THESIS Camille Bretz

UNIVERSITY OF AGRICULTURAL SCIENCES AND
VETERINARY MEDICINE
CLUJ-NAPOCA
FACULTY OF VETERINARY MEDICINE
ANIMAL PHYSIOLOGY DEPARTMENT
Camille BRETZ
GRADUATION THESIS
THE INFLUENCE OF A PROBIOTIC PRODUCT WITH

BACILLUS SUBTILIS AND PEDIOCOCCUS
ACIDILACTICI ON HEALTHY DOGS
Coordinators:
Prof. Dr. Laura Cristina ȘTEFĂNUŢ
Lecturer Dr. Andrei Radu SZAKACS
PhD Maria Cătălina MATEI
Cluj Napoca
2019
Camille BRETZ The influence of a probiotic product with Bacillus subtilis and Pediococcus acidilactici on healthy dogs
Acknowledgements
I would like to thank my thesis supervisor, Professor Dr. Cristina Stefanut, who supported me in
this project and who was always present to give me her best advice and enlightenment.
I would like to express my sincere gratitude to Lecturer Dr. Andrei Szakacs for his enthusiasm,
help, and the great role he played in this project.
Many thanks to the following persons for their contribution and wonderful help in this project:
Assistant Dr. Cristian Popovici and Lecturer Dr. Daniela Neagu.
My sincere thanks to the PhD Catalina Matei for her positivity and guidance along this year and
this work.
Many thanks to the PhD Victoria Buza for her assistance in the laboratory.
Thanks to Alexia Pintea for her help and availability during this year.
I would like to acknowledge everyone who played a role in my academic accomplishments and
the graduation committee.
I would like to express my deepest thanks and acknowledgement to my mother and father who
believed in my dreams and supported me with unconditional love. Without them, none of this
could have been possible.
Many thanks to my brother, sisters and their family for their care, support and motivation
throughout the years: Alex, Elo, Emma, Jack, Lou, Pauline, Mathieu, Arthur, Wojtek and
especially to my irreplaceable twin sister Clarence.
Many thanks to my grandparents for their enthusiasm and their life experience.
A great thanks to all my cousins: Chloé, Hugo, Flora, Adrien, Arthur and Victoria, my aunt Tati,
my uncle Jean-Luc who believed in me.
Thanks to my godmother Catherine-Isabelle Gros and her family Yann, Lise and Maud for their
care and trust.
To my love Alvaro that understand and take care of me. Thank you for walking by my side and
never letting me go.
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I would like to thanks Océanne, Jessie, Elea, their dogs and Maestro for their trust and
friendship.
Thanks to Lara my roommate for these 6 years of complicity.
Thanks to Marie, my colleague and dear friend during these years of university.
Thanks to Léa that supports me in the worst and the best moments.
Thanks to Nina for your sunny personality.
Thanks to my very special friend Océanne alias Chipy who always makes my days brighter.
Thanks to Késia who share her amazing fairy word and help me to keep my mind open.
Thanks to my wonderful Helene and destiny that brought us together.
Thanks to Alix for your positivity and your enthusiasm.
Thanks to Ariane who made me discover Cluj and helped me to live my dream.
Thanks to my oldest friend Claire who never let me down.
I am thankful to Geta who always kept her home and heart open.
Thanks to the pizza party team for our amazing diners and nice stories.
I would like to thank the 1st group English line: Marie, Léa, Nina, Georges, Maxence and Hanno
- the best group ever and all amazing memories we created together.
Thank you to all the people who crossed my life from close or far and make me smile and laugh.
Thank you to all the animals, small or big, which never judge you and always make your life
more wonderful.
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THE INFLUENCE OF A PROBIOTIC PRODUCT WITH BACILLUS

SUBTILIS AND PEDIOCOCCUS ACIDILACTICI ON HEALTHY DOGS
Camille BRETZ
Coordinators: Prof. Dr. Cristina STEFANUT, Lecturer Dr. Andrei SZAKACS, PhD
Catalina MATEI
University of Agricultural Sciences and Veterinary Medicine, Faculty of veterinary medicine, Calea
Manastur nr. 3-5, 400372, Cluj Napoca, Romania
camille.bretz@gmail.com
SUMMARY
The study was conducted at the University of Veterinary Medicine in Cluj-Napoca (Romania) in
the animal physiology department, between May 2018 and June 2019.
The aim of the study was to assess the influence of a probiotic product with Bacillus subtilis and
Pediococcus acidilactici on healthy dogs.
The probiotic was administered orally once a day to six healthy adult dogs from 1 year to 7 years
old for a period of 30 days. Before the treatment, we collected all the faeces for five days to perform
coprological examination and fecal digestibility. The dogs entering the study were submitted to a general
clinical examination. At day 1 and day 31/36 of the study, blood samples was collected and hematological
and biochemical parameters were evaluated. During the probiotic treatment, at day 1, day 7, day 15 and
day 30 the dog owners filled the Optidigest sheet (by Purina) to assess the fecal score weekly. Between
day 31 and day 35 of the study, an additional fecal collection was performed for coprological and
digestibility examination. The parameters studied for the digestibility were: dry matter, crude protein,
crude fat, crude cellulose and nitrogen free extract.
The objectives of the research were multiple. First of all, we observed whether the probiotic
influenced the general health of the dogs and changes in clinical parameters. Then, we identified the
dynamics of the hematological and biochemical parameters pre and post administration of the probiotics.
Finally, we measured the variation of the apparent digestibility with the probiotic treatment.
The in vivo study showed the safety of the probiotic product on healthy dogs and the beneficial
effect on the digestibility. In fact, no adverse reactions were observed.
Keys words: Bacillus subtilis, Pediococcus acidilactici, probiotic, digestibility, canine
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TABLE OF CONTENTS
INTRODUCTION ........................................................................................................................................................ 6
I. BIBLIOGRAPHIC PART ......................................................................................................................................... 7
1. PROBIOTICS- GENERALITIES ............................................................................................................................. 7
1.1. PROBIOTICS HISTORY AND IMPORTANCE ....................................................................................... 7
1.2. PROBIOTICS- HEALTH BENEFITS ........................................................................................................ 7
1.3. SAFETY CRITERIA FOR PROBIOTICS .................................................................................................. 8
1.4. PROBIOTICS- MECHANISMS OF ACTION ........................................................................................... 8
1.5. PROBIOTICS USED IN ANIMALS .......................................................................................................... 9
1.6. LEGISLATIVE PROBIOTICS STATUS ................................................................................................. 10
2. GASTROINTESTINAL MICROBIOTA- GENERALITIES ........................................................................... 10
2.1. IMPORTANCE OF THE MICROBIOTA ................................................................................................ 10
2.2. CANINE INTESTINAL MICROBIOTA .................................................................................................. 11
2.3. BACILLUS SPP. AS POTENTIAL PROBIOTIC ..................................................................................... 12
2.3.1 Bacillus species ........................................................................................................................................ 12
2.3.2 Bacillus spp. used as probiotics ................................................................................................................ 13
2.3.3 Bacillus spp. life cycle in gastrointestinal tract ........................................................................................ 14
2.3.4 Life cycle of Bacillus spp. in animal gastrointestinal tract ....................................................................... 15
2.3.5 Bacillus species safety .............................................................................................................................. 15
2.3.6 Bacillus spp. - Mechanism of action......................................................................................................... 16
2.3.7 Bacillus species as commercial products .................................................................................................. 16
2.3.8 Probiotic versus pathogenic Bacillus spp. ................................................................................................ 17
II. EXPERIMENTAL PART ...................................................................................................................................... 18
1. OBJECTIVES ......................................................................................................................................................... 18
2. MATERIAL AND METHODS .............................................................................................................................. 18
2.1. EXPERIMENTAL PROTOCOL............................................................................................................... 18
2.2. PRESENTATION OF CASES .................................................................................................................. 19
2.3. PROBIOTIC DESCRIPTION ................................................................................................................... 21
2.4. CLINICAL INVESTIGATIONS............................................................................................................... 22
2.5. FECAL SCORE ......................................................................................................................................... 22
2.6. PARACLINICAL INVESTIGATIONS .................................................................................................... 23
2.6.1 Hematology .............................................................................................................................................. 23
2.6.2 Biochemistry............................................................................................................................................. 25
2.6.3 Parasitology .............................................................................................................................................. 27
2.6.4 Digestibility .............................................................................................................................................. 28
3. RESULTS AND DISCUSSION ............................................................................................................................. 32
3.1. CLINICAL EXAMINATION ................................................................................................................... 32
3.2. FECAL SCORE ......................................................................................................................................... 33
3.3. HEMATOLOGY ....................................................................................................................................... 38
3.4. BIOCHEMISTRY ..................................................................................................................................... 44
3.5. PARASITOLOGY .................................................................................................................................... 51
3.6. DIGESTIBILITY ...................................................................................................................................... 52
4. CONCLUSIONS ..................................................................................................................................................... 58
BIBLIOGRAPHY ....................................................................................................................................................... 59
ANNEXES .................................................................................................................................................................. 62
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INTRODUCTION
These last decades probiotics have been described in many ways by a large panel of
scientists. In 2014, experts have established a common opinion about probiotics (Bernadeau et
al., 2017). Probiotics are usually described as live microorganisms (bacteria or yeast usually) that
confer a health benefit on the host when they are consumed in adequate quantity. Probiotics can
be integrated in various types of products such as feed/food, therapeutic drugs or feed
supplements (Amraii et al., 2014).
In 1999, in his book, the scientist Michael Gershon described intestine as a « second
brain ». In fact, the gastrointestinal tract is one of the most complex and important organ of the
body. It is representing a considerable ecosystem. A trillion (10^12- 10^14) microorganisms
composed this microbiota. It represents about ten times more than the number of all host cells
(Garcia-Mazcorro & Minamoto, 2013; Gershon M., 1999).
These microorganisms are numerous and specific to each species and even each
individual (Tomar et al., 2015). We count about 500 different species of microorganism in the
gastrointestinal tract (GIT). This complex organ has a great importance and submits many
questions mark (AISA, 2006).
Probiotics related to Bacillus species emerged 15 years ago. Bacillus subtilis, Bacillus
clausii, Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus indicus are the
predominant species (Cutting, 2011).
The aim of our study was to evaluate the influence of a probiotic product with Bacillus
subtilis and Pediococcus acidilactici on healthy dogs.
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I. BIBLIOGRAPHIC PART
1. PROBIOTICS- GENERALITIES
1.1. PROBIOTICS HISTORY AND IMPORTANCE

The use of probiotics in human has been reported hundred years ago (Endres et al.,
2009). They have worldwide utilization and are widely developed in Asia. As an example, the
Japanese fermented soybean dish Natto is famous for its probiotic’s properties. Many more
natural dishes associated with probiotics are found across the world such as TapaiUbi
(Malaysia), Douchi (China), Rabadi (India, Pakistan), Soibum (India), etc... (Elseghabee et al.,
2017)
Nowadays researches are currently performed to produce safer and healthier probiotics
products (Elseghabee et al., 2017).
The probiotic market is divided in five categories: Lactobacillus, Bifidobacterium, spore
formers, yeast and others (Elshaghabee et al., 2017).
The World Gastroenterology Organisation described the most used probiotics as Lactobacillus,
Bifidobacterium but also Saccharomyces cerevisiae yeast and few species of Escherichia coli
and Bacillus (http://www.worldgastroenterology.org/guidelines/global-guidelines/probiotics-
and-prebiotics).
1.2. PROBIOTICS- HEALTH BENEFITS

In 2015 Papadimitriou et al. record in vitro and in vivo assays that have been performed
to prove the diverse beneficial properties of probiotic. Different characteristics of probiotic have
been assessing (Papadimitriou et al., 2015).
The enhancement of intestinal barrier function is one of the properties of probiotics. That
capacity helps in fighting pathogenic microorganisms (Papadimitriou et al., 2015).
The immunomodulation of the probiotics has been performed showing their anti-
inflammatory immune-stimulating properties (Papadimitriou et al., 2015).
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Antimicrobial assays have been evaluated. The studies have shown the importance of
probiotics in production of antimicrobial metabolites and their aggregation with pathogens to
protect against infection (Papadimitriou et al., 2015).
Other assays related to their beneficial role in cardiovascular diseases and cancerous
patients have been conducted (Papadimitriou et al., 2015).
Additional health benefits are carrying by probiotics such as production of vitamins (K
vitamin, B2 & B12 vitamins) and oxalate-degradation preventing or solving kidney stones
(Papadimitriou et al., 2015).
A recent in vivo study shows the beneficial effect of a multi-strains probiotic product
with Lactobacillus spp. and Bifidobacterium spp. on improving canine feeding intake, gaining
weight but also improved the gut microbiota and reinforced the canine immune system (Xu et al.,
2019).
1.3. SAFETY CRITERIA FOR PROBIOTICS
Probiotics must fulfil some safety criteria to be used in animals and human health. They
should be non-toxic, non-pathogenic and be a normal inhabitant of the gastrointestinal tract
(GIT) host. Accurate taxonomic identification is required to avoid the use of unsafe bacteria
(Gaggia et al., 2010).
Other criteria such as antagonism towards pathogenic bacteria and be genetically stable
to avoid antibiotic resistance or other adverse effects. They should show stability during
processing, storage and delivery (Gaggia et al., 2010).
More assays must be performed to confirm the safety of probiotics such as antibiotic resistance,
hemolytic activity, and production of enzymes, toxins or biogenic amines (Gaggia et al., 2010).
1.4. PROBIOTICS- MECHANISMS OF ACTION

Mechanisms developed by probiotics to enhance human and animal health are variable.
Theses mechanisms of action tends to be strain specific (Schmitz & Suchodolski, 2016).
They must survive to stress within the host. In fact, following ingestion probiotics are
confronted to extreme low pH and the activities of digestive enzymes (Schmitz & Suchodolski,
2016).
Probiotics can compete with pathogens by interfering with their adherence to the
intestinal wall or by induction of mucus production (Schmitz & Suchodolski, 2016).
Moreover, probiotics microorganisms can produce antimicrobial substances like fatty
acids, lactic acid or acetic acid (Ryu & Chang, 2013).
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Studies, also shown that some strains of probiotics can absorbed and decreased toxins
produced by some pathogenic bacteria such as Salmonella, Escherichia coli and Clostridium
perfringens (Schmitz & Suchodolski, 2016).
The efficacy of probiotics depends of the dose, time and duration of administration
1.5. PROBIOTICS USED IN ANIMALS

In animals field probiotics have been used in dietary supplement but also as growth
promoters and competitive exclusion agents especially in pigs and chickens (Cutting, 2011). In
his review Cutting (2011), reports the use of probiotics in aquaculture (mainly shrimps) to
enhance the growth and disease-resistance (Cutting, 2011).
In addition, Cutting (2011), named few probiotics products use in veterinary medicine.
Bioplus® 2B- (Bacillus licheniformis and Bacillus subtilis) use in piglets, turkeys and chickens
for fattening. Toyocerin® is another example of probiotics containing B. cereus and use in
calves, poultry, rabbits, swine and in aquaculture. Other products are cited but not approved in
Europe (Cutting, 2011).
To date the European Food Safety Authority (EFSA) has identified four bacterial
strains/products safe and efficient as probiotics in dogs. These microbial organisms are two
Enterococcus faecium strains (approved in 2004), Lactobacillus acidophilus DSM 13241 25
(approved in 2004) and Bifidobacterium sp. animalis (approved in 2012)
(http://www.efsa.europa.eu/)
Other probiotics exist as nutritional supplements in dogs in Europe, but they haven't been
tested or approved by authorities.
Schmitz and Suchodolski (2016) listed others bacterial strains available for dogs over the
counter. They contain Lactobacilli strains, Bifidobacteria, Bacillus subtilis, Bacillus coagulans,
Saccharomyces cerevisiae or fungi such as Aspergillus oryzae. Nevertheless, their efficacy and
safety are not recorded (Schmitz & Suchodolski, 2016).
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1.6. LEGISLATIVE PROBIOTICS STATUS
The clinical benefits observed in human and animals have been proved in many
researches. Nevertheless, the regulatory system is unclear and the status of probiotics on
international level is not well established. The safety and wellbeing of consumers must be
measured with caution. The legislation across the world is different concerning probiotics. In
Europe probiotics has to follow the Food products Directive for the regulation of food labelling
(Elshaghabee et al., 2017).
Probiotics have shown interesting health benefits both for human and animals. However,
their application in immune compromised patients should be carefully applied. Indeed,
bacteraemia has been revealed in immune-compromised patient treated with spore formers and
other probiotics. Critically ill, new-born and old population is more at risk to suffer opportunistic
pathogen bacteria (Elshaghabee et al., 2017).
2. GASTROINTESTINAL MICROBIOTA- GENERALITIES
2.1. IMPORTANCE OF THE MICROBIOTA

The microbiota is the newest word defining all living microorganisms (bacteria, fungi,
protozoa, viruses) that inhabit the intestine. The term « microflora » was used in the old literature
(Suchodolski, 2016).
The most common commensal groups of microorganisms found in intestine are
Bifidobacterium, Bacteroides, Clostridium, Escherichia, Eubacterium, Enterococcus,
Streptococcus and Lactobacillus. However, the ratio between species and the number of
microorganisms vary greatly along the GIT and the host (Tomar et al., 2015).
The link between the intestinal microbiota and the health of the host has involved
researches to understand the importance of a balanced microbial ecosystem (Suchodolski, 2016).
There is an interaction between intestinal bacteria and the host immune system. Either by
direct contact between the bacteria and the innate immune system (toll-like receptors, NOD 2
receptors) either indirect through microbial metabolites (O’hara & Shanahan, 2006).
The intestinal microbiota helps in many functions of the body. It has biochemical and
physiological functions as nutrients fermentation, provides nutritional benefits, protective role
10
against pathogenic bacteria and stimulates the immune system (AISA, 2006). The presence of
bacteria confers also an important role in intestinal structure (Suchodolski, 2016).
Resident bacteria of the gastrointestinal tract play a role in the stimulation of mucosal
mechanisms of defense and insure immune response (O’hara & Shanahan, 2006).
Maintaining a healthy microbiota is the key to fight against intestinal pathogenic
infections. Studies have put in evidence that age, diet, host phylogeny, gut morphology and
health/pathological status influence microbial composition of the gastrointestinal tract (Ley et al.,
2008).
In monogastric species such as pig, chicken, rabbit or man the microbiota is essentially
composed of Bacteroides, Clostridium, Bifidobacterium, Eubacterium, Lactobacillus,
Enterobacteriaceae, Streptococcus, Fusobacterium, Peptostreptococcus and Propionibacterium.
In polygastric animals such as cow, sheep, lamb we find mainly Fibrobacter, Ruminococcus,
Butyrivibrio, Bacteroides, Prevotella, Selenomonas, Streptoccus, Lactobacillus and
Megasphaera (Gaggia et al., 2010).
An imbalance in microbial composition called dysbiosis can lead to a variation in
immunity and dysfunction of the gastrointestinal tract and much more. Changes in gut
microbiota can trigger pathological inflammation of the gut and results in increase of
pathological bacteria and decrease in health promoting-bacteria. For example, in stress
conditions the beneficial bacteria such as Lactobacilli and Bifidobacteria tend to decrease
Current researches are performed to better understand the relationship between a
dysbiosis and gastrointestinal disorders such as inflammatory bowel disease (IBD),
granulomatous colitis and irritable bowel syndrome (IBS) (Suchodolski, 2016).
2.2. CANINE INTESTINAL MICROBIOTA
In their recent review Schmitz and Suchodolski (2016) summarize knowledges about dog
intestinal microbiota and the interest of using probiotics. To a better understanding of intestinal
microbiota, mucosal samples, intestinal content of different segments of the GIT and fecal
samples have been made in healthy dogs. The main species found in stomach are Helicobacter
and Lactobacilli spp. The duodenum is composed by Firmicutes (46%), Proteobacteria (27%),
Lactobacilli (22%), Bacteroides (12%), Spirochaetes (10%), Fusobacterium (4%) and
Actinobacteria (1%). The jejunal microbiota is dominated by Proteobacteria (46%) and follows
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by Firmicutes, Actinobacteria, Spirochaetes, Lactobacilli, Bacteroidetes and Fusobacteria.

Finally, the ileum is composed by Fusobacteria, Firmicutes and Bacteroides (Schmitz &
Suchodolski, 2016).
Studies have been made to characterize the modification in microorganism’s composition
and the related gastrointestinal diseases in dogs. However, these relationships need to be
interpreted with caution. Fecal samples have been evaluated in acute diarrheic dogs. A change in
microbial composition is noticed with increase in Clostridium spp., E coli, Lactobacillus and
Enterococcus spp. And a decrease in the main normal bacterial groups like Faecalibacterium,
Ruminococcus and Blautia spp. In chronic diarrheic dogs, an increase in Bacteroides spp. is
present (Schmitz & Suchodolski, 2016).
Dogs presenting inflammatory bowel disease (IBD) and submit to duodenal brush
samples show a decrease in bacterial richness species and a higher amount of
Enterobacteriaceae. The analysis of duodenal mucosal biopsies revealed a higher proportion of
Proteobacteria and a decrease in Clostridia (Schmitz & Suchodolski, 2016).
The dogs suffering from intestinal diseases highlight a dysbiosis with a poor
microorganism’s diversity. Microbiota changes could be a cause or consequence of
dysfunctional GIT (Schmitz & Suchodolski, 2016).
2.3. BACILLUS SPP. AS POTENTIAL PROBIOTIC
2.3.1 Bacillus species

The Bacillus phylogeny belongs to the domain: bacteria, phylum Firmicutes: Bacillus
and Lactobacillus spp., class: bacilli, order: bacillales, family: bacillaceae, genus: Bacillus. The
genus is subdivided in 318 species (Elshaghabee et al., 2017).
Bacillus species are Gram-positive, rod shaped and spore-forming microorganisms. It is
defined as an aerobic or facultative anaerobic bacterium (Elshaghabee et al., 2017).
Bacillus is considered soil organisms but can be also found in air, water, vegetables, and food.
Additional studies have shown the presence of Bacillus in insects and animals GIT (Hong et al.,
2008).
Some Bacillus strains present two forms: vegetative and endospore. Both forms have
interests used in probiotics. They show different qualities and properties that could be used
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Spores depending of the species are spherical or ellipsoidal. Their length is between 0,8 –
1,4 µm. Spores are usually moderately hydrophobic. Spores are surrounded by a peptidoglycan
layer (cortex) and one or several layers of proteinaceous material (coat). These morphological
properties help the spore to ensure heat resistance up to 80-85 °C. It also protects spores against
enzymes or others environmental changes. In suitable conditions spores will germinate and result
in vegetative cell growth (Cutting, 2011).
Bacillus spore forming bacteria have the advantage to be more stable and can be store at
ambient temperature without suffering degradation. Moreover, the entire dose of probiotic based
ingested will reach the GIT and not be deteriorate before. This confers much more power to
spore forming probiotics (Cutting, 2011).
Due to its high stability Bacillus is more suitable for health promoting formulation
2.3.2 Bacillus spp. used as probiotics

In rabbit, studies showed the beneficial role of Bacillus subtilis in development of the
gut-associated lymphoid tissue (GALT) (Hong et al., 2008).
In their in vitro study Hong et al. (2008) have identified the various properties of Bacillus subtilis
such as anaerobic growth, biofilm formation, adhesion, antimicrobial activity (Hong et al.,
2008).
Bacillus coagulans show resistance to heat, low pH and bile salts permitting a better
chance to survive in the GIT (Hong et al., 2008).
A study shown beneficial effect of oral administration of Bacillus coagulans. It
ameliorates stool frequency, intestinal environment and skin condition (Hong et al., 2008).
Felix et al. (2010) studied the effect of Bacillus subtilis probiotic product on healthy
dogs. The results concerning the fecal characteristics seems positive (Felix et al., 2010).
Paap et al. (2016), seems also to obtain positive results concerning the effect of Bacillus
subtilis on dogs with chronic diarrhoea. Some fecal characteristics and the coat condition are
improved (Paap et al., 2016).
Spore forming bacteria play a considerable role in stimulating the immune system.
Bacillus spores have the property to germinate in jejunum and ileum (Hong et al., 2008; Biourge
et al., 1998).
Bacillus spores are used in a large amount of probiotic preparations. For example, the
famous Japanese product Natto contains Bacillus subtilis spores and show some beneficial effect
on health specially in cardiovascular field (Hong et al., 2009).
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Enterogermina® product contains Bacillus clausii spores and it is used in diarrhoeal

infection patients (Hong et al., 2009).
These followings bacteria have been used as probiotics: B. clausii, B. subtilis, B. pumilus,
B. coagulans and B. cereus. However, some of these bacteria raise the question of safety like B.
cereus known as pathogenic. Some strains of B. cereus are considered as a food poisoning
organism producing enterotoxins. Some other strains are used as probiotics in animal and human
(Hong et al., 2008).
In addition, bioactive secondary metabolites are produced by the vegetative cells and can
regulate growth processes or act as antimicrobial. The molecules produced are bacteriocins,
bacteriocin-like inhibitory substances or antibiotics (surfactin, iturins, bacilysin) (Bernardeau et
al., 2017).
Others secondary metabolites have been demonstrated to help in digestion like protein-
degrading enzymes (Bernardeau et al., 2017).
2.3.3 Bacillus spp. life cycle in gastrointestinal tract

Once the spores have been ingested, they need to resist the stomach barriers but in the
other hand needs to be metabolically activated in the intestine. That's why it is of interest to
understand the life cycle of Bacillus in the gut. In their review Bernardeau et al. (2017) are
studying the main characteristics of beneficial Bacillus spores and vegetative cells (Bernardeau
et al., 2017).
Bacillus spores once ingested are submitted to the gastrointestinal transit (enzymes, bile
salts, peristaltic movements). They have a high tolerance for gastric acidity due to their structure.
In the intestine, spores will germinate in presence of nutrients, lysozyme, salts, high pressures
and cationic surfactants (dodecylamine). These agents will bind to the spore’s receptors and will
release a concentration of dipicolinic acid (DPA). This mechanism will trigger the entrance of
water in the spore core. Subsequently rehydrated spores will start their germination. These
processes lead to the presence of live Bacillus bacteria in the gut. Vegetative cells will proliferate
and replicate. Finally, Bacillus will be present in stool and release in the environment
(Bernardeau et al., 2017).
The rate, the location and velocity of germination are important factors to take in
consideration as potential probiotics (Bernardeau et al., 2017).
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2.3.4 Life cycle of Bacillus spp. in animal gastrointestinal tract

The host species play a role in Bacillus life cycle. Moreover, the ratio between vegetative
cells and spores varies according to different parameters such as gut sections, transit time, gut
motility, microbiota, environmental conditions and nature of feed (Bernardeau et al., 2017).
Studies performed on mice shown the conditions encountered in the upper digestive tract are
necessary to trigger spore germination. Indeed, the low pH and the presence of nutrients in the
stomach are factors of activation. If the spores are administrated directly in stomach or if the
spores haven't been activated before entering in the large intestine they will never germinate
(Bernardeau et al., 2017).
The persistence of microorganisms in GIT varies between species and strains. This fact is
explained by differences of metabolism between strains. Studies in mice have shown B. cereus
more persistent than B. subtilis, B. pumilus and B. clausii (Bernardeau et al., 2017).
Studies shown than in birds, vegetative cells are in every part of the gut while in
mammals they are mainly in distal part of the tract (Bernardeau et al., 2017).
These data should be taken in consideration to assess the probiotic efficacy of Bacillus
strains.
2.3.5 Bacillus species safety

Hence Bacillus is a free-living organism the safety concern has been raised. The
importance to study their phenotype, genotype and life cycle in GIT of human and animals is
primordial (Elshaghabee et al., 2017).
Some Bacillus strains appear to be involved in human diseases. In fact, Bacillus anthracis
and Bacillus cereus are pathogenic. Concerning Bacillus cereus, it seems to be case-by-case
basis and result from opportunistic infections (Cutting, 2011).
Others extensive studies in human and animals have been performed. The safety of
several species has been recorded. B. subtilis var Natto, B. indicus, B. coagulans, B.
licheniformis 2336 show no adverse effects (Cutting, 2011).
Approved products with Bacillus spp. in Europe and USA are few. Authors described
incorrectly B. subtilis as GRAS (Generally Regarded As Safe). It is only the enzyme Nattokinase
purified from B. subtilis that present GRAS status. In 2008, B. coagulans was the first strain to
obtain GRAS approval (Cutting, 2011).
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2.3.6 Bacillus spp. - Mechanism of action

Bacillus spores carry capacity to survive to intestinal transit. (Cutting, 2011).
Studies have proved that Bacillus subtilis spores can germinate in the small intestine, grow,
proliferate and finally re-sporulate. Bacillus spores in feces can reach 10^4 spores/gram (Cutting,
2011).
Studies in mice models have shown an increase of immune system due to administration
of germinating spores (Cutting, 2011).
In poultry, administration of Bacillus subtilis had shown the eradication of infective
agents such as Salmonella enterica, Clostridium perfringens and Escherichia coli. Same
principle has been applied in mice and has shown suppression of Citrobacter rodentium a
pathogen involves in traveller's diarrhea syndrome in human (Cutting, 2011).
Others works in rabbit demonstrate the beneficial effect of probiotics mainly Bacillus
subtilis on gut-associated lymphoid tissue (GALT) (Cutting, 2011).
Moreover, works shown that vegetative cells play a greater role in immune response than
spores. Bacillus spp. can have different immune responses according to the strains and the host
and other factors such as the stage of Bacillus spp. (Bernardeau et al., 2017).
By secreting antimicrobials like coagulin, amicoumacin and subtilisin Bacillus species is
beneficial for the health. In fact, this property provides ability for the host to compete against
pathogenic microorganisms (Cutting, 2011).
Furthermore, Bacillus spp. produces enzymes such as amylase, glucoamylase, protease,
pectinase, cellulase. Bacillus spp. produces also vitamins such as riboflavin, cobalamin, inositol
and carotenoids (Elshaghabee et al., 2017).
2.3.7 Bacillus species as commercial products

Elshaghabee et al. (2017) in their review report few examples of probiotic supplements
containing Bacillus spp. available in the global market. In United States we have for example
NutriCommit or GanedenBC®. In Europe we can notice Enterogermina®, Anaban™,
Biosporin®, Calsporin®. This is a list non exhaustive of probiotics present on the market. These
products have been approved by regulatory agencies for human pharmaceutical formulations
16
2.3.8 Probiotic versus pathogenic Bacillus spp.

Researchers using phylogenetic and sequencing techniques have classified Bacillus
species in different groups. The pathogenic, non-pathogenic groups and the ones used in
probiotics have been identified. For example, B. anthracis and B. cereus belong to the group Xth.
B. cereus show pathogenic factors due to the expression of the plcR gene. This gene encodes for
pathogenic extracellular factors (Hbl, Nhe, cytK) and proteins (Elshaghabee et al., 2017).
Other associated pathogens in humans related to Bacillus spp. are the following; B.
weihenstephanensis and B. thuringiensis (Elshaghabee et al., 2017).
These bacteria and specially Bacillus cereus produce enterotoxin such as cereulide. Other
toxins such as protective antigen- lethal factor (PA-LF), PA- edema factor (PA-EF), Cry and Cyt
are produce by some pathogenic strains of Bacillus spp. (Elshaghabee et al., 2017).
Knowing that an identification and selection of Bacillus spp. used as probiotic is
necessary to avoid unsafe pharmaceutical products. Advanced molecular techniques such as
pulsed-field gel electrophoresis (PFGE), 16S rDNA sequencing, amplified fragment length
polymorphism (AFLP) and multilocus sequence typing (MLST) are important tools in the
characterization of Bacillus spp. probiotics to obtain a complete sequencing genome
17
II. EXPERIMENTAL PART
1. OBJECTIVES
Our study had the following specific objectives:

1. Evaluation of changes in clinical parameters.
2. The dynamics of hematological and biochemical parameters of the health state.
3. Influence on digestibility.
2. MATERIAL AND METHODS
The study was conducted between May 2018 and June 2019 at the University of
Veterinary Medicine in Cluj-Napoca (Romania). The investigations were performed in four
departments of the university: Animal physiology, Nutrition, Internal medicine and Parasitology
departments.
2.1. EXPERIMENTAL PROTOCOL
A total of six adults dogs aged between 1 and 7 years old entered in the study from 10th of
April till the 20th of May. The probiotic product composed of Bacillus subtilis and Pediococcus
acidilactici is given to the six dogs once a day with the meal during thirty days.
The previous figure (figure 2.1) summarized the different steps of the study. The
probiotic product is administered to the 6 dogs for 30 days. Day 1 is the first day of probiotic
administration and day 30 is the last day of probiotics treatment. Between day -5 and day 0 all
the feces are collected for digestibility examination and coprological examination. Day 1 of the
study a general clinical examination of the dogs is performed, three blood sampling at 6 hours
interval are performed (7 am, 1 pm, 7pm). A meal and the probiotic product are given to all dogs
after the first blood sampling. The probiotic treatment is given once a day with the meal during
30 days to all the six dogs. Day 31 of the study three blood samplings at 6 hours interval are
18
performed on Khalac, Maestro and Halva. Between day 31 and day 35 all feces of all dogs are
collected for parasitological examination and digestibility examination. Day 36 one blood
sampling is performed on Luna, Nessie and Holiday.
ACTIONS
Fecal collection for Probiotics treatment Fceal collection for digestibility
Clinical examination
digestibility & Optidigest sheet &
1st day probiotic
Blood sampling
Blood sampling
START
Coprology examination Coprology examination
END
Luna, Nessie,
sampling
K+M+H
holiday
Blood
-5 -3 0 1 30 31 35 36
DAYS
Figure 2.1 - Chronological steps of the study
2.2. PRESENTATION OF CASES
The study is based on real clinical cases. They are 6 adult’s dogs (figure 2.2) from
different age, sex, breed and diet. These dogs are healthy and are not under any medical
treatments. Each case with its individual characteristics is presented below.
Inclusion criteria for the healthy dogs are: without gastro-enterological manifestations
(diarrhea, vomiting), without antibiotic treatment in the last 6 months, clinically healthy, one
meal/day.
Exclusion criteria for this category of dogs are: gastro-enterological manifestations
(diarrhea, vomiting), antibiotic treatment in the last 6 months.
19
Figure 2.2 – The dogs included in the study
CASE 1: Khalac
Khalac is a 4 years old castrated male Czechoslovakian wolfdog. He is a healthy dog
weighing 40 kg. He has its antirabic vaccination and no specific external or internal treatment
against parasites. He is fed once a day with raw diet mainly composed of 750 g raw beef, raw
chicken, raw fish, bones and 200 g vegetables and fruits and some complementary minerals like
salmon oil and kefir.
CASE 2: Maestro
Maestro is 3 years old castrated male Swiss White Shepherd. He is a healthy dog
weighing 33 kg. He has its Distemper, Hepatitis, Parvovirus, Parainfluenza and antirabic
vaccination (DHPPi + R). For internal parasites he is treated every 3 months with Drontal Plus
and for external parasites is taking Bravecto every 3 months. He is fed with 290g of Acana
prairie poultry twice a day.
CASE 3: Luna
Luna is a 3 years old intact female Border collie. She is a healthy dog weighing 22 kg.
She has its DHPPi and antirabic vaccination. For external parasites she is taking Bravecto every
6 months. She is fed with 250g Purizon Single Meat Lamb once a day.
CASE 4: Nessie
Nessie is a 1-year old intact female Shetland shepherd dog. She is a healthy dog weighing 10 kg.
She has its DHPPi and antirabic vaccination. For external parasites she is taking Bravecto every
6 months. She is fed with 130g Purizon Single Meat Lamb once a day.
20
CASE 5: Holiday
Holiday is a 7 years old intact female Shetland shepherd dog. She is a healthy dog weighing 7
kg. She has its DHPPi and antirabic vaccination. For external parasites she is taking Bravecto
every 6 months. She is fed with 100g Purizon Single Meat Lamb once a day.
CASE 6: Halva
Halva is a 1,8-year-old intact female mixed dog breed. She is a healthy dog weighing 15 kg. She
has her antirabic vaccination and no specific external parasites treatment. For internal parasites
she is treated regularly with Drontal Plus. She is fed with 250g Purina Vitafit once a day.
2.3. PROBIOTIC DESCRIPTION

The probiotic product tested (figure 2.3) is composed of two strains of bacteria: Bacillus
subtilis HU58 and Pediococcus acidilactici. The probiotic product is presented as capsules.
During thirty days one capsule per day is given to each dog with the meal.
Figure 2.3 – The tested probiotic
Bacillus subtilis is already used in different probiotic products and researches

demonstrate its efficacy.
Pediococcus acidilactici is a Gram-positive coccus that has also emerged as a potential
probiotic. According to studies, it has shown promising results (Herstad et al., 2010; Joysowal et
al., 2018).
Moreover, an addition of liver powder is used to improve the taste of the probiotic
product and facilitates its taking.
21
2.4. CLINICAL INVESTIGATIONS

To ensure that the dogs entering the study are healthy we performed individual clinical
investigations. A general clinical examination and clinical parameters are performed on the six
dogs.
For these investigations we based the normal range values for dogs according to the
Merck Veterinary Manual (2016). The normal range value for the rectal temperature is between
37,9-39,9ºC. The normal range value for the resting heart rate is between 70-120 beats per
minute. The normal range value for the resting respiratory rate for dogs is between 18-34 breaths
per minute (Merck et al., 2016).
2.5. FECAL SCORE
During the probiotic’s treatment, the dog’s owners are asked to fulfill the Optidigest
clinical sheet made by Purina (Figure 2.4) (https://www.purina.ro/produse/proplan-optidigest).
This clinical sheet is divided in three parts; stool consistency, stool volume and smell,
Gut rambling and flatulence.
The stool consistency part is assessing the stool of the dog during one full day using the
fecal score scale. The scale is described as follow 1: very hard and dry, 2: firm but not hard, 3:
Log-like, 4: very moist, 5: very moist but has distinct shape, 6: has textured but no defined
shape, 7: watery no texture, flat.
The stool volume and smell are also recorded during a full day. The volume is graded as
1: small, 2: normal, 3: large, 4: very large. The smell is recorded as 1: very little odor, 2: low
odor, 3: moderately unpleasant smell, 4: unpleasant smell, 5: very unpleasant smell.
The gut rambling and the flatulence is evaluated during the day and their presence or
absence is recorded.
All these data have to be filled day 1, day 7, day 15 and day 30 of the study.
22
Figure 2.4 – Optidigest clinical sheet made by Purina
2.6. PARACLINICAL INVESTIGATIONS
2.6.1 Hematology
The hematology is performed on the six dogs before and after the period of the
administration of probiotics. The objective is to see if the probiotics administrated to the dogs
influence the blood results. The blood will be collected in hematologic tubes with an
anticoagulant (EDTA). The hematologic samples are analyzed via the device ABACUS JUNIOR
VET (figure 2.5). This apparatus is conceived for veterinary use and is programmed for more
than 30 animal species. The Abacus junior vet determines 18 hematology parameters including
three-parts WBC differential. The analyzer can process 30 blood samples per hour
(http://www.diatron.com/upload/producttab/92/VUM011_AJV_UM_2.76.pdf).
23
Figure 2.5 - Device ABACUS JUNIOR VET
The table presents few of the parameters given by ABACUS JUNIOR VET (table 2.1).
Table 2.1
Hematologic parameters and their methods*
Measure of
No. Parameter The method
Unit
Calculated from RBC and MCV,
1. Hematocrit (HCT) %
HCT= RBC x MCV x 100
2. Hemoglobin (HGB) Measured photometrically g/dl
3. Red blood cell count (RBC) Number of erythrocytes cells/L
Average volume of individual
4. Mean corpuscular volume (MCV) erythrocytes derived from the RBC Fl
histogram
5. Mean corpuscular hemoglobin (MCH) MCH= HGB/RBC Pg
Mean corpuscular hemoglobin concentration
6. MCHC= [HGB / HCT] x 100 g/dl
(MCHC)
7. Total White blood cell count (WBC) Number of leucocytes cells/L
8. Thrombocytes (PCT) PCT= PLT x MPV x 100 %
*- http://www.diatron.com/upload/producttab/92/VUM011_AJV_UM_2.76.pdf
24
Determination of the Leucocytes formula

The leucocyte formula is the differential white cell count. White blood cells are a
heterogeneous group of nucleated cells that can be found in circulation. The white cells types are
granulocytes (neutrophils, eosinophils, basophiles) and agranulocytes (lymphocytes, monocytes)
The leucocyte formula can be given as a percentage or as an absolute value. The absolute
number of each type of WBC is more accurate than its proportion (Meyer & Harvey, 2004).
Principle of the leucocyte’s formula

To determine the differential, a drop of blood is thinly spread over a glass slide, air dried,
and stained with a Diff-Quick Panoptic stain. Two hundred cells are then counted and classified.
The slide must be read entirely (Meyer & Harvey, 2004).
Formula:
 in percentage
Example:
%Neutrophils = [number of neutrophils counted / number of leucocytes counted] x 100
 in absolute value (109/L)

N = [% population / 100] x n
N – the number of each population per liter of blood

n – the total number of leucocytes per liter of blood
2.6.2 Biochemistry
The biochemistry is performed on the six cases before and after the period of
administration of probiotics. The objective is to see if the probiotics administrated to the dogs
influence the biochemistry results.
Vet Scan Chemistry Analyzer, using Comprehensive Tests (figure 2.6 A; B) witch
provide quantitative determinations of: alanine aminotransferase (ALT), albumin (ALB),
alkaline phosphatase (ALP), amylase (AMY) total calcium (CA++), creatinine (CRE), globulin
(GLOB), glucose (GLU), phosphorus (PHOS), potassium (K+), sodium (NA+), total bilirubin
(TBIL), total protein (TP), and urea nitrogen (BUN) in heparinized whole blood, heparinized
plasma, or serum (table 2.2).
25
Table 2.2
Biochemical parameters and their methods
No Parameter Indication Principle of method
ALT catalyzes the transfer of an amino group from L-alanine to
Alanine Liver diseases, including
α-ketoglutarate to form L-glutamate and pyruvate. The
1. aminotransferase viral hepatitis and cirrhosis;
absorbance value is directly linked with the amount of ALT in
(ALT) heart diseases.
the sample
Dye binding technique is used. Bromocresol green (BCG) is bind
2. Albumin Liver and kidney diseases.
to albumin.
Alkaline phosphatase hydrolyses p-NPP in a metal- ion buffer
Liver, bone, parathyroid,
3. Alkaline phosphatase and forms p-nitrophenol and phosphate. Absorbance is
and intestinal diseases.
measured.
The substrate 2-chloro-p-nitrophenyl-α-D-maltotrioside
Kidney and pancreatic (CNPG3) reacts with α-amylase of the sample releasing 2-
4. Amylase
disease. chloro-p-nitrophenol (CNP). The release of CNP creates a
change in color. Absorbance is measured.
Parathyroid, bone and
Calcium in sample binds with arsenazo III to form a calcium-dye
5. Calcium chronic renal disease;
complex. Absorbance is measured.
tetany.
Endogenous creatine is measured in the blank cuvette, which is
6. Creatinine Renal disease. subtracted from the combined endogenous creatine and the
creatine formed from the enzyme reactions in the test cuvette.
Globulin concentration will
increase with dehydration Globulin fraction is generally determined by subtracting the
7. Globulin
and should also increase albumin from the total protein.
with antigenic stimulation.
Diabetes, hyperglycemia,
Quantitative procedures using the enzymes hexokinase and
8. Glucose hypoglycemia, diabetes and
glucose oxidase.
liver disease.
Kidney disease, The method uses sucrose phosphorylase (SP) coupled with the
9. Phosphorus hypoparathyroidism and phosphoglucomutase (PGM) and glucose- 6-phosphate
nutritional disorders. dehydrogenase (G-6-PDH) reactions. Absorbance is measured.
Malnutrition and renal
disease. This electrolyte is
Enzymatic method is based on the activation of pyruvate kinase
10. Potassium used to diagnose the causes
(PK) with potassium. Absorbance is measured.
of vomiting, diarrhea and
cardiac symptoms.
Dehydration, and diabetes.
β-galactosidase is activated by the sodium in the sample. The
This electrolyte is used to
activated enzyme catalyzes the reaction of ο-nitrophenyl-β-D-
11. Sodium diagnose the causes of
galactopyranoside (ONPG) to ο-nitrophenol and galactose.
vomiting, diarrhea and
Absorbance is measured.
cardiac symptoms.
Bilirubin is oxidized by bilirubin oxidase into biliverdin.
12. Total bilirubin Hepatic disorders.
Dehydration, kidney, liver The Cu (II) ions react with peptide bonds between the carbonyl
13. Total protein disease, metabolic and oxygen and amide nitrogen atoms to form a colored Cu-Protein
nutritional disorders. complex (Biuret reaction). Absorbance is measured.
Urease hydrolyses urea into ammonia and carbon dioxide. Upon
combining ammonia with 2-oxoglutarate and reduced
14. Blood Urea Nitrogen Liver and kidney diseases. nicotinamide adenine dinucleotide (NADH), the enzyme
+
glutamate dehydrogenase (GLDH) oxidizes NADH to NAD .
26
A B
Figure 2.6 – A - Vet Scan Chemistry Analyzer; B - Comprehensive Tests
2.6.3 Parasitology
A coprology examination is performed on the six cases before and after the
administration of probiotics. The technique of flotation (Willis method) and sedimentation are
used for the six dogs. The objective is to see if the probiotics effect is not misinterpreted with the
presence of internal parasites.
Flotation procedure
Principle of the method: It exists many techniques of flotation methods, but the
principle is the same. After mixing the fecal sample and the flotation solution, the less dense
material floats on the top. It means common helminth and protozoa eggs and cysts will float
(Zajac & Conboy, 2012).
Material: mortar and pestle, fecal sample, flotation solution, a sieve, a flotation tube, a
slide and a coverslip, a microscope (Zajac & Conboy, 2012).
Method: in a mortar mix 3-5 g of feces with a small amount of flotation solution
(saturated sodium chloride). The mixture is grinded with a pestle. The mixture will be filtered,
and the flotation tube will be fill with the filtered mixture till obtaining a reverse meniscus.
Above the flotation tube a slide is added. The process will take 15-20 minutes. After the flotation
process is done, we add a coverslip on the slide and examine it under the microscope. The
flotation slide should be entirely scanned using the 10x objective of the microscope (Zajac &
Conboy, 2012).
27
Sedimentation procedure
Principle of the method: This procedure is used to isolate eggs of flukes,
acanthocephalans and some other tapeworms and nematodes (Zajac & Conboy, 2012).
Material: mortar and pestle, fecal sample, tap water, a sieve, 2 centrifuge tubes, a
centrifuge machine, a pipette, a slide and a coverslip, a microscope (Zajac & Conboy, 2012).
Method: in a mortar mix 1 g of feces with about 10 mL of tap water. The mixture is
grinded with a pestle. The mixture will be filtered, and the centrifuge tube will be fill with the
filtered mixture till it is one-half full. To balance the centrifuge a second centrifuge tube is filled
with the same amount of tap water. The tubes are placed in the centrifuge. Centrifuge for 3-5
minutes 2000-3000 rpm. With a pipette remove most of the supernatant and resuspend the
sediment. Place one or two drops on a slide and add a coverslip. Examine the slide under the
microscope using the 10x objective of the microscope (Zajac & Conboy, 2012).
2.6.4 Digestibility
Feeds digestibility or digestion efficiency is expressed in relative figures (percent) and
represents the proportion in which the nutritive principles from feeds are absorbed from the
intestines. In other words, digestibility expresses the composition of feeds in nutrients.
The fodder which contains in higher proportion the digestible substances is considered to have a
better nutritive value (Macri & Szakacs, 2014).
I = intake (feed)
E = excretion (fecal) D=I-E
D = digestion
The digestibility of fodders is measured using the digestibility coefficient (D.C.) which
represents the portion of a feed or nutrient of feed which is not recovered in feces, i.e., the
portion which has been absorbed by the animal.
UDC (%) = Utilization Digestibility Coefficient UDC can be:

INTAKE – EXCRETION
1. Apparent - ADC (%) = X 100
INTAKE
I – (E - ”methabolic residues”)
2. Real - UDC (%) = X 100
INTAKE
28
The methods used for digestibility determination are (Macri & Szakacs, 2014):
- experiences on animals (“in vivo”)
- direct methods – with one or two control periods
- indirect methods – experiences with one or two markers (inert substances from the
digestive point of view are used to evaluate digestibility).
- experiences in artificial conditions (“in vitro” – replicating the conditions from the
digestive tract)
A digestibility experience implies specific conditions

- the animal groups have to be made with animals from the same species which feeds with
the investigated fodder. The animals must be uniform in size and grown in similar
conditions, treated for parasites.
- Digestibility with 1 control period (1 C.P.) – when the fodder can be used alone in animal
feeding; with 2 control periods (2 C.P.) – when the fodder cannot ensure the appropriate
ration.
The digestibility experience with 1 control period is composed of 2 steps:
- a training period, in which the analyzed fodder is administered for the animal organism to
accommodate.
The length of the training period has to be equal to the time past from the ingestion of
fodders to the elimination of feces (table 1).
- a control period, in which - feces are collected, weight of intake, excretion and
unconsumed fodder are measured, samples from each are taken for further chemical
composition investigations.
2.6.4.1 Determination of dry matter (DM)
The principle of the method for the determination of the dry matter is based on the
vaporization of water from fodders, in thermo-adjustable air ovens (Macri & Szakacs, 2014).
In order to determine the humidity and the dry matter at the fodders that have a higher content in
water than 15-16%, the next stages are coming up:
29
a) determining relative dry matter (RDM) at 65°C;

b) determining absolute dry matter (ADM) at 105°C;
II. In the case of fodders, that have predictable water content lower than 15-16%, ADM is
determined directly at 105°C.
2.6.4.2 Determination of crude protein (CP)
The current method for the determination of the protein in the laboratory is the Kjeldahl
method.
The principle of the method: determination of N x 6,25
16 g N _ _ _ _ _ _ _ _ _ _ _ _ _ 100 g proteins
1 g N _ _ _ _ _ _ _ _ _ _ _ _ _ 6,25 g proteins
The Kjeldahl method is carried out in three stages:
I. Mineralization
II. Alkalization and distillation of ammonia
III. Titration
(ml H2SO4 sol 0,1 n x F1 – ml NaOH sol. 0,1 n x F2) x 0.0014 x 6,25
Crude protein (%) = X100
m(g)
F1 – Factor of correction for H2SO4
F2 – Factor of conrrection for NaOH
m (g) – Weight of the sample
2.6.4.3 Determination of crude fat (CF)
Crude fat includes all soluble substances in an organic solvent (petroleum ether, acetone,
petrol). In solvent there are extracted besides pure fat also substances like waxes, steroids,
phosphatides, fat-soluble vitamins, carotenoids (Soxhlet extraction) (Macri & Szakacs, 2014).
M1 – M2
Crude fat (%) = X100
m (g)
M1 – weight of the flask with fat
M2 – weight of the empty flask
m (g) – weight of the sample
2.6.4.4 Determination of crude cellulose (CC)
The method (Weende method) of measuring cellulose is based on removing the

associated substances through successive acid and basic hydrolysis, crude cellulose and crude
ash being separated afterwards by calcination at 550oC (Macri & Szakacs, 2014).
M1 – M2 – M3
Crude cellulose (%) = X100
m (g)
m (g) – weight of the sample
M1 - weight of the crucible, filter, cellulose and ash
M2 – weight of the crucible with ash
M3 – weight of paper filter
30
2.6.4.5 Determination of nitrogen free extract (NFE)
The nitrogen free extract is represented mainly by glucids (starch, simple glucids etc.). The
value of NFE, measured in percent is calculated using the following formula (Macri & Szakacs,
2014):
- If N.F.E. is calculated in correlation with total mass of sample:
NFE (%) = DM (%) – (CP % + CF % + CC % + CAsh %),
31
3. RESULTS AND DISCUSSION
3.1. CLINICAL EXAMINATION
A general clinical examination is performed on all the six dogs before to start the
probiotics treatment. The table (table 3.1) shows the values obtained for the main physiological
parameters for the six dogs (Annexes 1 to 6).
Table 3.1
Physiological parameters of the six cases
Respiratory
Cardiac
frequency* A
Temperature* Frequency* Clinically Vomiting/ Antibiotic
Parameter (18-34 meal/
(37,9 – 39,9° C) (70-120 healthy Diarrhoea treatment**
Breaths/ day
Beats/min)
min)
Khalac 38,5 45 89 + - - +
Maestro 38,5 64 43 + - - -
Luna 38,3 84 52 + - - +
Nessie 39,0 90 150 + - - +
Holiday 38,5 88 48 + - - +
Halva 38,7 116 200 + - - +
*- (Merck et al.2016); **- In the last 6 weeks
All the dogs present no significant modifications at general examination and are clinically
healthy. The temperature for all the dogs is normal between 37,9-39,9 °C. The cardiac frequency
is a bit low for Khalac and Maestro without significant importance. For the other dogs the
cardiac frequency is normal between 70-120 beats per minute. All the dogs present a higher
value for the respiratory rate between 43 breaths per minute for Maestro and 200 breaths per
minute for Halva. The normal value at rest for the respiratory frequency is between 18 -34
breaths per minute. The fact that all the dogs have an increased in respiratory rate is not alarming
it could be due to the excitement, stress and/or the too high temperature in the room. However,
the dogs are clinically healthy; they don’t present any signs of illness, diarrhea or vomiting.
Moreover, they are not under any kind of medication especially not antibiotics.
All the dogs are feed once a day except Maestro that is fed twice a day.
We can also notice that Luna present a poor fur coat quality since she gave birth to puppies three
months ago.
32
3.2. FECAL SCORE
During the probiotic’s treatment the dog’s owners are asked to fulfil the Optidigest
clinical sheet made by Purina. The following table (table 3.2) present the results for each dog
(Annexes 7 to 11) (https://www.purina.ro/produse/proplan-optidigest).
Table 3.2.
Optidigest clinical sheet for the dogs
KHALAC Day 1 Day 7 Day 15 Day 30
Stool Consistency 4 3 2 2
Stool Volume 2 2 2 1
Stool Smell 2 2 2 1
Gut rumbling - - - -
Flatulence - - - -
MAESTRO Day 1 Day 7 Day 15 Day 30
Stool Volume 3 3 2+ 2+
Stool Smell 3 3+ 4 3
Flatulence - - - -
LUNA Day 1 Day 7 Day 15 Day 30
Stool Smell 3 3 3 3
Flatulence + + - -
NESSIE Day 1 Day 7 Day 15 Day 30
Stool Smell 3 3 3 3
Flatulence - - - -
HOLIDAY Day 1 Day 7 Day 15 Day 30
Stool Smell 2 2 3 3
Flatulence - - - -
HALVA Day 1 Day 7 Day 15 Day 30
Stool Smell 3 3 3 3
Flatulence - - - -
The results for Khalac shows an increase in stool consistency from day 1 to day 30 of the
treatment. It shows also a slight decrease in stool volume and a slight decrease in unpleasant
smell. From day 1 to day 30 Khalac doesn’t presents any signs of gut rumbling or flatulence.
33
The results for Maestro show a slight decrease in stool consistency from day 1 to day 30
of the treatment. It shows also a slight decrease in stool volume and a variation of stool smell
from 3 to 4 to 3. From day 1 to day 30 Maestro doesn’t presents any signs of gut rumbling or
flatulence.
The results for Luna show an increase in stool consistency from day 1 to day 30 of the
treatment. The stool volume and smell show no variation during the treatment. From day 1 to
day 30 Luna doesn’t presents any signs of gut rumbling. However, Luna presents a diminution of
flatulence in the second half period of the treatment. The owner noticed a good improvement of
the coat for Luna during the treatment.
The results for Nessie show no variation in stool consistency, volume and smell from day
1 to day 30 of the treatment. From day 1 to day 30 Nessie doesn’t presents any signs of gut
rumbling or flatulence.
The results for Holiday show an increase in stool consistency from day 1 to day 30 of the
treatment. It shows no variation in stool volume but a slight increase in unpleasant smell in the
second half of the probiotic treatment. From day 1 to day 30 Holiday doesn’t presents any signs
of gut rumbling or flatulence.
The results for Halva show an increase in stool consistency starting day 7 of the
treatment. It shows also a slight decrease in stool volume but no variation in stool smell. From
day 1 to day 30 Halva doesn’t presents any signs of gut rumbling or flatulence.
In most of the dogs the stool consistency and stool volume slightly improved along the
probiotic treatment. According to the Optidigest sheet results the stool’s smell didn’t improve
with the probiotic’s treatment. The gut rumbling wasn’t present in any of the six dogs before,
during and after treatment. However, the flatulence seems to improve in the only dog that
presented flatulence before the treatment.
In their study, Paap et al. (2016), noticed no changes in dog fecal characteristics
(consistency, volume, color, appearance of blood and mucus) after Bacillus subtilis treatment.
Nevertheless, in their study the fecal odor, flatulence and coat condition was significantly
improved. On the contrary Felix et al. (2010), noticed an improvement in dog fecal consistency
with Bacillus subtilis treatment. The difference between these two studies is that Paap et al.
(2016), tested the probiotic on dogs with chronic diarrhea while Felix et al. (2010) tested the
probiotic on healthy dogs like in our study (Felix et al., 2010; Paap et al., 2016).
The table 3.3 records the food quantities and the feces quantities for each dog five
consecutive days before the probiotic’s treatment (table 3.3; 3.4; figure 3.1).
34
Table 3.3
Food and fecal quantity before probiotics treatment 5 days
Food quantity
Total
and Feces Day -5 Day – 4 Day -3 Day -2 Day -1
Feces
quantity (g)
Khalac
112 62 609 206 126 1115
(4750g/ 5 days
Maestro
329 226 247 143 161 1106
(2175g/ 5 days)
Luna
64 308 120 62 247 801
(1250g/ 5 days)
Nessie
85 117 90 45 105 442
(650g/ 5 days)
Holiday
116 129 75 17 100 437
(500g /5 days)
Halva
65 196 193 223 129 806
(1250g/ 5 days)
Khalac is eating about 4.75 kg of food (750g of raw meat: 450g chicken breast, 200g
sprat, 100g beef and 200g of fruits/vegetables: carrot, salad, radish, apple, banana per day) in
five days and the feces collected in five days are about 1,11 kg.
Maestro is eating about 2,17 Kg of Acana kibbles and its feces weight 1,10 kg for in five
days.
Luna is eating 1,25 kg of Purizon kibbles and its feces weight 0,80 kg for five days.
Nessie is eating 0,65 kg of Purizon kibbles and its feces weight 0,44 kg for five days.
Holiday is eating 0,5 kg of Purizon kibbles and its feces weight 0,44 kg for five days.
Halva is eating 1,25 kg of Purina kibbles and its feces weight 0,80 kg for five days.
The table 3.4 records the food quantities and the feces quantities for each dog five
consecutive days after the probiotic’s treatment.
Table 3.4
Food and fecal quantity after probiotics treatment, 5 days
Food quantity
Total
and Feces Day 31 Day 32 Day 33 Day 34 Day 35
Feces
quantity (g)
Khalac
129 106 162 0 161 558
(4750g/ 5 days
Maestro
127 211 93 467 260 1158
(2900g/ 5 days)
Luna
70 97 70 102 95 434
(1250g/ 5 days)
Nessie
34 86 29 69 24 242
(650g/ 5 days)
Holiday
23 14 55 50 31 173
(500g /5 days)
Halva
116 29 137 83 194 559
(1250g/ 5 days)
35
Khalac’s feces collected in five days are about 0,56 kg.

Maestro is feeding more this time about 2,9 Kg instead of 2,17 Kg of kibbles in five days
and its feces weight 1,16 kg for five days. Luna feces weight 0,43 kg for five days. Nessie feces
weight 0,24 kg for five days. Holiday feces weight 0,17 kg for five days. Halva feces weight
0,56 kg for 5 days.
The Figure 3.1 represents the evolution of the fecal quantity before and after the
probiotic’s treatment for the six cases.
1400
1200
1000
Feces quantity (g)
800
600
400
200
0
Khalac Maestro Luna Nessie Holiday Halva
Day -5 to Day 0 Day 31 to Day 35
Figure 3.1 - Evolution of the fecal quantity before and after the probiotic’s treatment
36
The table 3.5 summarizes the difference of fecal quantity before and after the probiotic’s
treatment.
Table 3.5
Variation in fecal quantity before and after probiotics treatment
Khalac* Maestro** Luna Nessie Holiday Halva
Difference in
faeces -557 + 52 -367 -200 -264 -247
quantity (g)
* To notice day 32 khalac ate about 200g of kibbles (Bosch petfood concept, dog premium plus) that is not part of
his usual diet. We must take in consideration this data on the day 33 for Khalac, the amount of feces is increased.
Moreover day 33 of the study Khalac was put to starve that’s why there are no feces on day 34 of the study.
**For Maestro we must take in consideration that is eating 145g more kibbles than before the treatment. That’s
mean is eating 725g more kibbles in five days than before the probiotic’s treatment.
According to the Figure 3.1 and the table 3.5 we observe a large variation of feces
quantity before and after the probiotic’s treatment. For the six dogs there is an important
diminution of feces production after the treatment.
About 560g less feces for khalac, 370g less feces for Luna, 200g less feces for Nessie,
264g less feces for Holiday and 247g less feces for Halva in five days feces collection. For
Maestro, we observe a difference of +52g of feces but we must take in consideration that is
eating 145g more kibbles every day that means 725g more kibbles in 5 days than before the
probiotic’s treatment. So, if we do the math, he is supposed to produce 1474g of feces
(2900*1106)/2175 = 1474 g) but he is producing 1158 g of feces in five days that means 316g
less than expected.
The diminution of feces for the six dogs has an average of 325g, it is representing a
significative change and this diminution can be correlated to the increase of absorption of
nutrient. As we will see in the digestibility part, we observed an increase in apparent digestibility
after probiotic treatment.
In the literature, Felix et al. (2010), studied the effect of Bacillus subtilis probiotic
product on dogs. Felix et al. (2010) noticed a higher fecal score after probiotic treatment. .For
precision, the fecal score was evaluated always by the same researcher in a 1 to 5 scale as: 1 =
very soft feces; 2 = soft, unshaped feces that took the shape of the collection vessel; 3 = soft,
shaped and moist feces that left a mark on the floor; 4 = shaped and solid feces that did not
adhere to the floor; 5 = shaped, dry, and hard feces.
37
3.3. HEMATOLOGY
The following table (table 3.6) represents the hematological results before and after the
probiotics treatment for all the six cases. For Khalac, Maestro and Halva blood samplings have
been realized day 0 and day 31 of the study. For Luna, Nessie and Holiday blood samplings have
been realized day 0 and day 36 of the study.
Table 3.6
Results for hematological parameters before and after probiotics treatment
Hematologic Khalac Maestro Luna Nessie Holiday Halva
Parameters
Day 0 31 0 31 0 36 0 36 0 36 0 31
HCT 46.74 47.01 43.91 39.07 48.00 48.08 42.76 48.94 47.67 47.53 45.25 42.71
(37-55%)
HGB 21.30 21.70 19.80 19.20 20.60 22.00 18.20 23.60 21.10 21.40 21.40 21.70
(12-18 g/dL)
RBC 7.11 7.13 6.70 6.25 6.86 7.30 5.82 6.74 7.05 6.85 7.22 6.84
(5.5-
8.5*10^12/L)
MCV 66.00 66.00 66.00 63.00 70.00 66.00 73.00 73.00 68.00 69.00 63.00 62.00
(60-77 fL)
MCH 30.00 30.50 29.50 30.70 30.00 30.20 31.20 35.00 29.90 31.20 29.60 31.70
(19.5-24.5 pg)
MCHC 45.60 46.20 45.00 49.10 42.90 45.80 42.50 48.20 44.20 45.00 47.20 50.80
(32-36 g/dL)
WBC 13.35 12.95 12.13 15.22 16.29 12.43 11.43 17.28 9.75 16.99 18.80 19.69
(6-17*10^9/L)
PCT 0.11 0.21 0.15 0.18 0.15 0.33 0.37 0.45 0.51 0.37 0.25 0.30
(%)
For a better visual understanding of these parameters, we can observe the hematological
results on the figures 3.2 till 3.9.
50
45
HCT(37-55%)
40
35
30
Day 0 Day 31/36
Figure 3.2 - Evolution of the hematocrit before and after the probiotic’s treatment
38
25
HGB (12-18 g/dL) 20
15
10
Day 0 Day 31/36
Figure 3.3 - Evolution of the hemoglobin before and after the probiotics’s treatment
7.5
RBC (5,5-8,5*10^12/L)
7
6.5
6
5.5
5
Day 0 Day 31/36
Figure 3.4 - Evolution of the red blood cells before and after the probiotic’s treatment
75
MCV (60-77 fL)
70
65
60
Day 0 Day 31/36
Figure 3.5 - Evolution of the mean corpuscular volume before and after the probiotic’s treatment
34
MCH (19.5-24.5 pg)
29
24
19
Day 0 Day 31/36
Figure 3.6 - Evolution of the mean corpuscular hemoglobin before and after the probiotic’s treatment
39
52
MCHC (32-36 g/dL) 47
42
37
32
Day 0 Day 31/36
Figure 3.7 - Evolution of the mean corpuscular hemoglobin concentration before and after the probiotic’s
treatment
20
WBC (6-17*10^9/L)
15
10
5
Day 0 Day 31/36
Figure 3.8 - Evolution of the white blood cells before and after the probiotic’s treatment
0.6
0.5
0.4
PCT (%)
0.3
0.2
0.1
0
Day 0 Day 31/36
Figure 3.9 - Evolution of the plateletcrit before and after the probiotic’s treatment
The results show for Khalac, Luna, Nessie, an increase of the hematocrit. For Maestro,
Holiday and Halva there is a decrease in hematocrit. However, the differences are not
significative (figure 3.2). For all the dogs except Maestro we observe a slight increase in
hemoglobin production after the treatment (figure 3.3). We observe a slight increase of the red
blood cells for Khalac, Luna, Nessie but a slight decrease for Maestro, Holiday and Halva.
However, the differences are not significative (figure 3.4).
The MCV presents no variation in Khalac and Nessie while we observe a decrease in
MCV after probiotics treatment for Maestro, Luna Halva. Holiday presents an increase of the
MCV after the probiotic’s treatment (figure 3.5). For all dogs we observe an increase of the
40
MCH (figure 3.6) and the MCHC after the probiotic’s treatment (figure 3.7). These parameters
are in the normal range of value and their variations are not significant.
We observe for Khalac and Luna a decrease of the white blood cells while for Maestro,
Nessie, Holiday and Halva there is an increase in white blood cells (figure 3.8).
All the dogs except Holiday show an increase in platelets after the probiotic’s treatment
(figure 3.9).
Even if some of the results show values outside of the normal range it is not alarming
because it can be due to the device. The hematological results are considered in the normal range
for dogs and don’t represent any signs of diseases.
The hematological parameters for our six dogs are quite heterogenous but are always in
the normal range of values for dogs. Nevertheless, we observe a slight increase in hemoglobin
(Hb), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration
(MCHC), plateletcrit (PCT) and white blood cells (WBC) for almost all the dogs after the
probiotic treatment.
These results are not significant and confirm the study of Hong et al. (2008). In fact, in
their study, they tested the effect of oral administration of Bacillus subtilis for 30 days in rabbit.
After the probiotic treatment, rabbits don’t show any significant changes in hematological
parameters (Hong et al. 2008).
The table 3.7 record the percentage of leucocytes before and after the probiotic’s
treatment for all the six cases.
Table 3.7
Percentage of leucocytes before and after the probiotic’s treatment
Leucocytes Khalac Maestro Luna Nessie Holiday Halva
Day 0 31 0 31 0 36 0 36 0 36 0 31
Neutrophil (58-
43 51 63 40 47 65 62 56 61 57 54 59
85%) *
Eosinophil (0-
7 7 6 30 5 6 8 5 4 6 6 7
9%) *
Basophil
0 0 0 0 0 0 0 1 0 0 0 0
(0-1%) *
Monocyte (2-
17 12 18 13 20 12 10 18 18 16 9 11
10%) *
Lymphocyte
33 30 13 17 28 17 20 20 17 21 31 23
(8-21%) *
*Merck Veterinary Manual, 2016
The leucocytes values are graphically represented in the figures 3.10, 3.11, 3.12, 3.13 and
3.14.
41
80
Neutrophils (58-85%)
60
40
20
0
Days of probiotics treatment
Day 0 Day 31/36
Figure 3.10 - Evolution of the neutrophils before and after the probiotic’s treatment
Eosinophils (0-9%)
30
20
10
0
Day 0 Day 31/36
Figure 3.11 - Evolution of the eosinophils before and after the probiotic’s treatment
1.2
1
Basophils (0-1%)
0.8
0.6
0.4
0.2
0
Day 0 Day 31/36
Figure 3.12 - Evolution of the basophils before and after the probiotic’s treatment
25
Monocytes (2-10%)
20
15
10
5
0
Day 0 Day 31/36

-
Figure 3.13 - Evolution of the monocytes before and after the probiotic’s treatment
42
40
Lymphocytes (8-21%)
30
20
10
0
Day 0 Day 31/36
Figure 3.14 - Evolution of the lymphocytes before and after the probiotic’s treatment
For Khalac, Luna and Halva there is an increase of the neutrophils. For Maestro, Nessie
and Holiday there is a decrease in neutrophils (figure 3.10).
The figure 3.11 represents the evolution of the eosinophils before and after the probiotic’s
treatment. There is no variation of eosinophils in Khalac. Maestro presents an important increase
of the eosinophils from 6 to 30% after probiotics treatment. Luna, Holiday and Halva show a
slight increase in eosinophils. While Nessie presents a slight decrease in eosinophils.
Concerning Maestro, the important increase in eosinophils can correlates with the second
coprological examination confirming the presence of intestinal parasites. Even if the mechanism
is not very clear, the probiotics influence and modifies the gastrointestinal environment. The
process involves a stimulation of immune system cells to produce cytokines, which play a role in
the induction and regulation of the immune response. The environment became less adequate for
parasites and a possible release of toxins by dead parasites is a valuable hypothesis. Eosinophils
increase in the presence of parasitic toxins and in allergic reactions (Travers et al., 2011).
Usually in dogs, basophils are very rare and that why there are not present in our results.
We found one basophil in Nessie after probiotics treatment. However, it is not significative
(figure 3.12).
For Khalac, Maestro, Luna and Holiday there is a decrease of the monocytes while for
Nessie and Halva there is an increase in monocytes (figure 3.13).
We observe for Khalac, Luna and Halva a decrease of lymphocytes. For Maestro and
Holiday there is an increase in lymphocytes (figure 3.14). Nessie show no variation in
lymphocytes.
Even if the monocytes and lymphocytes seem a bit elevated, it is not alarming. The
leucocytes values before and after probiotic treatment present heterogenous results but are in the
normal range of values for dogs. The eosinophils tend to slightly increase in most of the dogs
43
after treatment. Neutrophils, basophils, monocytes and lymphocytes increase or decrease

according to the individual. These results confirm the study of Hong et al. (2008) and conduct to
no significant changes of the leucocytes after probiotic treatment.
3.4. BIOCHEMISTRY
The table 3.8 presents the results for the biochemical parameters before and after the
probiotic’s treatment for all the six cases. For Khalac, Maestro and Halva blood samplings have
been realized day 0 and day 31 of the study. For Luna, Nessie and Holiday blood samplings have
been realized day 0 and day 36 of the study.
Table 3.8
Results of the biochemical parameters before and after the probiotics treatment
Biochemistry
Parameters
Day 0 31 0 31 0 36 0 36 0 36 0 31
ALT
41 46 33 38 28 25 38 38 32 23 51 58
(10-118 µ/L)
ALB
36 39 39 39 40 42 38 38 39 39 41 39
(25-44 g/L)
ALP
38 33 24 28 33 38 27 21 21 38 46 39
(20-150 µ/L)
Amylase
520 526 473 542 404 418 509 421 508 495 499 400
(200-1200 µ/L)
Calcium
(2.15-2.95 2.55 2.65 2.70 2.63 2.68 2.70 2.58 2.74 2.62 2.59 2.64 2.59
mmol/L)
Creatinine
132 102 109 97 92 62 53 51 64 42 93 84
(27-124 µmol/L)
Globulin
30 24 31 28 25 26 22 24 25 26 25 23
23-52 g/L)
Glucose
4 4 4.4 3.9 4.3 5.5 4.8 6 2.0 5.7 5.1 4.9
(3.3-6.1 mmol/L)
Phosphorus
(0.94-2.13 2.10 1.99 1.88 1.77 1.49 1.42 2.08 1.43 1.57 1.22 1.53 1.77
mmol/L)
Potassium
4.7 5.2 4.9 4.7 5.3 4.6 5.2 4.8 6.1 5.1 4.7 5.1
(3.7-5.8 mmol/L)
Sodium
(138-160 146 142 145 145 144 148 145 146 144 146 145 142
mmol/L)
Total Bilirubin
5 7 5 5 4 6 4 8 5 9 5 4
(2-10 µmol/L)
Total protein
66 63 70 67 65 68 60 63 64 65 66 63
(54-82 g/L)
Blood Urea
Nitrogen (BUN) 7.1 10.3 7.9 7.2 4.7 7.3 9.0 9.4 6.4 9.9 8.1 7.9
(2.5-8.9 mmol/L)
Khalac, Maestro and Halva show an increase of the ALT. Luna and Holiday present a
decrease in ALT after the treatment. While for Nessie there is no variation of the ALT (figure
3.15). Nevertheless, the values are in the normal range. The ALT are produce mainly by the
hepatocytes and the results show a normal function of the liver (Poli G., 2016).
44
We observe in Khalac and Luna an increase of the ALB. While for Maestro and Nessie
there is no variation of the ALB. For Halva there is a decrease in ALB after the treatment (figure
3.16). The values are still in the normal range and translate a good functioning of the liver (Poli
G., 2016).
The ALP results for Khalac, Nessie and Halva show a decrease of the ALP. The other
dogs; Maestro, Luna and Holiday present an increase in ALP. However, the differences are not
significative (figure 3.17). This enzyme is produced mainly by bile canicular membranes, bones
and kidney and the normal range of values obtained confirm a good state of these organs (Poli
G., 2016).
The amylase increases for Khalac, Maestro and Luna while decreases for Nessie, Holiday
and Halva. The differences are not significative (figure 3.18). The normal values show a healthy
pancreas and liver (Poli G., 2016).
We observe an increase in calcemia for Khalac, Luna and Nessie but a decrease in
calaemia for Maestro, Holiday and Halva (figure 3.19). The values are in the normal range of
value. This parameter usually is representative of tumoral process or endocrinological diseases
(Poli G., 2016).
All the dogs present a decrease in creatinine after the probiotic’s treatment (figure 3.20).
Before the probiotic treatment Khalac show a creatinine value of 132 µmol/L, slightly above the
normal range (27-124 µmol/L) but this result is not alarming especially that it decreases after the
treatment. Creatinine parameter is used to evaluate mainly the kidney function (Poli G., 2016)
For Khalac, Maestro and Halva there is a decrease of the globulin while it is increasing
for Luna, Nessie and Holiday after treatment (figure 3.21). For Nessie the value is slightly above
the normal range but it is not significant. The protein is representative of the state of the liver
(Poli G., 2016).
There is no variation in glucose for Khalac before and after the treatment. For Maestro
and Halva there is a decrease of the glucose after the treatment and Luna, Nessie and Holiday
present an increase in glucose (figure 3.22). Before the probiotic treatment Holiday shows a
glucose value under the normal range but it increased in the normal range after the treatment.
These results are not significant and translate a good functioning of the pancreas for the dogs
(Poli G., 2016).
All the dogs except Halva show a decrease in phosphorus after the probiotic’s treatment.
The phosphorus is increasing for Halva after the treatment (figure 3.23). This parameter can be
modified in some endocrinal diseases, bones diseases or renal diseases (Poli G., 2016).
45
The potassium increases for Khalac and Halva while for the other dogs it decreases after
the treatment (figure 3.24). For Holiday, we observe a potassium value slightly above the normal
range but it come back to the normal range after the probiotic treatment. These normal values
translate a good functioning of the heart and muscle (Poli G., 2016).
For Khalac and Halva there is a decrease in sodium after the treatment. No variations are
recorded for Maestro before and after treatment. For Luna, Nessie and Holiday there is an
increase of the sodium after the treatment (figure 3.25). The values obtained are in the normal
range. This parameter is representative of the kidney function (Poli G., 2016).
We observe for Khalac, Luna, Nessie and Holiday an increase of the total bilirubin. No
variations are shown for Maestro while for Halva there is a decrease in total bilirubin. However,
the results are not significative (figure 3.26). It shows a good functioning of the liver and bile
duct (Poli G., 2016).
The total protein decreases for Khalac, Maestro and Halva while there is an increase for
Luna, Nessie and Holiday after the treatment (figure 3.27). However, the values are in the
normal range. An increase of total protein can represent: a dehydration, inflammatory or
neoplastic diseases or an infection. While a decrease of this parameter can show an over
hydration, liver malfunctioning, a nephropathy, a hemorrhage or an acute tissue injury (Poli G.,
2016).
Khalac, Luna, Nessie and Holiday have an increase of the BUN. Maestro and Halva
present a decrease in BUN after treatment (figure 3.28). The values are still in the normal range
and reflect a good functioning of the kidney and liver (Poli G., 2016).
60
ALT (10-118 u/L)
50
40
30
20
10
Day 0 Day 31/36
Figure 3.15 - Evolution of the alanine aminotransferase before and after the probiotic’s treatment
46
45
ALB (25-44 g/L) 40
35
30
25
Day 0 Day 31/36
Figure 3.16 - Evolution of the albumin before and after the probiotic’s treatment
50
ALP (20-150 u/L)
40
30
20
10
0
Day 0 Day 31/36
Figure 3.17 - Evolution of the Alkaline phosphatase before and after the probiotic’s treatment
600
Amylase (200-1200 u/L)
500
400
300
200
Day 0 Day 31/36
Figure 3.18 - Evolution of the amylase before and after the probiotic’s treatment
2.95
Calcium (2.15-2.95 mmol/L)
2.75
2.55
2.35
2.15
Day 0 Day 31/36
Figure 3.19 - Evolution of the calcium before and after the probiotic’s treatment
47
140
Creatinine (27-124 umol/L)

120
100
80
60
40
20
Day 0 Day 31/36
Figure 3.20 - Evolution of the creatinine before and after the probiotic’s treatment
32
Globulin (23-52 g/L)
30
28
26
24
22
20
Day 0 Day 31/36
Figure 3.21 - Evolution of the globulin before and after the probiotic’s treatment
8
Glucose (3.3-6.1 mmol/L)
0
Day 0 Day 31/36
Figure 3.22 - Evolution of the glucose before and after the probiotic’s treatment
2.5
Phosphorus (0.94-2.13
2
1.5
mmol/L)
1
0.5
0
Day 0 Day 31/36
Figure 3.23 - Evolution of the phosphorus before and after the probiotic’s treatment
48
Potassium (3.7-5.8 mmol/L)

6
3
Day 0 Day 31/36
Figure 3.24 - Evolution of the potassium before and after the probiotic’s treatment
150
Sodium (138-160 mmol/L)
148
146
144
142
140
138
Day 0 Day 31/36
Figure 3.25 - Evolution of the sodium before and after the probiotic’s treatment
10
Total Bilirubin (umol/L)
2
Day 0 Day 31/36
Figure 3.26 - Evolution of the total bilirubin before and after the probiotic’s treatment
75
Total protein (54-82g/L)
70
65
60
55
50
Day 0 Day 31/36
Figure 3.27 - Evolution of the total protein before and after the probiotic’s treatment
49
12
BUN (2,5-8,9 mmol/L)

10
8
6
4
2
Day 0 Day 31/36
Figure 3.28 - Evolution of the blood urea nitrogen before and after the probiotic’s treatment
The hematological and biochemical parameters have shown slight changes pre and post
administration of probiotics with values in the normal range.
That’s means the probiotic product tested doesn’t provoke any paraclinical adverse
effects on dogs. It can be considered as a safe probiotic product on healthy dogs.
50
3.5. PARASITOLOGY
The first flotation and sedimentation tests are negative for Maestro, Luna, Nessie,
Holiday and Halva. For Khalac, the flotation test is positive for hookworms. In fact, we found
eggs of Uncinaria spp./ Ancylostoma. spp. Khalac has been treated with Drontal + before to start
the probiotics treatment.
The second flotation and sedimentation tests are negative for Khalac, Luna, Nessie,
Holiday and Halva. For Maestro, the flotation test is positive for Uncinaria spp./ Ancylostoma
spp. (figure 3.29). Hookworms such as Uncinaria spp. and Ancylostoma spp. don’t cause
significant clinical signs in mature dogs. Febendazole, moxidectin or pyrantel are efficient in the
treatment of hookworms. As measure of prevention in dogs a deworming treatment is
recommended every three months (Merck et al., 2016).
Figure 3.29 - Eggs of Uncinaria/Ancylostoma found in Khalac coprological examination
51
3.6. DIGESTIBILITY
The following table 3.9 represents the Apparent Digestibility Coefficient (ADC) in
percent before and after the probiotic treatment. Five digestibility parameters are observed: the
ADC dry matter, the ADC crude protein (CP), the ADC crude fat (CF), the ADC crude cellulose
(CC) and the ADC nitrogen free extract (NFE).
Table 3.9
Results of Apparent Digestibility Coefficients (ADC) before and after the probiotic’s treatment (%)
KHALAC Dry matter Crude protein Crude fat Crude cellulose NFE
Pre-administration 80.96 88.88 98.38 5.63 59.59
Post -administration 92.19 95.94 99.30 6.24 89.74
MAESTRO Dry matter Crude protein Crude fat Crude cellulose NFE
LUNA Dry matter Crude protein Crude fat Crude cellulose NFE
NESSIE Dry matter Crude protein Crude fat Crude cellulose NFE
HOLIDAY Dry matter Crude protein Crude fat Crude cellulose NFE
HALVA Dry matter Crude protein Crude fat Crude cellulose NFE
For a better visual understanding of these results, we can observe the figures 3.29 till
3.33.
100
90
80
70
60
50
40
30
20
10
0
Pre-administration Post -administration
Figure 3.29 – Dynamic evolution ADC of dry matter (%)
52
100
90
80
70
60
50
40
30
20
10
0
Figure 3.30 – Dynamic evolution of ADC of crude protein (%)
100
98
96
94
92
90
88
86
Figure 3.31 – Dynamic evolution of ADC of crude fat (%)
53
10
9
8
7
6
5
4
3
2
1
0
Figure 3.32 – Dynamic evolution of ADC of crude cellulose (%)
100
90
80
70
60
50
40
30
20
10
0
Figure 3.33 – Dynamic evolution of ADC of nitrogen free extract (%)
The apparent digestibility coefficient for dry matter after probiotic administration is
increasing for all the six dogs. We observe an improvement of dry matter from 3% for Maestro
till 19% for Holiday. As an average we observe an increase of dry matter of 10% after the
treatment.
Concerning the results for the apparent digestibility coefficient for the crude protein, we
observe for Khalac, Maestro, Luna and Holiday an increase from 7% to 18% (Holiday) and a
decrease in crude protein from 4% to 6% for Nessie and Halva. There is an average of increase
of 5% crude protein’s digestibility after treatment.
54
The apparent digestibility coefficient for crude fat has an increase average of 3% after the
probiotic treatment. The values are between 1% to 6% (Holiday).
We observe an increase average of 1,5% for the apparent digestibility coefficient for
crude cellulose after the treatment. The values are between 0,5% (Halva) to 3% (Luna).
The apparent digestibility coefficient for nitrogen free extract (N.F.E.) after probiotic
administration is increasing for all the six dogs. We observe an increase of 3% for Maestro till
30% for Khalac. The improvement of digestibility for NFE after the treatment has an average of
13%.
For all the six dogs, we clearly observe an improvement of the apparent digestibility
coefficient for all the parameters: Dry matter, crude protein, crude fat, crude cellulose and
nitrogen free extract.
Daumas et al. (2014), have studied the digestibility of different commercial diets in dogs.
For the apparent digestibility, the range of values of crude protein and crude fat were about 70–
82 % crude protein and 76–95 % crude fat. Comparing our results, the range for the crude
protein is 69-88% and for the crude fat 91-98% before the probiotic treatment. Post-probiotic
administration the range of values increases: 71-95% for the crude protein and 93-99% for the
crude fat (Daumas et al. 2014).
In their study, Hagen-Plantinga et al. (2014) have determined the apparent digestibility of
canine diets. The average values obtained for the apparent digestibility of dry matter, crude fat,
and nitrogen are 77%, 94% and 78%. If we compare our results pre-administration we obtain an
apparent digestibility of dry matter, crude fat and nitrogen of 77%, 94% and 80%. The results
that we found are closely similar to the ones from literature (Hagen-Plantinga et al. 2014).
Felix et al. (2010), studied the digestibility of dogs fed with Bacillus subtilis in their diet.
The apparent digestibility except for the dry matter, shown an improvement of digestibility with
the probiotic treatment. For the control dogs (without probiotics) they obtain an average value of
dry matter of 82,1% and for the dogs with a probiotic’s treatment an average value of 82,4%.
The improvement is not really significant. However, the crude protein digestibility didn’t
improve. We observed that the dogs were fed with the probiotic only 25 days instead of 30 days
like in our trial (Felix et al. 2010).
Biourge et al. (1998) and Felix et al. (2010), studied the effect of Bacillus subtilis
probiotic product on dogs. In their studies, Biourge et al. (2010) found a slight improvement in
digestibility but no really significative differences in apparent digestibility coefficient (ADC) of
dry matter (DM) and crude protein (CP) (Biourge et al., 1998; Felix et al., 2010).
55
In their study Joysowal et al. (2018), have tested the effect of Pediococcus acidilactici
FT28 probiotic product on pigs. Their results shown the improvement in growth, feed intake and
digestibility of crude protein after probiotic treatment. However, the digestibility of dry matter,
crude fiber and nitrogen free extract did not show any significant effect (Joysowal et al., 2018).
According to Xu et al. (2019) the use of probiotics in dog can improve feed intake,
weight gain, immunity and intestinal microbiota. Moreover, the elderly dogs showed the
strongest response to the probiotics. In fact, Holiday the oldest dog of our study presents the best
increase in digestibility for dry matter, crude protein and crude fat (Xu et al. 2019).
In the literature, the data about the effect of probiotics on digestibility are variable. We
could consider, that the combination between Bacillus subtilis and Pediococcus acidilactici
could be a possible explanation for a such improvement in digestibility for our study. Even if the
interaction between these two bacterial strains is not very well understood, it could result in an
efficient combination for a probiotic product.
It needs to be noted that the study conducted has several limitations.
First of all, the data observed in this study were not a standardized method using
laboratory dogs. Indeed, the heterogeneity of our cases can alter the results. The dogs have an
important difference of age, size, weight. The breed and the difference in environmental
conditions (type of food, owner, amount of exercise, habits) can play a significative role in the
results. This may reduce the reliability of the data found in this study.
The dog’s owners have participated in the study, scoring and filling the Optidigest sheet.
A more careful instruction of the owners could have been a solution for a better compliance and
reliability of the data. To improve the homogeneity of the data, the same person must fill the
Optidigest sheet for all the dogs.
According to the literature, the fecal collection for digestibility has been made on a period
of five days and seems a representative sample. A shorter period of collection will not be
significant.
The period of the probiotic treatment was 30 days, a shorter period will not be vey
significant while a longer period could have been interesting.
The literature comprises only few data concerning the effect of probiotic product on the
hematological, biochemical parameters and the digestibility. In our study, we observe a
significant improvement in fecal weight and digestibility parameters for all the dogs after
administration of probiotics that can be explained by the increase in total apparent digestibility of
the consumed feedstuffs. Other tests, like intestinal biopsies, PCR or bacterial examinations
could be helpful for a better understanding of the results.
56
Nevertheless, the study proved the safety of the probiotic product used on healthy dogs.
Clinically, dogs didn’t show any signs of sickness, diarrhea or vomiting. Before, during and after
the period of the probiotic treatment, dogs show a good general status. No adverse effects have
been observed in healthy dogs.
57
4. CONCLUSIONS
The clinical appearance of dogs is not altered by the treatment however one of the dogs
presented an improvement of the coat. No symptoms such as diarrhea or vomiting have been
observed.
The fecal score is slightly improved and a decrease in flatulence was noticed. The fecal
weight was widely decreased after treatment due to digestibility increase.
The hematological and biochemical parameters have shown slight changes pre and post
administration of probiotics with values in the normal range.
The study of the digestibility of feedstuffs by measuring the apparent digestibility

coefficients shows an important improvement of digestibility of all the studied components (dry
matter, crude fat, crude cellulose and N.F.E.) with the exception of crude protein where the
values decreased in two cases.
The use of the probiotic with Bacillus subtilis and Pediococcus acidilactici presents no
adverse effects in healthy dogs.
The combination of these two strains of bacteria: Bacillus subtilis and Pediococcus
acidilactici used as probiotic shows promising results when used in dogs.
Further studies and researches must be conducted with this probiotic product on
challenged dogs, especially in dogs with inflammatory bowel disease or chronic gastrointestinal
diarrhea.
58
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61
ANNEXES
Annexe 1: Clinical sheet for Khalac
62
Annexe 2: Clinical sheet for Maestro
63
Annexe 3: Clinical sheet for Luna
64
Annexe 4: Clinical sheet for Nessie
65
Annexe 5: Clinical sheet for Holiday
66
Annexe 6: Clinical sheet for Halva
67
Annexe 7: Optidigest sheet for Maestro and Khalac
68
Annexe 8: Optidigest sheet for Luna
69
Annexe 9: Optidigest sheet for Nessie
70
Annexe 10: Optidigest sheet for Holiday
71
Annexe 11: Optidigest sheet for Halva
72
Faculty of Veterinary Medicine Cluj-Napoca
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I CAMILLE BRETZ student attending the University of Agricultural Sciences and

Veterinary Medicine in Cluj-Napoca, Faculty of VETERINARY MEDICINE (USAMV),
Specialization VETERINARY MEDICINE ENGLISH LINE, solemnly declare, knowing the
regulations in the art. 292 of the Penal Code regarding falsified information present in the
Dissertation/Diploma project, that this Dissertation/Diploma project with the title THE
INFLUENCE OF A PROBIOTIC PRODUCT WITH BACILLUS SUBTILIS AND
PEDIOCOCCUS ACIDILACTICI ON HEALTHY DOGS does not contain any plagiarised
material or falsified information or data and the research done and outcomes from it are truly my
own.
The project has been developed by myself and has never been presented or defended at
other national or international universities/faculties or other post-secondary institutions.
I also declare that all bibliographic material, including studies on the internet or electronic
journals, are properly mentioned and cited in the bibliographic list, abiding by the regulations
preventing plagiarism, respectively:
- All text take from any bibliographic work must be placed in quotation marks, even if
translated from one language to another, and detain the proper citation.
- Rephrasing, using your own words, of text written by other authors should be also
properly mentioned and cited.
- Summarization of ideas belonging to other authors, must contain reference to the original
text of the article or journal used.
By this statement, I have understood that if plagiarism is proven, with concrete evidence,
I will be disqualified and expelled from the license exam and the dissertation/diploma project
presentation.
Cluj-Napoca
Graduate student name,
CAMILLE BRETZ
Date 21/06/2019
73

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UNIVERSITY OF AGRICULTURAL SCIENCES AND

FACULTY OF VETERINARY MEDICINE

ANIMAL PHYSIOLOGY DEPARTMENT

THE INFLUENCE OF A PROBIOTIC PRODUCT WITH

Thanks to Lara my roommate for these 6 years of complicity.

Thanks to Nina for your sunny personality.

Thanks to my wonderful Helene and destiny that brought us together.

Thanks to Alix for your positivity and your enthusiasm.

Thanks to my oldest friend Claire who never let me down.

THE INFLUENCE OF A PROBIOTIC PRODUCT WITH BACILLUS

Keys words: Bacillus subtilis, Pediococcus acidilactici, probiotic, digestibility, canine

1.1. PROBIOTICS HISTORY AND IMPORTANCE

1.2. PROBIOTICS- HEALTH BENEFITS

1.3. SAFETY CRITERIA FOR PROBIOTICS

1.4. PROBIOTICS- MECHANISMS OF ACTION

1.5. PROBIOTICS USED IN ANIMALS

1.6. LEGISLATIVE PROBIOTICS STATUS

2. GASTROINTESTINAL MICROBIOTA- GENERALITIES

2.1. IMPORTANCE OF THE MICROBIOTA

2.2. CANINE INTESTINAL MICROBIOTA

by Firmicutes, Actinobacteria, Spirochaetes, Lactobacilli, Bacteroidetes and Fusobacteria.

2.3. BACILLUS SPP. AS POTENTIAL PROBIOTIC

2.3.1 Bacillus species

2.3.2 Bacillus spp. used as probiotics

Enterogermina® product contains Bacillus clausii spores and it is used in diarrhoeal

2.3.3 Bacillus spp. life cycle in gastrointestinal tract

2.3.4 Life cycle of Bacillus spp. in animal gastrointestinal tract

2.3.5 Bacillus species safety

2.3.6 Bacillus spp. - Mechanism of action

2.3.7 Bacillus species as commercial products

2.3.8 Probiotic versus pathogenic Bacillus spp.

II. EXPERIMENTAL PART

Our study had the following specific objectives:

2. MATERIAL AND METHODS

2.1. EXPERIMENTAL PROTOCOL

Coprology examination Coprology examination

Figure 2.1 - Chronological steps of the study

2.2. PRESENTATION OF CASES

Figure 2.2 – The dogs included in the study

2.3. PROBIOTIC DESCRIPTION

Figure 2.3 – The tested probiotic

Bacillus subtilis is already used in different probiotic products and researches

2.4. CLINICAL INVESTIGATIONS

2.5. FECAL SCORE

Figure 2.4 – Optidigest clinical sheet made by Purina

2.6. PARACLINICAL INVESTIGATIONS

Figure 2.5 - Device ABACUS JUNIOR VET

Determination of the Leucocytes formula

Principle of the leucocyte’s formula

 in absolute value (109/L)

N – the number of each population per liter of blood

UDC (%) = Utilization Digestibility Coefficient UDC can be:

A digestibility experience implies specific conditions

The digestibility experience with 1 control period is composed of 2 steps:

a) determining relative dry matter (RDM) at 65°C;

2.6.4.3 Determination of crude fat (CF)

2.6.4.4 Determination of crude cellulose (CC)

The method (Weende method) of measuring cellulose is based on removing the

2.6.4.5 Determination of nitrogen free extract (NFE)

3. RESULTS AND DISCUSSION

3.1. CLINICAL EXAMINATION

3.2. FECAL SCORE

Khalac’s feces collected in five days are about 0,56 kg.