EVALUATION OF THE ACTIVITY OF TWO GARLIC COMPOUNDS (PTS and

PTSO) AND ITS COMERCIAL PREPARATION AGAINST Salmonella enteric

IN LAYING HENS (Oral Presentation)
Fatemeh Hasanshahi (DVM, Veterinary Researcher, Educated from Shiraz Veterinary Faculty),
1 1
E. Arandilla (PhD Veterinary, University of León), C. Alvarez (DVM, Complutense University of Madrid),
2 2
E. Guillamon (PhD Chemistry, University of Granada), A.Baños (MsC Microbiologist, University of
1
Granada), R. López (Nutritionist, Agronomist Engineer, Polithecnic University of Madrid)
1
Prebia Feed Extracts SL. Talavera de la Reina, Toledo (Spain)
2
DOMCA SAU. Alhendín, Granada (Spain).
Author adress: rlopez@prebia.es

Abstract
Salmonella enterica is involved in many human
food borne diseases, being eggs and poultry meat strong antimicrobial potency of this type of
important sources of this pathogen. The aim of this molecules.
study, is to evaluate the antimicrobial activity of This results point Garlicon® as a promising tool
two garlic compounds, PTS (propyl propyl for poultry farmers, combining efficacy and food
tiosulphinate) and PTSO (propyl propyl safety.
thiosulphonate) and a commercial preparation Key words: Food Safety, Salmonella, Garlic,
containing both principles (Garlicon ®, Prebia PTS, PTSO.
Feed Extracts SL, Spain).
Two experiments were carried out to evaluate the Introduction
antimicrobial activity of Garlicon® using it at 100 At the present time Salmonella enterica is
mL/1000 L drinking water during 7 days in 36.000 one of the major pathogens implicated in human
laying hens or applying it as a nebulizate, using a food-borne disease (EFSA, 2010). Eggs and
thermonebulizator machine. poultry meat remain as the main sources of
Total Enterobacteriaceae and Salmonella content Salmonella, as stated in Commision Regulation
dramatically decrease in faeces after the drinking (EC) No 1168/2006 of 31 july 2006. According to
treatment. In the same samples, we found a Regulation (EC) No 2160/2003 and amendments
significant increasement in the counts of (regarding reduction of the prevalence of certain
Lactobacilli and Bifidobacteria, showing a Salmonella serotypes in laying hens of Gallus),
different antimicrobial activity in this groups that Salmonella control programs have to be
could lead to a higher number of colonies due to implemented in broiler chickens, laying hens and
competitive exclusion. porcine in different countries, including all the
Regarding environmental treatment, all microbial steps of the food chain from the primary
groups studied (Fungi, Total Bacteria and production, the slaughterhouses, distribution and
Enterobacteriaceae) have a significant retail following the "from stable to table" approach
decreasement in surfaces and air, showing the to food safety as stated in the EU regulations
(Zoonoses Directive, 2003/99/EC). On the other In the present study, a patented and
hand, the use of antibiotics has been restricted standardized commercial garlic/onion extract,
during the latest years in animal production. Garlicon® (Prebia Feed Extracts SL), have been
In a study conducted by the USDA evaluated as potential antimicrobial substances in
National Integrated Fool Safety Initiative (NIFSI) an assay in layers against Salmonella enteric.
from 1998 to 2008, data were collected for Differents studies have shown that Garlicon®
observed percentage of positive Salmonella tests in presents broad antimicrobial spectrum against
the Pathogen Reduction/Hazard Analysis and different bacterial groups as Brachyspira
Critical Control Point Verification Testing hyodysenteriae (Hidalgo A. 2009.IPVS),
Program. According to the “Progress Report on Methicillin resistant Staphylococcus
Salmonella Testing of Raw Meat and Poultry aureus),methicillin-sensitive Staphylococcus
Products, 1998-2008” broilers, turkeys, ground aureus, (Arandilla E. 2010 ICAR), Salmonella
chicken, and ground turkey demonstrated the enteric (Arandilla E. 2010 ICAR), E.coli
highest prevalence of Salmonella (7.3, 6.2, 25.5, (Arandilla E. 2011 ITEA), and Campylobacter sp.
and 15.4% respectively) compared with cows or (Hidalgo A. 2011.ITEA).
bulls, steers or heifers, and ground beef (0.5, 0.2, Materials and Methods:
and 2,4% respectively). Antibiotics are used in the A farm of 36.000 Hy Line Brown laying
commercial poultry industry to promote growth hens with 52 weeks of age (older animals excrete
and reduce pathogenic bacteria; however, more Salmonella) was selected for the study. The
consumer awareness has increased in recent years laying hens are housed in cages of 5 animals.
with regard to the relationship of the food Animals had access to drinking water in bowls,
production process and the possible presence of and the feeder was run ad-libitum. The faeces fall
antibiotic-resistant bacteria in the final product. to a conveyor tape. Diet composition and nutrient
This movement has caused many consumers to level of experimental diet is showed in table 1.
question the use of antibiotics in animal Table 1
production, resulting in efforts to search for natural Ingredient (g/Kg)
alternatives (Lilly KGS, 2011). Yellow corn 200.0
Reducing the presence of Salmonella pre- Soybean meal 230.6
slaughter can aid in reducing Salmonella post- Barley 240.06
slaughter. Some antibiotic-alternative strategies to Wheat 200.17
combat this include dietary inclusion of different Soy Oil 10.0
natural compounds (Garlic). In this situation, new Dicalcium Phosphate 93.0
tools and strategies for the control of Salmonella at Limestone 8.78
farm level are of great interest. Garlic not only has Salt 10.0
a well known antibacterial activity, also has Premix1 10.0
antiviral activity, antifungal and antiprozotozoal ME2 (MJ/Kg) 12.46
activity (Harris et al, 2001).
 CP (g(Kg) 215.8 evaluate the decrease of Salmonella enteric in
Ca (%) 12.46 surfaces, and faeces. Once the treatment in water
AP3 215.8 with Garlicon® 7 days finished, it was made the
Lys (%) 1.16 nebulization with a formula based on Garlicon®
Met + Cys (%) 0.93 and the right carriers to obtain a homogenous
1
Provided per kilogram of diet: vitamin A, 1,600 IU; vitamin E, 10 nebulizate. It was made a cold nebulization of
mg; vitamin D3, 220 IU; niacin, 35.0 mg; d-pantothenic acid, 12 mg;
riboflavin, 3.6 mg; pyridoxine, 3.5 mg; thiamine, 2.4 mg; menadione, Garlicon® Nebulo, producing an aerosol with a
1.3 mg; folic acid, 1.4 mg; biotin, 0.15 mg; vitamin B12, 0.03 mg;
Mn, 60 mg; Zn, 40 mg; Fe, 80 mg; Cu, 8 mg; I, 0.3 mg; Se, 0.1 mg.
2
particle size between 0.5 and 20 microns. The
The ME value is calculated; the other nutrition levels are analyzed.
3
AP = available phosphorus. application rate of the product was recommended
by the machine manufacturer at 1:9 (1 L
1. Sampling procedure
Garlicon® + 9 L of water).
A rigorous microbiological study was
3. Culture medium used
conducted by sampling before and after treatment
For the isolation and enumeration of
with Garlicon®. We proceeded to the collection of
Salmonella spp. SESAME (Salmonella
fresh stool in sterile containers before and after
chormogenic medium) was used as pre-
treatment with the product. In addition, samples of
enrichment medium from Bioko Diagnostics
sewage and surface in plant and machinery were
(Beauvais, France). For the growth of
taken. It were used sterile swabs (Deltalab, Spain)
Enterococcus and Enterobacteriaceae groups it
previously soaked in sterile diluents. After the
was used commercial media Agar Conckey Rothe
collection procedure, samples were preserved
and Mac respectively, of the same company.
properly identified and shipped refrigerated to the
Quantification of total anaerobes in the
laboratory for analysis. Samples of all surfaces
faeces was performed with Wilkins- Chalgren
were taken (cages, feeders, egg belt walls, floor)
business environment (Bioko Diagnostics). This
before treatment and after treatment: 50 samples
medium does not require the addition of blood to
from pool of faeces before and 50 after, and 15
the successful growth of the vast majority of
samples from sewer before and 15 samples after
anaerobic organisms and contains yeast extract
treatment. Analytical samples were taken and
that provides the vitamins, purines and pyrimidines
surface air before and after the nebulizer treatment.
necessary for the growth of Peptostreptococcus
The air collection procedure was based on the agar
spp, Prevotella spp. It is also supplemented with
impaction method (ISO 24-698) by
arginine, hemin, sodium pyruvate, and vitamin K
aerobiocolector air Test Omga (LCB, France). The
to ensure the growth of Eubacterium, Veillonella
volume of air sampled in each unit was 100 L.
and Bacteroides.
2. Test
The selective growth of bifidobacteria was
Test consisted of administering 100 ppm
held amid enrichement to propound acid cysteine
of Garlicon® (natural product from Garlic/onion
described by Beerens, 1990. Finally, for
whith contrasted antimicrobial activity) in drinking
lactobacillus medium count, it was used
water during 7 days to 36.000 laying hens to
LAMVAB described by Hartemink et al
(Hartemink et al., 1997). This is a highly selective Results and Discussion:
medium for this group due to their low pH and
added vancomycin that are sensitive to The prevalence of Salmonella enteric in
Bifidobacterium, Streptococcus, Enterococcus and the faeces of the animals of study before treatment
other groups present in faeces and which are was 17.8%, and after treatment was 2%. This 2%
resistant lactobacilli. It is also added bromocresol is the prevalence of Salmonella in faeces that it
green as indicator to identify colonies that utilize were taken on the tape. Really, it is possible that
sugars from the medium by turning the indicator. the final prevalence of Salmonella was near 0%, in
the faeces of the treated group. Samples faeces
4. Microbiological analysis of stools were taken from a dirty tape, probably previously
Pretreatment of stools for microbiological contaminated. Anyway, results show a significant
analysis was performed in phosphate-buffered drop of 15,8% in the incidence of Salmonella after
saline (PBS: 8 g / L NaCl, 0.2 g / L KCl, 1.15 g / L treatment with Garlicon® which proves its
Na2HPO4, and 0.2 g / L KH2PO4 pH 7.3) with effectiveness.
added cysteine hydrochloride (0.5 g / L) to
promote the survival of anaerobic species. The
A B
initial dilution (1:10) Stomacher mechanically 17,8
2%
%
homogenized for 2 minutes. Since this, different
dilutions were made in sterile saline to 1%. After
that, were seeded by triplicate in different
media. The plates were incubated at 37 ° C
aerobically for Mac Conckey and Rothe and Figure 1. Prevalence of Salmonella on the farm (faeces
anaerobic chamber for the determination of total count). A before treatment with Garlicon®. B after
anaerobes and Bififobacterium. Different treatment with Garlicon®.
incubation times were adapted to the demands of
growth for each microbial group and were 24, 48 BEFORE AFTER Total nº
® ®
GARLICON GARLICON samples
or 72 hours.
5. Research Presence / absence of Faeces 8/47 1/50 97

Salmonella Sewers 3/15 0/15 30

To investigate the presence / absence of
Surfaces 2/30 0/30 60
Salmonella in fecal samples and the surface taken
with swabs, we proceeded to pre-enrichment in Table 2. Research of Salmonella spp. confirmed
positive samples analyzed.
BPW at 37 ° C for 18 ± 2 hours followed by a
subsequent enrichment in broth SESAME final
Regarding evolution of Enterobacteriacea
confirmation between Bioko COMPASS
population, there was a significant decreasement in
Salmonella agar Diagnostics (Beauvais, France).
total enterobacteria in stools of hens after receiving
 10

®
Garlicon treatment with a reduction of more than 9

two log units per gram of faeces. Garlicon® 7

LOG ufc / g heces

treatment causes a reduction of total enterobacteria 6

in faeces, so, apart from the importance in 4

reducing the prevalence of Salmonella sp. and 3

other foodborne pathogens, we expect a preventive 1

0
effect against secondary digestive diseases related Enterococcus spp. Anaerobios totales Lactobacillus spp. Bifidobacterium spp. Enterobacteriaceae

with E.coli presence, and probably Clostridium sp,

Figure 2. Microbial counts of enterococci, total
as it has been observed in other in vitro trials.
anaerobes, lactobacilli, bifidobacteriaceae and

BEFORE SD AFTER SD enterobacteriaceae in feces of laying hens before and

GARLICON GARLICON ®
after treatment with Garlicon in drinking water during
Enterococcus 6,01 0,59 6,17 0,85 7 days. Results are expressed as logarithm of colony
forming units per gram of fecces. Black: Log CFU/g of
Total 7,69 0,62 7,72 0,78
Anaerobics feces before treatment. White: log CFU/g of feces after
®
treatment with Garlicon .

Lactobacillus 5,62a 0,71 8,56b 0,68 UFC/m

3
Bacteria Fungi

Bifidobact. 6,88a 0,52 8,35b 0,64

Before treatment 3.350 2.500
Enterobact. 7,51a 0,80 5,18b 0,59 ®
with Garlicon

After treatment 250 47

Table 3. Counts of major microbial groups present in with Garlicon
®

faeces before and after treatment with Garlicon®.

Results are expressed in logarithmic units of colony
forming units per gram of faeces (Log CFU/g). Table 4. Prevalence of Bacteria and fungi in the
environment before and after nebulization with
As can be observed in table 3, after
®
Garlicon (CFU/g)
treatment with Garlicon® there is a significant
increasement in the prevalence of beneficial flora
Treatments did not affectt feed or water intake,
(Lactobacillus and Bifidobacterium).
neither egg laying rate or any other productive
Nebulization with Garlicon®, significantly
parameter, while it is detected a trend regarding
improved the microbiological quality of the
decreasing mortality during treatment (See table
installation as is shown in table 3. Total bacteria
5).
counts and fungi were decreased. Maybe the most
important notice, is that treatment was made on
animals presence, without any negative effect on
yielding rate, health status or feed/water
comsuption.
 pathogens but without negative effects of
Treatment Drinking Mortality
day water (nº birds) conventional antibiotics.
(m3) - Higher mucus secretion that results in a impaired
Day 0 5.8 10 adhesion of pathogens and thus to produce an
overall benefit to the birds.
Day 1 6.3 17
Furthermore, environmental disinfection with this
Day 2 6.5 15
molecules, leads to a lower pathogen pressure
Day 3 7.1 1 making it easier to control Salmonella prevalence
Day 4 6.4 2 in farms, animals and animals products.

Day 5 6.5 2
Day 6 5.4 3
Day 7 6.7 4 efficacy and food security and all this qualities are
Day 8 6.5 4 important to maintain productivity and to offer a
Table 5.
Conclusions:
Antimicrobial properties of plant extracts, spices
and herbs are well known, less studied are
secondary effects at gut level that help to
understand the overall activity on animals
performance and health. As phytogenic feed
additives, garlic/onion and other plants may
favourably affect gut functions (passage rate of
ingesta, activity of digestive enzymes, and nutrient
absorption). Other authors reported improved gut
mucus secretion after use garlic extracts (Jamroz et
al., 2006), and this mucus secretion decrease
pathogens pressure, because microorganisms have
less adhesion chances. So, it is pointed that at
least two mechanisms could be involved regarding
benefical effects observed after use of the product
Garlicon®:
- High antimicrobial effect on enterobacteriaceae
and less effect on Lactobacilli and Bifidobacteria,
highlighting a selective activity against enteric
This results shows how Garlicon® could be a
promising tool for poultry producers, keeping
better products for consumers.
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