with those not received probiotics. In past, similar studies were also carried out by (Ghafoor et al. 2005; Talebi et al. 2008; Asghar et al. 2016). Immuno-modulatory effects of probiotics against NDV vaccine in different poultry groups presented in table 4.16. Those groups which were supplemented with probiotics at day 01 to 35 showed high GMT titer against NDV in the end of trial. GMT titer of groups fed with IKP 23-PM, IKP111-PM, IKP333-PM and Protexin were 112, 108, 98.5, and 96 respectively. The highest GMT titer was recorded by IKP 23-PM among all experimental groups at day 35. These results are in agreement with (Ghahri et al. 2013). The antibody titers against NDV were increased at day 28 and 35 after probiotics supplementation in broiler as compared to control group.

Immuno-modulatory effects of probiotics against AIV H9 vaccine in different poultry groups are presented in table 4.17. Similar type of trend was observed in antibody titer between groups against AIV H9 and NDV. Poultry groups which were supplemented with IKP23-PM, IKP11-PM, IKP333-PM and Protexin had higher antibody titers 110, 122, 103, 102 respectively against AIV H9 at day 35 as compared to negative control group, positive control group and antibiotic supplemented group. Similar trend of antibody titer against AIV H9 was reported in broiler fed with probiotics by (Ghafoor et al. 2005; Talebi et al. 2008; Asghar et al. 2016). Antibody titers were higher in those poultry groups which were administrated with probiotics before challenge bacteria. PM of probiotics was better than TM in terms of immune response in broiler.

Gut function play an important role in birds health and gut function is directly influenced by intestinal morphology (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 GIT and reduced gut performance (Xu et al. 2003). In the present study, supplementation of Lactobacillus fermentum (IKP 23), Lactobacillus fermentum (IKP 111) and Lactobacillus 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. Our results are in accordance with many of the previous studies (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 (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 provide information that Lactobacillus fermentum (IKP 23), Lactobacillus fermentum (IKP 111) and Lactobacillus salivarius (IKP 333) have potential of improving gastrointestinal morphology in broiler chicken as growth promotor. Present study also claims 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 8.10um was measured in IKP 111-PM in all experimental groups and lowest ratio 6.09um was measured in antibiotic supplemented

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Discussiongroup. Significant difference (p˂0.05) was observed in villus height and crypt depth ratio in probiotics group (IKP 23-PM, IKP111-PM and IKP 333-PM and other experimental groups. In jejunum the highest villus height and crypt depth ratio 11.88 was measured in IKP 23-PM and lowest ratio 6.04um was measured in antibiotic supplemented group among all groups. In ileum the highest villus height crypt depth ratio 8.53um was measured in IKP111-PM and significantly different (P˂ 0.05) from negative group, positive group and antibiotic group. The lowest villus height and crypt depth ratio 6.09um was measured in ileum in negative group. It was concluded, that IKP 23, IKP111 and IKP 333 in PM and TM 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 (Kahn 2005; Rutgers 2005; 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 difference 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 and 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 capacity which was supplemented with probiotics at day 01 to 35 (PM) as compared to those groups in which probiotics were supplemented at day 07 to 35 (TM). The highest concentration of D-xylose 58.06 mg was measured in plasma of broiler group supplemented with IKP 23-PM 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 groups both in PM and TM in comparison with negative control group, positive group and antibiotic supplemented group.

5.1. Conclusions

It is concluded that L. fermentum IKP 23, L. fermentum IKP 111 and L. salivarius IKP 333 may be used as potential probiotics in poultry to control and mitigate Salmonellae as an alternative of antibiotics. Study will help to cope with insufficiency of indigenous anti- Salmonella probiotics for poultry. L. fermentum IKP 23, L. fermentum IKP 111 and L. salivarius IKP 333 may also increase weight gain, enhance nutrients absorption and antibody response against NDV and AIV H9 virus vaccine in broiler chick.

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CHAPTER 6SUMMARY

Poultry provides cheaper source of protein and employment for more than 1.5 million people. It is facing several problems due to microbial diseases. Salmonella is one of the major pathogen of poultry. Its transmission to human through food chain is one of the major public health issues. Use of antibiotics to control Salmonella in poultry has resulted in emergence of antibiotic resistant strains. So, it is dire need of time to develop and explore alternate ways to control transmission of Salmonella from poultry to human beings. Lactobacilli of indigenous poultry origin have anti-Salmonella activity and potential to reduce Salmonella in broiler chicks. Lactobacilli (n=84) were isolated from the droppings, ileum and caecum of back-yard poultry birds on De Man Rogosa and Sharpe medium. Lactobacilli isolates were screened and selected on the basis of their antimicrobial activity (6.33±0.57-20.33±1.15 mm) against Salmonella Enteritidis by well diffusion assay. In vitro characterization it was revealed that IKP23, IKP 111 and IKP 333 had pH tolerance, survival and growth in bile salts, no acquired antibiotic resistance, good auto-aggregation and co-aggregation capacity. Therefore, these three isolates were selected as potential probiotics. IKP23, IKP 111 and IKP 333 were identified as L. fermentum, L. fermentum and L. salivarius, respectively on the basis of homology of their 16S rRNA sequences. Selected isolates (IKP23, IKP 111 and IKP 333) were evaluated in vivo. In vivo characterization, lactobacilli were screened for lactobacilli count in dropping, reduction of Salmonella count in dropping, increased weight gain, immuno-modulatory effects, better intestinal morphological parameters (villi height and villus/crypt depth ratio) and improved D -xylose absorption capacity of broiler. Lactobacilli counts were significantly higher (P˂ 0.05) in groups administrated with IKP 23-PM, IKP 111-PM and IKP 333-PM (6.19±0.11, 6.83±0.22, 6.30±0.41 respectively) prior to Salmonella challenge (PM). Salmonella count was significantly increased (P˂ 0.05) in positive control group as compared to groups administrated with IKP 23, IKP 111 and IKP 333 (PM and TM), Protexin and antibiotic supplemented groups. Coliform counts were significantly low (P˂ 0.05) in groups administrated with IKP 23-PM, IKP 111-PM and IKP 333-PM (3.09±0.63, 3.21±0.90, 3.18±0.41 respectively) prior to Salmonella challenge. Poultry groups which were supplemented (day 01 to 35) with selected probiotics in PM and Protexin had higher antibody titers 112, 108, 98.5, 96 respectively against NDV vaccine at day 35 as compared to negative control group, positive control group and antibiotic supplemented group. Poultry groups which were supplemented (day 01 to 35) with selected probiotics in PM and Protexin had higher antibody titers 110, 122, 103, 102 respectively against AIV H9 vaccine at day 35 as compared to negative control group, positive control group and antibiotic supplemented group. Immuno-modulatory effects of probiotics were higher in PM as compared to TM. Weight gain of broilers were significantly higher (P˂ 0.05) at day 35 in groups administrated with IKP 23-PM, IKP 111-PM and IKP 333-PM (1640±48.1, 1608±59.7, 1590±49.0, respectively) prior to Salmonella challenge (PM) as compared to groups administrated with IKP 23, IKP 111 and IKP 333 (1569±45.1, 1515±47.8, 1530±51.7 respectively) post Salmonella challenge (TM). Villus height crypt depth ratio in duodenum was significantly higher (P˂ 0.05) in groups administrated with IKP 23-PM, IKP 111-PM and IKP 333-PM (8.03 um, 8.10 um, 8.07 respectively) prior to Salmonella challenge (PM). In jejunum villus height crypt depth ratio of broiler challenged with Salmonella significantly increased (P˂ 0.05) in selected probiotics IKP 23-PM, IKP 111-PM groups (PM) and IKP 333-TM groups as compared to all experimental groups. Villus height crypt depth ratio of broiler challenged with Salmonella significantly increased (P˂ 0.05) in selected probiotics IKP 111-PM, IKP 333-PM groups (PM) and IKP 23-TM groups as compared to other experimental groups. D-xylose concentration was higher but non-significantly different (P˂ 0.05) in groups administrated with IKP 23-PM, IKP 111-PM and IKP 333-PM (58.06 mg, 52.75 mg, 53.09 mg, respectively) and groups administrated with IKP 23-TM, IKP 111-TM and IKP 333-TM (48.81 mg, 46.71 mg, 48.02, respectively). These isolates (IKP 23, IKP 111 and IKP 333) may be used for the commercial production of poultry probiotic products.

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Annexure

Annexure 1: Poultry dropping Samples collected from different areas of Punjab

Sample Source Location Coordinates/LatitudeLongitude

PDSO1 Dropping Lyton road Lahore 31.5543/74.3112

PDSO2 Dropping Multan road Lahore 31.464/74.2264

PDSO3 Dropping Wagah border Lahore 31.6047/74.5741

PDSO4 Dropping Harbans-pura Lahore 31.5745/74.4247

PDSO5 Dropping Kala shah Kaku Sheikhupura 31.725/74.2677

PDSO6 Dropping Muriddke Sheikhupura 31.8094/74.2534

PDSO7 Dropping Sadhoke Gujranwala 31.9064/74.237

PDSO8 Dropping Kamoke Gujranwala 31.9723/74.2192

PDSO9 Dropping MorEmanabad Gujranwala 32.0539/ 74.2091

PDS10 Dropping Shahdra Lahore 31.6277/74.2825

PDS11 Dropping Narang Mandi 31.9014 /74.517

PDS12 Dropping Narowal 32.1071/74.8685

PDS13 Dropping Raiwind 31.2355/74.2171

PDS14 Dropping Changa Manga 31.0838/73.9944

PDS15 Dropping Pattoki 31.0838/73.9944

PDS16 Dropping Phool nagar 31.2057/73.937

PDS17 Dropping Kasur 31.1176/74.45

PDS18 Dropping Hafizabad 32.068/73.6844

PDS19 Dropping Jaranwala 31.3453/73.4298

PDS20 Dropping FaisaIKPad 31.4181/73.0776

PDS21 Dropping Sahiwal 30.6612/73.1086

PDS22 Dropping Arifwala 30.2866/73.0582

PDS23 Dropping Jhang 31.2681/72.337

PDS24 Dropping Kabirwala 30.401/71.863

PDS25 Dropping Multan 30.1978/71.4697

PDS26 Dropping Khanewal 30.2998/71.9309

PDS27 Dropping Sheikupura 31.714/73.9843

PDS28 Dropping Gujrat 32.5738/74.0789

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Annexure

PDS29 Dropping Kharian 32.807/73.8944

PDS30 Dropping Jehlum 32.9335/73.7207

PDS31 Dropping Rawalpindi 33.6058/73.0437

PDS32 Dropping Rawat 33.4951/73.1969

PDS33 Dropping Okara 30.8014/73.4483

PDS34 Dropping Mandi Bahudin 32.5833/73.5

PDS35 Dropping Malakwal 32.5507057/73.208709

PDS36 Dropping Bhera 32.4792/72.9114

PDS37 Dropping Phalia 32.4327/73.577

PDS38 Dropping Kallar kehar 32.7833 /72.7

PDS39 Dropping Kot Momin 32.1883/73.0286

PDS40 Dropping Chakwal 32.9303/72.8556

PDS41 Dropping Sargodha 32.0791/72.6718

PDS42 Dropping Mianwali 32.5833/71.55

PDS43 Dropping Khushab 32.2955/72.3488

PDS44 Dropping Sukheke 31.8602/73.5064

PDS45 Dropping Rahim Yar khan 28.4164/70.2998

PDS46 Dropping Kot Radha kishan 31.171/74.1063

PDS47 Dropping Sangla Hill 31.7167/73.3833

PDS48 Dropping Nankana Sahib 31.4475/73.6972

PDS49 Dropping Feroz Pur Road Lahore 31.4988/74.334

PDS50 Dropping Lyton road Lahore 31.5543/74.3112

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Annexure

Annexure 02: Poultry Cecum Samples collected from different areas of Punjab


PCSO1 Cecum Lyton road Lahore 31.5543/74.3112

PCSO2 Cecum Multan road Lahore 31.464/74.2264

PCSO3 Cecum Wagah border Lahore 31.6047/74.5741

PCSO4 Cecum Harbans pura Lahore 31.5745/74.4247

PCSO5 Cecum Kala shah Kaku Sheikhupura 31.725/74.2677

PCSO6 Cecum Muriddke Sheikhupura 31.8094/74.2534

PCSO7 Cecum Sadhoke Gujranwala 31.9064/74.237

PCSO8 Cecum Kamoke Gujranwala 31.9723/74.2192

PCSO9 Cecum Mor Emanabad Gujranwala 32.0539/74.2091

PCS10 Cecum Shahdra Lahore 31.6277/74.2825

PCS11 Cecum Narang Mandi 31.9014 /74.517

PCS12 Cecum Narowal 32.1071/74.8685

PCS13 Cecum Raiwind 31.2355/74.2171

PCS14 Cecum Changa Manga 31.0838/73.9944

PCS15 Cecum Pattoki 31.0838/73.9944

PCS16 Cecum Phool nagar 31.2057/73.937

PCS17 Cecum Kasur 31.1176/74.45

PCS18 Cecum Hafizabad 32.0682/73.6844

PCS19 Cecum Jaranwala 31.3453/73.4298

PCS20 Cecum FaisaIKPad 31.4181/73.0776

PCS21 Cecum Sahiwal 30.6612/73.1086

PCS22 Cecum Arifwala 30.2866/73.05

PCS23 Cecum Jhang 31.2681/72.337

PCS24 Cecum Kabirwala 30.401/71.863

PCS25 Cecum Multan 30.1978/71.4697

PCS26 Cecum Khanewal 30.2998/71.9309

PCS27 Cecum Sheikupura 31.714/73.9843

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Annexure

PCS28 Cecum Gujrat 32.5738/74.0789

PCS29 Cecum Kharian 32.807/73.8944

PCS30 Cecum Jehlum 32.9335/73.7207

PCS31 Cecum Rawalpindi 33.6058/73.0437

PCS32 Cecum Rawat 33.4951/73.1969

PCS33 Cecum Okara 30.8014/73.4483

PCS34 Cecum Mandi Bahudin 32.5833/73.5

PCS35 Cecum Malakwal 32.5507057/73.208709

PCS36 Cecum Bhera 32.4792/72.9114

PCS37 Cecum Phalia 32.4327/73.577

PCS38 Cecum Kallar kehar 32.7833 /72.7

PCS39 Cecum Kot Momin 32.1883/73.0286

PCS40 Cecum Chakwal 32.9303/72.8556

PCS41 Cecum Sargodha 32.0791/72.6718

PCS42 Cecum Mianwali 32.5833/71.55

PCS43 Cecum Khushab 32.2955/72.3488

PCS44 Cecum Sukheke 31.8602/73.5064

PCS45 Cecum Rahim Yar khan 28.4164/70.2998

PCS46 Cecum Kot Radha kishan 31.171/74.1063

PCS47 Cecum Sangla Hill 31.7167/73.3833

PCS48 Cecum Nankana Sahib 31.4475/73.6972

PCS49 Cecum Feroz Pur Road Lahore 31.4988/74.334

PCS50 Cecum Lyton road Lahore 31.5543/74.3112

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Annexure

Annexure 03: Poultry Ileum Samples collected from different areas of Punjab


PISO1 Ileum Lyton road Lahore 31.5543/74.3112

PISO2 Ileum Multan road Lahore 31.464/74.2264

PISO3 Ileum Wagah border Lahore 31.6047/74.5741

PISO4 Ileum Harbans pura Lahore 31.5745/74.4247

PISO5 Ileum Kala shah Kaku Sheikhupura 31.725/74.2677

PISO6 Ileum Muriddke Sheikhupura 31.8094/74.2534

PISO7 Ileum Sadhoke Gujranwala 31.9064/74.237

PISO8 Ileum Kamoke Gujranwala 31.9723/74.2192

PISO9 Ileum Mor Emanabad Gujranwala 32.0539/ 74.2091

PIS10 Ileum Shahdra Lahore 31.6277/74.2825

PIS11 Ileum Narang Mandi 31.9014 /74.517

PIS12 Ileum Narowal 32.1071/74.8685

PIS13 Ileum Raiwind 31.2355/74.2171

PIS14 Ileum Changa Manga 31.0838/73.9944

PIS15 Ileum Pattoki 31.0838/73.9944

PIS16 Ileum Phool nagar 31.2057/73.937

PIS17 Ileum Kasur 31.1176/74.45

PIS18 Ileum Hafizabad 32.0682/73.6844

PIS19 Ileum Jaranwala 31.3453/73.4298

PIS20 Ileum FaisaIKPad 31.4181/73.0776

PIS21 Ileum Sahiwal 30.6612/73.1086

PIS22 Ileum Arifwala 30.2866/73.0582

PIS23 Ileum Jhang 31.2681/72.337

PIS24 Ileum Kabirwala 30.401/71.863

PIS25 Ileum Multan 30.1978/71.4697

PIS26 Ileum Khanewal 30.2998/71.9309

PIS27 Ileum Sheikupura 31.714/73.9843

93

Annexure

PIS28 Ileum Gujrat 32.5738/74.0789

PIS29 Ileum Kharian 32.807/73.8944

PIS30 Ileum Jehlum 32.9335/73.7207

PIS30 Ileum Rawalpindi 33.6058/73.0437

PIS32 Ileum Rawat 33.4951/73.1969

PIS33 Ileum Okara 30.8014/73.4483

PIS34 Ileum Mandi Bahudin 32.5833/73.5

PIS35 Ileum Malakwal 32.5507057/73.208709

PIS36 Ileum Bhera 32.4792/72.9114

PIS37 Ileum Phalia 32.4327/73.577

PIS38 Ileum Kallar kehar 32.7833 /72.7

PIS39 Ileum Kot Momin 32.1883/73.0286

PIS40 Ileum Chakwal 32.9303/72.8556

PIS41 Ileum Sargodha 32.0791/72.6718

PIS42 Ileum Mianwali 32.5833/71.55

PIS43 Ileum Khushab 32.2955/72.3488

PIS44 Ileum Sukheke 31.8602/73.5064

PIS45 Ileum Rahim Yar khan 28.4164/70.2998

PIS46 Ileum Kot Radha kishan 31.171/74.1063

PIS47 Ileum Sangla Hill 31.7167/73.3833

PIS48 Ileum Nankana Sahib 31.4475/73.6972

PIS49 Ileum Feroz Pur Road Lahore 31.4988/74.334

PIS50 Ileum Lyton road Lahore 31.5543/74.3112

94

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