GENETIC APPROACHES TO COMBAT BOVINE PARATUBERCULOSIS

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GENETIC APPROACHES TO COMBAT BOVINE
PARATUBERCULOSIS
SEMINAR
ON

• SATISH KUMAR
• M.V.Sc scholar
• Division of AGB,IVRI

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OUTLINES

• Introduction
• Prevalence
• Economic loss
• Method to combat
• Medicinal
• Genetic
• Conclusion

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PARATUBERCULOSIS

• OIE in List B OIE., 2001
  
• Caused by Mycobacterium avium subspecies
  paratuberculosis
  Thorel et al., 1990
• Chronic granulomatous enteritis of ruminants
• weight loss, wasting diarrhoea
• and death from cachexia

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What a Johnes Cow Looks Like
Regardless of how they look, they are a constant
source of new infection - 95 of infected cows
show no signs
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• Johnes Disease Transmission Cycle

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PREVALENCE OF JOHNES DISEASE

• Globally prevalent 15 to 78
  NAHMC,1997
• 68.1 of US dairy farms are infected with MAP
• 29.0 of sero-prevalence of JD is reported in
  northern India.
• UP 31.9 and Punjab 23.3
  Singh et al., 2008
• Dairy cattle herds of Mathura district 28.3 by
  feacal culture and 20.8 by ELISA
  Mishra et al., 2009
• Endemic in India
  Tripathi et al., 2007

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PREVALENCE OF BOVINE JD Source http//crohn.ie/archive/johne.htm PREVALENCE OF BOVINE JD Source http//crohn.ie/archive/johne.htm PREVALENCE OF BOVINE JD Source http//crohn.ie/archive/johne.htm
COUNTRY PREVALENCE REFERENCE
UK 3.5 of cattle Cetinkaya et al., 1996
USA 18 of cattle Chiodini et al., 1986
CANADA 6.1 of cattle NcNab et al., 1991
SWITZERLAND 5.99 of cattle Meylan et al., 1995
DENMARK 47 of cattle Nielsen et.al,2000
NETHERLAND 55 of cattle Muskens et.al, 2000
AUSTRALIA 17 of cattle Jordan et al., 1996
SWEDEN Free of disease Viske et al., 1997
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ECONOMIC LOSSES

• Economic losses to the cattle industry in the
  United States due to PTB reaching 1.5 billion
  annually
  MICHIGAN STATE UNIV, 2012
• 15-20 less milk in
• subclinical carriers

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DIRECT LOSS
Mortality (genetic death)
Decreased milk production (15-20)
Culling of animals and their calves
Decreased fertility
Decreased productive age length
Decreased feed conversion ratio
Groenendaal, 2005
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INDIRECT LOSS

• Herd replacement
• Diagnostic testing
• Ineffective vet care
• Control programme
• Loss due to trade restrictions
• Loss of reputation of farm

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METHOD TO COMBAT

• VACCINATION
• MEDICINAL
• MANAGEMENTAL PRACTICES
• GENTIC APPROACH

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• VACCINATION
• Heat killed (strain 18)
• Heat killed (5889 Bergey)
• Live attenuated (316 F)
• MEDICINAL
• No drugs are approved
• Clofazimine or Isoniazid
• Rifabutin or Ethambutol
• D-cycloserine, amikacin
• Clarithromycin

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GENETIC APPROACHES
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CANDIDATE GENE APPROACH

• Gene suspected of being involved in the
  expression of a trait such as a disease
• Can be identified by
• Association with the phenotype
• linkage analysis to a region of the genome
• Potential source of markers for MAS

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STEPS
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CANDIDATE GENES FOR PTB

• SLC11A1
• TLR
• CARD15
• IL-10
• IL-10Ra
• IL-10Rß
• IFN-?
• TGFß1
• TGFßR class I/II

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SOLUTE CARRIER FAMILY 11 MEMBER A1 (SLC11A1)

• Natural resistance associated macrophage protein
  1, NRAMP1
  Blackwell et al., 1995
• NRAMP1 is located in the BTA2 and comprises 15
  exons
• Size 10926bp
• Expessed in phagosomes of macrophages, monocytes
  and in neutrophils.
  
  Cellier et al., 1994
  
• Acidification of the phagosomes, regulation of
  nitric oxide and vacuolar iron concentrations
  Awomoyi, 2007
  

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SLC11A1 POLYMORPHISM

• A microsatellite in the 3UTR region called HORIN
  has a genetic association between this
  polymorphism and resistance to BPT in Friesian
  cattle
  Estonba et al., 2005
• SNP N23 was found to be genetically associated
  with resistance to the PTB(p0.0478) Ruiz
  et.al 2007
• Transmembrane domain8(1067CgtG)
• Intron11-12(1157-91AgtT)
• Suggest a link with JD susceptibility

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Toll Like Receptors 2 (TLR2)

• Pattern recognition receptors
• Chromosome Location BTA 17
• Size13223 bp
• Expressed in monocytes, DC, macrophages and
  polymorphonuclear cells
• Function Recognition of mycobacteria,
  stimulating phagocytosis, development of cell
  mediated immunity Quesniaux
  et al., 2004

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TLRs POLYMORPHISM

• SNPs found in
• TLR1(G685A)
• TLR2(2038AgtG)
• TLR4(892GgtY)
• These SNPs suggest a link with JD susceptibility
  in several breeds of cattle.
• 
  Mucha et al., 2009

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CASPASE RECRUITMENT DOMAIN 15 (CARD15)

• Earlier known as Nucleotide Oligomerisation
  domain (NOD2)
• Bovine Card15 is located in BTA18 and comprises
  12 exons
• Size 30636bp
• Expressed in monocytes, macrophages, dendritic
  cells and epithelial cells
• Function Induce cytokine production by
  activating NF-?B

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CARD15 POLYMORPHISM

• SNPs found in
• LRR domain (2197TgtC)
• 3-UTR domain (1908CgtT)
• Significantly related to paratuberculosis
  susceptibility
• Breed effect is noticed with highest frequency of
  variant allele in Brahman Angus cattle
• SNP C12 of Card15 ,
• Frequency of the G allele was significantly
  higher In control animal
• SNP C25 of Card15
• minor allele, T, is significantly more frequent
  in control
• 
  
  Ruiz O et.al,2007

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Interleukin 10 receptor alpha(IL-10Ra)

• Determinant of IL10 responsiveness
• IL10 production is decreased by increased iNOS
  expression

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IL-10Ra polymorphism

• Four IL-10Ra related synonymous SNPs
• 984G gt A
• 1098C gt T
• 1269T gt C
• 1302A gt G
• Statistically significant association with MAP
  infection with a strong additive and dominance
  relationship at the GCTA allele
• 
  
  Verschoor et al., 2010

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IL-10Ra polymorphism

• Three of the SNPs in the IL-10Ra gene
• SNP IL-10Ra 1047CgtA
• SNP IL-10Ra 1398GgtA
• SNP IL-10Ra 1599CgtT
• AAT haplotype in three of the SNPs in the
  IL-10Ra gene showed a significant effect in the
  negative cohort.
• AGC haplotype is most frequent haplotype in the
  positive cohort
• Patent US20110039918 - Single Nucleotide
  Polymorphisms (SNPs) in Genes Associated with
  Inflammatory disease Feb 17, 2011

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IFNG-?

• Interferons constitute a multi-gene family of
  inducible cytokines. IFNG Type II interferon
• Chromosome Location BTA 5
• Size 4824bp
• Function Interferes with viral RNA and protein
  synthesis, increase antigen presentation, potent
  activator of macrophages and has anti-tumour
  properties

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IFN-? polymorphism

• A significant association of MAP infection is
  found for SNP(2781GgtT) in exon1 region of IFN ?
  gene
• 
  
  Pinedo et al. 2009

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GENE POLYMORPHISMS ASSOCIATED WITH JD IN CATTLE GENE POLYMORPHISMS ASSOCIATED WITH JD IN CATTLE GENE POLYMORPHISMS ASSOCIATED WITH JD IN CATTLE GENE POLYMORPHISMS ASSOCIATED WITH JD IN CATTLE GENE POLYMORPHISMS ASSOCIATED WITH JD IN CATTLE
Gene Microsatellite SNP Location Reference
CARD15 - 2197TgtC LRR Pinedo et al. 2009 Pinedo et al. 2009
- 1908CgtT 3UTR Ruiz-Larranaga et al., 2010
IFN? - 2781G/T Exon 1 Pinedo et al. 2009
SLC11A1 275/ - 3UTR Pinedo et al. 2009
279/ - 3UTR Pinedo et al. 2009
- 1067CgtG Transmembrane domain8 Ruiz-Larranaga et al.,2010
- 1157-91AgtT Intron11-12 Ruiz-Larranaga et al.,2010
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Gene SNP Location Reference
TLR1 G658A Ectodomain Mucha et al., 2009
TLR2 2038AgtG Toll/IL-1R domain Mucha et al., 2009
1903TgtC Putative LRR Koets et al., 2010
TLR4 892GgtY Ectodomain Mucha et al., 2009
895GgtA Ectodomain Mucha et al., 2009
1165GgtA Ectodomain Mucha et al., 2009
1167TgtC Ectodomain Mucha et al., 2009
IL10RA 984GgtA Coding Verschoor et al., 2010
1098CgtT Coding Verschoor et al., 2010
1269TgtC Coding Verschoor et al., 2010
1302AgtG Coding Verschoor et al., 2010
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Genome wide association studies (GWAS)

• Studies of genetic variation across the entire
  genome
• Designed to identify associations between genetic
  markers observable traits, or the
• presence/absence of a disease or
• condition

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Polymorphism

• 22 SNPs on 7 different chromosomes are
  significantly asssociaated with Johnes disease
• Ex
• rs41962859, rs29012842, rs41579048,
  rs109621404 and rs110490432 rs110002076
  
  http //www.animalgenome.org/cgi-bin/QTL
  db

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WORK DONE AT IVRI

• Three genotype were detected at SNPrs41945014
  TT(294bp),GG(196bp,98bp) and GT
  (294bp,196bp,98bp)
• The proportion of GG and GT genotypes were
  significantly higher in PTB positive animals
  suggested that selection against these 2
  genotypes may confer resistance against bovine
  paratuberculosis
• 
  Yadav et.al.,2014

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CONCLUSION

• Advances in techniques used to study livestock
  genomes has increased the use of candidate gene
  and GWAS based discoveries
• There is risk of inaccurate classification of
  infected and non infected animals
• With further potential marker evaluation, marker
  assisted breeding approaches can lead to
  protection of animal populations against paraTB

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