Genetic Evaluation of TLR4 and CaLA-DRB Genes for Mastitis Susceptibility in Dairy Goats and the Response of Mastitis-Bacteria to Garlic and Black pepper Extracts
GENETIC EVALUATION OF TLR4 AND CaLA-DRB GENES
FOR MASTITIS SUSCEPTIBILITY IN DAIRY GOATS AND
THE RESPONSE OF MASTITIS-CAUSING BACTERIA TO
GARLIC AND BLACK PEPPER EXTRACTS
MOLEFE PETLANE
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
2012
DECLARATION
I, Molefe Petlane, hereby declare that the work on which this thesis
entittled;
GENETIC EVALUATION OF TLR4 AND CaLA-DRB GENES FOR
MASTITIS SUSCEPTIBILITY IN DAIRY GOATS AND THE RESPONSE
OF MASTITIS-CAUSING BACTERIA TO GARLIC AND BLACK PEPPER
EXTRACTS
is based on my original work and produced through the guidance of my academic
advisors and that neither the whole work nor part of it has been submitted for
another degree at this or any other University.Where other sources of information
have been used, they have been acknowledged in the text as well as in the
references.
Bogor, July 2012
MOLEFE PETLANE
D151108141
List of Acronyms and Abbreviations
ANOVA
Analysis of Variance
BAU
Bogor Agricultural University
BMSCC
Bulk Milk Somatic Cell Counts
BoLA
Bovine Leukocyte Antigen
BPA
Baird-Parker Agar
BPW
Buffered Peptone Water
CaLA
Caprine Leukocyte Antigen (see GoLA)
CD4
Cluster of Differentiation4
CFU
Colony Forming Units
CMT
California Mastitis Test
DCM
Dichloromethane
DMSCC
Direct Microscopic Somatic Cell Count
DMSO
Dimethyl Sulfoxide
DMRT
Duncan’s Multiple Range Tests
DNA
Deoxyribonucleic Acid
dNTP
Deoxyribonucleotide Triphosphate
DRB
Designation for a region in CaLA/GoLA/BoLA/MHC
EDTA
Ethylenediaminetetraacetic Acid
EMBA
Eosin Methylene Blue Agar
EtBr
Ethidium Bromide
FAOSTAT
Food and Agricultural Organization Statistics
FDA
Food and Drugs Administration
FEZL
Forebrain Embryonic Zinc-Like
GLM
General Linear Model
GoLA
Goat Leukocyte Antigen (see CaLA)
H-W
Hardy Weinberg Equilibrium
IPB
Institut Pertanian Bogor
IRAK
Interleukin- Receptor –Associated Kinase
LP
Lipopolysacchirdes
LSCS
Lactation Average of Somatic Cell Scores
MDR
Multi Drug Resistant
MgCl2
Magnesium Chloride
MHA
Mueller Hinton Agar
MHC
Major Histocompatibility Complex
MIC
Minimum Inhibition Concentration
MyD88
Myeloid Differentiation88
NA
Nutrient Agar
NB
Nutrient Broth
NFKB
Nuclear Factor-kappa-B
PAMP
Pathogen Associated Molecular Patterns
PCR
Polymerase Chain Reaction
PIC
Polymorphic Information Content
RFLP
Restriction Fragment Length Polymorphism
RNA
Ribonucleic Acids
SCC
Somatic Cell Count
SCS
Somatic Cell Scores
SNF
Solid Non-Fat
TCR
T-Cell Receptor
TLR4
Toll-like Receptor4 gene
TNF
Tumor Necrosis Factor
TRAF5
TNF Receptor Associated Factor6; see TNF
UV
Ultraviolet
ABSTRACT
MOLEFE PETLANE. D151108141. Genetic Evaluation of TLR4 and CaLADRB Genes for Mastitis Susceptibility in Dairy Goats and the Response of
Mastitis-Bacteria to Garlic and Black pepper Extracts. Supervised by
RONNY R. NOOR and RARAH R.A. MAHESWARI.
Mastitis is a bacterial disease that affects mammary glands of nursing animals. It
results in serious production and economic losses due to costs attached to its
treatment and loss of animals through culling. This study was aimed at evaluating
TLR4 and CaLA-DRB genes for mastitis susceptibility in Saanen, PE and PESA
using PCR-RFLP and also to investigate the antibacterial effects of garlic and
black pepper extracts on mastitis causing bacteria. TLR4 is a transmembrane
protein that recognizes PAMPs, convey signals into nucleus thus causing
transcription factors that inhibit primary infection while DRB is a cell surface
protein that recognizes antigens and present such antigens to T-cells and CD4.
Allicin is an active ingredient in garlic; it confers antibiotic properties by
interfering with the bacterial polymerase enzyme system thus inhibiting the
formation of nucleic acids whereas piperine is an alkaloid that forms active
ingredient in black pepper. Piperine alters the permeability of bacterial membrane
and inhibits NorA efflux pump mechanism thus causing leakage and accumulation
of extracellular substances into the cell which may cause cell death. A total of 68
lactating dairy goats; consisting of animals from Balitnak, Caprito farm, Cordero
dairy farm and Bangun dairy farm were used in this study, the population
consisted of both mastitic and healthy animals. Genetic polymorphism was
detected by digesting TLR4 PCR product with AluI while DRB amplicon was
digested with PstI and TaqI in two separate reactions. Antimicrobial tests were
performed using agar well diffusion technique. The study found that TLR4|AluI
locus was monomorphic and fixed with allele “T” in all breeds analyzed. Allele P
had high frequency in PE (0.7069) followed by PESA (0.6900) then Saanen
(0.6705) while allele T had high frequency in PESA (0.9205), followed by PE
(0.8976) then Saanen (0.7188). DRB|PstI and DRB|TaqI loci were found
polymorphic where Saanen exhibited most heterozygosity. DRB|PstI had moderate
to high PIC in all breeds while DRB|TaqI had low PIC in PE and PESA. χ2 tests
indicated that with the exception of DRB|TaqI locus in PE and in Saanen, all
breeds were still in Hardy-Weinberg equilibrium. Staphylococcus aureus was the
most frequently (80%) isolated bacteria in mastitic animals while Escherichia coli
was less frequent (
FOR MASTITIS SUSCEPTIBILITY IN DAIRY GOATS AND
THE RESPONSE OF MASTITIS-CAUSING BACTERIA TO
GARLIC AND BLACK PEPPER EXTRACTS
MOLEFE PETLANE
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
2012
DECLARATION
I, Molefe Petlane, hereby declare that the work on which this thesis
entittled;
GENETIC EVALUATION OF TLR4 AND CaLA-DRB GENES FOR
MASTITIS SUSCEPTIBILITY IN DAIRY GOATS AND THE RESPONSE
OF MASTITIS-CAUSING BACTERIA TO GARLIC AND BLACK PEPPER
EXTRACTS
is based on my original work and produced through the guidance of my academic
advisors and that neither the whole work nor part of it has been submitted for
another degree at this or any other University.Where other sources of information
have been used, they have been acknowledged in the text as well as in the
references.
Bogor, July 2012
MOLEFE PETLANE
D151108141
List of Acronyms and Abbreviations
ANOVA
Analysis of Variance
BAU
Bogor Agricultural University
BMSCC
Bulk Milk Somatic Cell Counts
BoLA
Bovine Leukocyte Antigen
BPA
Baird-Parker Agar
BPW
Buffered Peptone Water
CaLA
Caprine Leukocyte Antigen (see GoLA)
CD4
Cluster of Differentiation4
CFU
Colony Forming Units
CMT
California Mastitis Test
DCM
Dichloromethane
DMSCC
Direct Microscopic Somatic Cell Count
DMSO
Dimethyl Sulfoxide
DMRT
Duncan’s Multiple Range Tests
DNA
Deoxyribonucleic Acid
dNTP
Deoxyribonucleotide Triphosphate
DRB
Designation for a region in CaLA/GoLA/BoLA/MHC
EDTA
Ethylenediaminetetraacetic Acid
EMBA
Eosin Methylene Blue Agar
EtBr
Ethidium Bromide
FAOSTAT
Food and Agricultural Organization Statistics
FDA
Food and Drugs Administration
FEZL
Forebrain Embryonic Zinc-Like
GLM
General Linear Model
GoLA
Goat Leukocyte Antigen (see CaLA)
H-W
Hardy Weinberg Equilibrium
IPB
Institut Pertanian Bogor
IRAK
Interleukin- Receptor –Associated Kinase
LP
Lipopolysacchirdes
LSCS
Lactation Average of Somatic Cell Scores
MDR
Multi Drug Resistant
MgCl2
Magnesium Chloride
MHA
Mueller Hinton Agar
MHC
Major Histocompatibility Complex
MIC
Minimum Inhibition Concentration
MyD88
Myeloid Differentiation88
NA
Nutrient Agar
NB
Nutrient Broth
NFKB
Nuclear Factor-kappa-B
PAMP
Pathogen Associated Molecular Patterns
PCR
Polymerase Chain Reaction
PIC
Polymorphic Information Content
RFLP
Restriction Fragment Length Polymorphism
RNA
Ribonucleic Acids
SCC
Somatic Cell Count
SCS
Somatic Cell Scores
SNF
Solid Non-Fat
TCR
T-Cell Receptor
TLR4
Toll-like Receptor4 gene
TNF
Tumor Necrosis Factor
TRAF5
TNF Receptor Associated Factor6; see TNF
UV
Ultraviolet
ABSTRACT
MOLEFE PETLANE. D151108141. Genetic Evaluation of TLR4 and CaLADRB Genes for Mastitis Susceptibility in Dairy Goats and the Response of
Mastitis-Bacteria to Garlic and Black pepper Extracts. Supervised by
RONNY R. NOOR and RARAH R.A. MAHESWARI.
Mastitis is a bacterial disease that affects mammary glands of nursing animals. It
results in serious production and economic losses due to costs attached to its
treatment and loss of animals through culling. This study was aimed at evaluating
TLR4 and CaLA-DRB genes for mastitis susceptibility in Saanen, PE and PESA
using PCR-RFLP and also to investigate the antibacterial effects of garlic and
black pepper extracts on mastitis causing bacteria. TLR4 is a transmembrane
protein that recognizes PAMPs, convey signals into nucleus thus causing
transcription factors that inhibit primary infection while DRB is a cell surface
protein that recognizes antigens and present such antigens to T-cells and CD4.
Allicin is an active ingredient in garlic; it confers antibiotic properties by
interfering with the bacterial polymerase enzyme system thus inhibiting the
formation of nucleic acids whereas piperine is an alkaloid that forms active
ingredient in black pepper. Piperine alters the permeability of bacterial membrane
and inhibits NorA efflux pump mechanism thus causing leakage and accumulation
of extracellular substances into the cell which may cause cell death. A total of 68
lactating dairy goats; consisting of animals from Balitnak, Caprito farm, Cordero
dairy farm and Bangun dairy farm were used in this study, the population
consisted of both mastitic and healthy animals. Genetic polymorphism was
detected by digesting TLR4 PCR product with AluI while DRB amplicon was
digested with PstI and TaqI in two separate reactions. Antimicrobial tests were
performed using agar well diffusion technique. The study found that TLR4|AluI
locus was monomorphic and fixed with allele “T” in all breeds analyzed. Allele P
had high frequency in PE (0.7069) followed by PESA (0.6900) then Saanen
(0.6705) while allele T had high frequency in PESA (0.9205), followed by PE
(0.8976) then Saanen (0.7188). DRB|PstI and DRB|TaqI loci were found
polymorphic where Saanen exhibited most heterozygosity. DRB|PstI had moderate
to high PIC in all breeds while DRB|TaqI had low PIC in PE and PESA. χ2 tests
indicated that with the exception of DRB|TaqI locus in PE and in Saanen, all
breeds were still in Hardy-Weinberg equilibrium. Staphylococcus aureus was the
most frequently (80%) isolated bacteria in mastitic animals while Escherichia coli
was less frequent (