Baymix Grobig BS ®

Balance is
Performance
Bayer CropScience – Biologics
Delivering products that meet emerging market drivers
The same drivers in Agriculture are pushing
Animal Health
Regulatory Resistance
Reduced Pressure Management
Residue Health &
Increased Yield

Driven by Regulators & Driven by Manufacturers

Driven by Consumers, NGOs
Retailers & Global trade Driven by Manufacturers
Regulatory authorities are in Biologic products make ideal & Growers
Biologic products replace the process of de-listing partners to conventional
conventional products some of the more toxic, products to reduce New technologies to boost
allowing growers to meet older pesticides development of resistance the health and quality of
MRLs plants/animals leading to
increased yields
Core Business Product Line
Performance Proven and Accepted
T
Untreated Serenade
Efficacy of SERENADE® on Grape
(Chardonnay) Powdery Mildew,
Bunch Rot, Sour Rot (Yolo County,
CA - 1999)
Resistance Management Tool
Farms using conventional chemistry with
resistance issues applied Serenade 12/52
times a year for Mycosphaerella control .
Those farms responded better to
conventional systemic fungicides than farms
that did not use Serenade.
Quality & Consistent Manufacturing
• World Class Manufacturing site in Tlaxcala, Mexico
• Quality
• Bio-pharmaceutical plant purchased from Abbott*
• Rigorous QC delivering batch consistency
• Continuous improvement, over 1,400 runs
• Record yields continue into 2012

* Erythromycin for human health was originally the

primary product produced that the plant.
Process Optimization
Average lipopeptide concentration in AQ713 by production year
Bacillus subtilus, QST 713
Diverse Chemistry Directed at Specific Applications
140 Species in the Bacillus Collection

140 Species
Represented in the
Bacillus Collection
Commercial Use of Bacillus spp.

• Bacillus
• Genus is diverse, cosmopolitan
• Normal commensals of plants and animals
• Produce tough, highly stable spores compatible
with industrial formulation and processing
• Chemistry is as interesting as actinomycetes but
less exploited for technical reasons
Bacillus subtilis QST 713
 Patented growth performance enhancing strain
of B. subtilis
 Effective at managing necrotic enteritis
 Economical, efficacious alternative to AGPs
 Pricing comparable to mid-range/premium
AGP’s
 Commercially launched in the U.S. and India
for poultry
 Launched in 2010 with Alpharma as
Proflora: 713 combined with a MOS
 Grobig® BS is sold by Bayer in India;
registered in Korea & Thailand; registration
in process in over eight other countries
 QST 713 is now available in the US (2012)
as Optibac-S via HUVEPHARMA
 Spores Survive Feed Mill Processing
Kansas State University, Pilot Scale Feed Mill ,
2008
~90% recovery of spore germination following
pelleted feed formulation at 70°, 75°, 80 °or 85°C.
Extruded Aquaculture Feed Comercial Mill
2009
Extruder :210°F (99° C) for 2 minutes; RH > 25%
Dryer: 10 minutes @ 285°F (140°C) or, 20 minutes
@ 240°F (116°C); RH <10%
Loss of ½ log CFU during pellet formation.
Loss of ½ log CFU over 2 years at 25C°.
Quality Assurance Assays Commercial Trials
2010-2013
Pelleted feed targeting 106th CFU consistently test at
specification
Spores vs. vegetative cells
Poultry GIT and Transit Times
Multiple Attributes of Grobig BS
QST 713 Natural Product Chemistry

QST 713
Circular Genome Map of Grobig BS

Predicted Genes

Proteins

Chemistry
Clostridium perfringens Inhibition

MIC Cell free filtrate challenge: QST 713 grown overnight in a shake flask, spun and filtered (0.45µm).
75 µL of 1 X 105 challenge organism + 75 µL of diluted supernatant. Incubated overnight using

Anaerobe Gas Pack at 37°C .

Inhibition of Pathogens In vitro
O2,
CO2, Zone of
Culture Pathogen N2 inhibition
Name Bacteria ºC (mm)2
Clostridium Cp-1
perfringens Avirulent
necrotic 0
enteritis Cp-1
(NE)
Campy 3mm
Cp-2 gas; 3 Zone of Cp-2
ATCC 6-16% inhibition
O2
2-10%
Cp-3 Bacillus Cp-3
C02 0
Virulent
41°C swarming
(NE)
Cp-4
Cp-4 Bacillus
Virulent 0
swarming
(NE)
Campylobacter
jejuni
Human

Campylobacter
jejuni
ATCC

Campylobacter
jejuni
Poultry

Campylobacter
jejuni
Poultry
Transcriptional Profiling of Cell to Cell Interaction

C. jejuni C. perfringens
 Antibacterial Agent

Peptides Polyketides Bacteriocins

Surfactin Difficidins Subtilin
Mycosubtilin Mycobacillin Ericin S and A
Bacillomycins Bacillaene Mersacidin
Subtilosin Bacilysocin Epidermin
TasA Macrolactins Sublancin 168
Bacylsin Chlorotetain
Amicoumacins
Coreynebactin
Patent application on B. subtilis QST 713

Non antibiotic growth enhancer used as a potential replacement

for Antibiotic Growth Promoters, effective in controlling enteric
diseases.
Research Challenge Studies
Dose Efficacy Examination of QST713 in the Feed for the
Control of Necrotic Enteritis Caused by Clostridium perfringens
(Cp) in Broiler Chickens (IPE 2011)
Tmt # Treatment Name g of • 28-day cage study
Final Grobig/ • Mash fed ad-libitum, One-day-
Ton Feed old male Cobb-Vantress broiler
chicks.
T1 UTC 0g
• 100 cages, ten birds/cage Ten
T2 UTC Challenged 0g complete randomized blocks.
(Cp) • Day 14 inoculation with 5,000
T3 BMD (Cp) E. maxima cysts,
T4 GroBig (Cp) • 3 doses 108 cfu/ml C.
100 g perfringens (Cp) on days 19,
T5 GroBig (Cp) 20,&21
50 g
T6 Grobig (Cp) 10 g
Necrotic Enteritis Lesion; Ileum 200X
Population Ecology with QST 713
• IDEXX Labs Inc.
1) Quantitative Real time PCR methods
used to quantify the amount of QST
713 in the gut based on a strain
specific contig
2) SYBER Green-based real time PCR
used to assess the abundance of
signature 16S rDNa from key bacterial
populations in the back ground
community
Five probe based rtPCR tests;
1) Clostridium perfringens enterotoxin A gene (CPEA)
2) Salmonella spp.
3) Bacillus QST713
4) Pan Bakt internal control
5)Internal positive controls
Four SYBER Green based rtPCR tests;
1) Lactobacillus
2) Enterobacteriacea spp
3) Bacteriodales spp
4) Pan Bakt SYBER internal control
 Percent Mortality
Detection of Vegetative QST 713 via qRT-PCR

• Southern Poultry Research, Athens, Georgia USA

• Dr. Greg Mathis, Dr.Charles Hofacre
• LSD P=0.05 : Tukey’s HD
Detection of Enterotoxin A genes
C. perfringens Enterotoxin A
80000.0

70000.0

60000.0

50000.0

40000.0

30000.0

20000.0

10000.0

0.0

• Christian Lutenegger
• IDDEX Laboratories Sacramento CA
Lactobacillus and Bacteriodiales
Necrotic Enteritis Challenge
560 day-old broiler Cobb x Cobb male chicks (Cobb-Vantress
Hatchery, Cleveland, GA)
7 Treatments, 10 Replications, 8 birds/pen

1. Negative Control 5. Calsporin

2. BMD (50 ppm) 6. Gallipro
3. Grobig BS 100g 7. Gallipro-Tect
4. Clostat
• D14 inoculated with 5000 oocyst (E. maxima/bird)
• D19, 20, and 21 with C. perfringens (108 CFU/ml)
• Production performance was recorded throughout the 28 day
trial period
Southern Poultry Research, Georgia, USA
Body Weight Gain

Table 1. Bodyweight gain of broilers from 0-28 d

Body Weight Gain (g)
Treatment
0-28 Improvement (%)
Negative Control 570a 31.6
BMD 433c -
Grobig BS (100g) 557ab 28.6
Clostat 518ab 19.6
Calsporin 511ab 18.0
Gallipro 511ab 18.0
Gallipro-Tect 548ab 26.6
Feed Conversion Ratio

Table 2. Feed conversion ratio of broilers from 0-28 d

Feed Conversion Ratio
Treatment
0-28 Improvement (%)
Negative Control 1.479a 26.1
BMD 1.865c -
Grobig BS (100g) 1.605ab 16.2
Clostat 1.639b 13.8
Calsporin 1.643b 13.5
Gallipro 1.732bc 7.7
Gallipro-Tect 1.677b 11.2
Non-Challenge - Performance
1080 day-old broiler Cobb x Cobb male chicks (Cobb-
Vantress Hatchery, Cleveland, GA)
3 Treatments, 8 replications, 45 birds/replicate
1. Control
2. Baymix® Grobig® BS ( 100g = 1 x106 CFU/g)
3. Bacitracin Methylene Disalicylate, BMD (50 ppm)
Chicks received routine vaccination at the hatchery
• Dirty litter build up (~4 inches)
• Production performance was recorded during periods 0-
21, 0-35, and 0-42

Southern Poultry Research,

Georgia, USA
Body Weight Gain

Mean body weight gain of broilers on periods 0-21, 0-35, and

0-42 d.
Parameter Body Weight Gain (kg)

Period (d)
Treatment
0-21 0-35 0-42

Control 0.604 1.720 2.284b

Grobig BS 0.602 1.744 2.337a

BMD 0.597 1.737 2.356a

Data within a row that do not share superscripts are significantly different (P<0.05)
Feed Conversion Ratio

Adjusted feed conversion ratio of broilers on periods 0-21, 0-35, and 0-42 d.

Parameter Adjusted Feed Conversion Ratio

Period (d)
Treatment
0-21 0-35 0-42

Control 1.590a 1.728a 1.780a

Baymix® Grobig® BS 1.547b 1.679b 1.740b

BMD 1.539b 1.681b 1.733b

Data within a row that do not share superscripts are significantly different (P<0.05)
Taiwanese Commercial Farm

• Starting date：20130321
• Experimental target：ROSS broilers
• Experimental numbers：
- Control group：Up floor-19,250 heads
- Grobig group：Down floor-19,250 heads
• Immune plan：
- Day 4：ND+IB (spray)
- Day 12：IBD (water)
- Day 17-19：ND (water)
Taiwanese Commercial Farm

Grobig group Control group

First stage feed Butyric acid product(500 Butyric acid product(500
(D1~D11) g/ton feed) g/ton feed)
Grobig(100 g/ton feed)

Second stage feed Butyric acid product(500 Butyric acid product(500

(D12~D21) g/ton feed) g/ton feed)
Grobig(100 g/ton feed) Bio three*(500 g/ton feed)

Third stage feed Grobig(100 g/ton feed) Bio three(500 g/ton feed)
(D22~slaughter
house)

* Bio three: Probiotics product contains Bacillus mesentericus,

Streptococcus faecalis and Clostridium butyricum
Average Body Weight

Grobig group Control group ROSS standard BW

First week
213 g 199 g 185 g
(D7 measure)
Second week
597 g 562 g 473 g
(D14 measure)
Third week
1170 g 1130 g 916 g
(D21 measure)
Forth week
1868 g 1787 g 1479 g
(D28 measure)
Fifth week 2113 g
Mortality numbers by week

Grobig group Control group

First week 108 108

Second week 105 151

Third week 89 116

Forth week 81 100

The mortality rates all are reasonable (<1/1000), but Grobig group
has lower mortality numbers
Final Metrics
Grobig group Control group
Carcass weight 40,800 kg 40,060 kg
Feed intake 58,075 kg 61,035 kg
Live numbers 19,060 heads 18,900 heads
Survival rate 97% (99%)* 96% (98%)*
Average body weight 2.14 kg (31th Day) 2.11 kg (32th Day)
FCR 1.42 1.52
BW-FCR (g) 720 590

* Breeding farm provides 2% more heads to broiler farm, so the actual

survival rate has to minus this part.
Ancillary Metrics
Grobig: Feces are dry.
GI tracts are healthy.

Control: bloody materials, indicates

enteritis in the control group.
Commercial Poultry Farm – Northern India

 71,696 day-old broilers, 3 treatments

Treatments:
1. Basal diet + Grobig BS (1 x 106 CFU/g of feed)
(n=23,857)
2. Basal diet + Enramycin (90 g/ton of feed) (n=23,428)
3. Basal diet + other probiotics (100 g/ton of feed)
(n=24,411)
 Treatments were fed for 42 days
 Cumulative body weight, total feed consumption, feed
conversion, cumulative mortality, and total monetary gain
per bird.
Cumulative Mortality (%)
9.00 8.54
7.98
8.00

7.00
6.24
6.00

5.00

4.00

3.00

2.00

1.00

0.00
Grobig BS Enramycin Other Probiotics
Cumulative Body Weight (g/bird)

2215

2210
2210

2205

2200
2200

2195

2190
2190

2185

2180
Grobig BS Enramycin Other Probiotics
Feed Conversion
1.77
1.76
1.76

1.76
1.75
1.75

1.75

1.74

1.74
1.73
1.73

1.73

1.72

1.72
Grobig BS Enramycin Other Probiotics
Total Monetary Gain Per Bird
(Indian Rupees)
121.80
121.60 121.55
121.40
121.20
121.00
121.00
120.80
120.60
120.45
120.40
120.20
120.00
119.80
Grobig BS Enramycin Other Probiotics
Total Investment in the Product (Rps)
8400
8200
8200

8000

7800

7600 7515
7400
7200
7200

7000

6800

6600
Grobig BS Enramycin Other Probiotics

1 Indian Rupee = 0.015 US Dollar (4.7 cents; 15 cents)

1 Indian Rupee = 0.035 Brazilian Real (11 centavos; 35 centavos)
CARGILL, USA - Necrotic Enteritis
 Day-old broiler chicks, randomized into 6 treatments, 11 replicates
,
1. Control
2. BMD (50 ppm)
3. Baymix Grobig BS (1 x 106 CFU/g)
4. BMD (25 ppm) + Baymix Grobig BS (5 x 105 CFU/g)*
5. BMD (50 ppm) + Baymix Grobig BS (1 x 106 CFU/g)**
6. Tylosin (200 ppm) + Baymix Grobig BS (1 x 106 CFU/g)

 Day 8 – all birds/treatment were challenged with live vaccine (10x normal
dose)
 Day 14, 15, 16, and 17 - challenged (via feed) with Clostridium perfringens
 Production parameters were determined after 29 days

Cargill, Minnesota, USA

Feed conversion ratio 0-29 d
1.34

1.32a 1.32a
1.32

1.3
1.29abc
1.28abc
1.28
1.27abc

1.26

1.24c
1.24

1.22

1.2
Control BMD Grobig BS Grobig BS + Grobig BS + Grobig +
BMD* BMD** Tylosin

• ½ Rates for Grobig BS and BMD

• Full Rates of Grobig BS and BMD
Body Weight Gain
1900.0 1883.6a

1850.0 1839.7ab 1837.2ab

1821.5ab
1800.9b
1800.0

1750.0

1695.4c
1700.0

1650.0

1600.0
Grobig BS: Footpad Dermatitis
 Footpad dermatitis (FPD) causes
necrotic lesions on the footpads
 FPD have significant financial and
welfare implications
 In 2009 - US$ 280 million worth of
chicken paw exported to China
 Other European countries may
follow Sweden and Denmark in
adding FPD as measure
of broiler welfare
for national legislation
Commercial Integrator, USA
Paw Quality/Footpad Dermatitis Measurement
 480 day-old broiler Ross x Ross 708 male chicks
 3 Treatments, 8 replications, 20 birds/pen, for 40 days
1. Control
2. Baymix® Grobig® BS (100 g/ton, 1 x 106 CFU/g)
3. BMD (50 ppm)

 All diets were pelleted

 Chicken paws were assessed using the modified Ekstrand
score on periods 0-26 and 0-40 d:
0 = no lesion, slight discoloration of the skin
1 = mild lesion, superficial discoloration of the skin
2 = severe lesion, epidermis is affected, blood scabs,
hemorrhage, and severe swelling of the skin.
Paw Lesion: 26 days
0.700
0.620
0.600
0.539
0.500 0.462

0.400

0.300 Impr.
-13.1%*
Impr.
0.200 +16.7%*

0.100

0.000
Control Grobig BS BMD
Paw Lesion
0.700
0.626
0.600

0.500
0.439
0.401
0.400

0.300
Impr. Impr.
0.200 +42.6%* +56.1%*
0.100

0.000
Control Grobig BS BMD
Questions?
Opportunity: NH4+, Footpad dermatitis

 Baymix Grobig BS reduced the severity of FPD in

broiler chickens throughout the production period
 Baymix Grobig BS may be a more reliable dietary
intervention than BMD in reducing severity of FPD in
broiler chickens
The potential role of Baymix Grobig BS in improving
nutrient digestibility, reducing Ammonia, and
improving litter quality (known risk factors in the
etiology of FPD) - are areas that needs to be further
explored and investigated
Recommended Dose Rate

 Optimal dose recommendation – 100 g/ton of feed or

equivalent to 1 x 106 CFU/g of feeds
 Can be added to compound feeds prior to pelleting
 Available in 20 kg/box (5 kg x 4)
Grobig BS Advantage

 Highly stable during feed processing (pelleting process)

 Ability to maintain viability (acidic pH, biliary activity,
enzymatic activity) during transit in the GIT
 Highly defined mechanism of actions (competitive
exclusion, and antibacterial activity)
 Proven results in controlled research and field studies on
key performance parameters
 Complementary with common AGPs and coccidiosis
control program
 Price competitive
Period of Supplementation
The efficacy of Baymix Grobig BS depends on when it is
used:
 When young – day old chicks, the GIT microflora is not
yet established
 Period of stress – handling, shipping, vaccination, and
other situations that can be stressful to an animal
 Dietary changes or when the feed composition
encourages the proliferation of pathogenic micro-
organisms
 Antibiotic therapy – antibiotic treatments can lower
the number and growth of beneficial microbes in the
GIT
 In broilers we recommend from day old to harvest
Alphitobius (Poultry Darkling Beetle)
• Thailand spends ~ $300/year
/house on OPs, pyrethroids,
slaked lime, ‘combos’, etc.
• South Aftrica, Brazil (2,000 sq
m/house) are similar but the
acceptable cost is lower.
• In the US growers want a $60 to
$100.00 treatment for a 20,000
sq. ft. house.
• Imidicloprid was labeled and Ant Control
Darkling
Beetle Fly Control
sold into that range and now the Control Mite Control
southern beetles are showing
resistance ratio that run from 60
to 3600 (6x).
*Dr J.J. Arend’s view Roach
Wasp and
Spider Hornets Bed Bugs
Control Control
Mortality (lb/1000Sq. ft.) Laboratory Bioassay
100 94
91.5 SDS-Bt502
90 85.5
80 74.5
77
70
60 67.5
43.64 63.5
50
40
30 26.69 44.5
17.37
20
17.8
10 7.2
0 15.68 Larvae
5.08 9.75
Bt WDG Bt TP
3.000 0.300 Bt TP
Bt TP
Bt TP
Adults
0.300 +
0.150 + Bt TP
2808 5.2 0.075 +
2808 2.6 Bt TP
2808 1.3 0.150 + Bt WP
0.600 +
10%AI 2808 2.6
2808 5.2
3.000 +
+ CX 5.2
131lb/acre + CX
Turf Model 1.5 lb/20K Sq.ft.

13lb/acre
6 lb/20K Sq.ft.
Controlling Poultry Darkling Beetle
) SDS-502 Bt bacterium:
• Produces toxin protein known as “Cry8Da”
• Highly toxic to Turf Beetles and Emerald Ash borer larvae and adults
Laboratory Bioassay: Biologics Actives
Mortality (lb/1000Sq. ft.)
90
79.5 87
80
70
70
60 56.5
50
40
30 16.95
20 17.37 28.39
10 15.25
0

AQ20614 FD 5.5
AQ6121 FD 7.1
S. cinnabarinus AQ8798 FD 6.8
S. microflavus
B. amyloliquifaciens AQ2808 FD 5.2
B. pumilus
Culture QRD Lot# Conversion Factor lb/1000sqft
adult % control larval % Control
AQ20614 55.001 394.46 0.04g/ml 5.506608
AQ6121 31.002 468-19-1 0.05g/ml 7.15859
AQ8798 20.001 RF1936 0.05g/ml 6.88326
AQ2808 2.001 RF1940 0.038g/ml 5.231278
MIC Studies: Common AGPs
Minimum Inhibitory Concentration (MIC) µg/mL or ppm
Condition
Antibiotic
Aerobic Anaerobic Microaerophilic
Bacitracin >64 >64 >64
Chlortetracycline 1 0.25 0.25
Lincomycin 4 2 8
Oxyteracycline 8 2 2
Tiamulin >64 >64 >64
Tylosin 0.25 0.25 0.5
Amoxicillin <0.06 <0.06 <0.06
Colistin 32 16 64
Enrofloxacin <0.06 <0.06 <0.06
Neomycin 0.5 1 1
Penicillin <0.06 <0.06 <0.06
Spectinomycin 16 16 32
Sulfathiazole 16 8 32
Flavomycin 4 1 1
Apramycin 8 4 4
Imaging of Bacterial Cultures
• Imaging Mass Spectroscopy
using Matrix Assisted Laser
Desorption Ionization

• Useful tool to observe the

production of natural products
under varying conditions.

• Uniquely suited to provide Color

critical insights into the spatio- Scale is
temporal production of [Conc.]/
metabolites involved in Location
communication between of
colonies of competing/ Surfactin in
Bacillus subtilis
collaborating microbes. 168
Chemical Interaction
: Grobig BS vs. Pseudomonas

Pseudomonas aeriginosa Aerobic Grobig BS

Zones of inhibition are measured from the edge of

the bacillus to the beginning of the pathogen
 MALDI: Molecular Mass over Time
At 12 hours At 24 hours At 36 hours At 48 hours At 60 hours At 72 hours
we can see we can see we can see we can see we can see we can see m/z over time
m/z m/z m/z m/z m/z m/z 4500.0
427 PK 427 PK 427 PK 427 PK 427 PK 842 4000.0
466 465 466 713 617 1037 3500.0

Molecular Weights
642 578 PK 579 PK 842 714 1051 3000.0
580 617 618 1042 PEP 843 1058 2500.0
618 655 656 1059 PEP 843 1065 12hr
2000.0
656 842 844 1074 1044 PEP 1076 1500.0 24hr
714 1045 PEP 1046 PEP 1080 1045 1081
1000.0 36hr
843 1059 PEP 1053 PEP 1094 1060 1096
500.0
1045 PEP 1066 1061 1108 1076 1109
0.0
1060 1074 1067 1122 1094 1124 0 5 10 15 20 25 30
1075 1082 1076 1447 1110 1472
Number of Peaks
1088 1096 1083 1461 1124 362
1462 1110 1095 1476 1464 1486 m/z over time
1476 1124 1111 1500 1477 1500 4500.0
1486 1486 1125 1514 1488 1514 4000.0
1501 1501 1488 1528 1502 1528 Molecular Weights 3500.0
1515 1515 1515 1542 1516 1542 3000.0
1529 1529 1531 1556 1530 1988 2500.0
1543 1543 1545 1948 1545 3019 48hr
2000.0
4299 2224 1559 1986 1990 3374Bact 1500.0 60hr
4338 2979 1990 3019 3022 1000.0 72hr
3017 2020 3066 3060 500.0
3055 3023 3335 3348 0.0
3335 3379Bact 3372Bact 3379Bact 0 5 10 15 20 25 30
3374Bact 3947 Number of Peaks
3385
3412
Questions?