Mycobacterium paratuberculosis avium
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Transcript of Mycobacterium paratuberculosis avium
Mycobacterium avium : A food
borne pathogen of concern
History
.
The organism was cultured from cattle in 1910 and
was classified as a Mycobacterium by Twort and
Ingram (1910, 1912)
Paratuberculosis was first described in
1895 by Johne and Frothingham
Identified in granulomatous lesions in the
intestines of affected cattle that stained
acid-fast indicating of Mycobacterial
organism
Mycobacterium avium subspeciesparatuberculosis (MAP)
Gram positive rod (0.5 x 1.5 micron)
Acid fast
Facultative intracellular
Obligate parasitic pathogen
Requires iron for growth
Virulence Factors
Intracellular pathogen
Grow and multiply inside macrophages
Chemically resistant Mycobacterial cell wall that is
resistant to destruction or penetration
Ability to neutralize antibacterial chemicals produced inside
macrophages
Toxic chemical components of the Mycobacterial cell wall
Complete Genome Sequence of MAP K-10
Single circular chromosome of 4.8 Mb and encodes 4,350
predicted ORFs, 45 tRNAs, and one rRNA operon.
In silico analysis identified >3,000 genes with homologs
M .tuberculosis
Availability of the complete genome sequence offers a
foundation for the study of the genetic basis for virulence
and physiology of MAP
Enables the development of new generations of
diagnostic tests for Johne's disease.
(Bannantine et al., 2005)
Natural Reservoirs
Natural reservoir – wild animal population
(Ruminants, Swine, Rabbit, Deer, Bison, Elk)
MAP can survive – 250 days in water feces, cattle
slurry
Manure from infected animal most common mode of
contamination
Vertical transmission during pregnancy
Disease
Digestive tract route of entrance of MAP
Multiplication of MAP in Intestinal mucosa
Phagocytosis by macrophages
Via lymph channels
Infiltration regional lymph node
Cause
Decreased absorption & chronic diarrhea
Muscle wasting and loss of weight
Severity leads to death
Leads to : Reduced milk yield
Reduced meat yield
Reduced reproductive performance
Potential Human Exposure to MAP
Presence of MAP in Milk & Milk Products
MAP has been detected in the following :
Colostrum
Raw Milk
Pasteurized Milk
Powdered Milk
Cheese
Goat and Sheep Milk
MAP in Colostrum
Colostrum good sample for MAP isolation
Early potential exposure of dairy calf
MAP detected in udder tissue supramammary lymph nodes (Chiodini et al, 1984)
(Streeter et al, 1995)
Cow’s MAP Positive Colostrum
Clinically normal but MAP infected 16%
Subclinically Infected 22 %
Clinically Infected 36%
MAP in Raw Milk
Sources of contamination:
• Direct shedding in milk
• Fecal contamination
• Mixing contaminated milk
MAP isolated from – Supramammary lymph node; deep udder tissue
(Sweeney et al ., 1992)
Country Samples Results Reference
UK 244 raw milk PCR –7.8 %Culture –1.6%
Grant et al, 2002
UK 816 raw milk Culture –3.3% Grant et al, 2005
Swiss 1384 bulk milk PCR – 19.7 % Corti and Stephan, 2002
Ireland 389 bulk raw milk IMS-PCR – 12.9 % Culture – 0.3 %
O’ Reilly et al, 2004
USA 1 493 bulk raw milk Culture –2.8% Jayarao et al, 2004
702 raw milk Culture –2.8% Ellingson et al, 2005
Czech Republic
244 raw milk Culture –1.6% Ayele et al, 2005
Australia 26 raw milk Culture –35.0% Taylor et al, 1981
Argentina 25 raw milk Culture –8.3% Paolicchi et al, 2003
Incidence of MAP in Raw Milk
Pasteurized Milk
MAP survival depends on initial load
12/27 HTST pasteurized milk MAP positive
(Grant et al, 2005)
Standard pasteurization temperature fails toguarantee full inactivation of milk
6log10 - 85% reduction (Doherty et al, 2002)
MAP isolated from milk treated at 82.5 °C
(Slana et al, 2008)
Homogenization and pasteurization
(Grant et al,2005)
Survival: Heat Resistance
Country VolumeTested (ml)
Map SurvivalObserved
Decontamination Reference
UK 50 Yes None (2002)HPC
Grant et al, 2002, Grant et al, 2005
GER 50-100 Yes None Hammer et al, 2002
NZ 50 No HPC Pearce et al, 2001
AUS 1500 Yes HPC McDonald et al, 2005
CZ 50 Yes HPC Ayele et al, 2005
NL 40 No HPC Rademaker et al, 2007
In most cases a 3 - 4 log kill Achieved with Pasteurisation
Survival depends on initial contamination level
MAP in Powdered milk
Coffee cream, whole milk powder, half-fat milk, skimmedmilk,and baby food can also be contaminated
Children's at higher risk
Crohn’s disease in children's in Europe, 2004
Baby food contamination - 51 different samples, from 7European countries were examined in which 25 (49.0%) sampleswere found positive. (Hruska et al, 2005)
MAP in cheese
MAP has been detected from market cheese
(Clark et al,2006)
Sub pasteurization temp treatment of milk forcheese production insufficient for MAPinactivation (Pearce et al,2001)
Occurrence of MAP in Cheese by PCR
Greece 50 %
CZ 12 %
USA 5 % (Ayele et al, 2004)
MAP in Sheep & Goat Raw Milk
104 sheep and goat milk sample analyzed in UK
PCR - 1% (Grant et al, 2001)
340 goat milk sample analyzed in Norway
IMS-PCR- 7.1 % (Djanne et al, 2003)
In India, MAP isolated from milk and feces ofinfected goat (Singh and Vihan 2004)
Effect of Food Processing Steps on MAP
Clarification, centrifugation, separation,standardization and homogenization
Homogenization – increases MAP count
Centrifugation and microfiltration –removes MAP 95-99.9% (Grant et al, 2005)
Homogenization and Pasteurization – moreeffective for MAP inactivation (Grant et al, 2005)
Processing of Dairy Products
NaCl has little or no effect in cheese
Low pH significantly contribute MAP inactivation
Ripening of cheese significantly lower MAP
Temp and low pH – most important factor in MAPinactivation during ripening
Persistence of MAP in cheeseHigh conc. of MAP in raw milkShort ripening period
(Spahr and Schafroth, 2001)
Authors Country Samples Results (% + tive)
PCR Culture
Gao et al. 2002 Canada 710 retail pasteurized milk samples 15 0
Grant et al. 2002
UK 567 commercially pasteurized milk samples 11.8 1.8
O ‘ Reilly et al. 2004
Ireland 357 pasteurized milk samples 9.8 0
Ayele et al. 2005
Czech Republic 244 commercially pasteurized milk samples NA 1.6
Ellingson et al. 2005
USA 702 retail pasteurized whole milk samples NA 2.8
Ikonomopouloset al. 2005
Greece and Czech Republic
Retail cheeses (Feta, soft hard and semihard cheese )
31.7 3.6
Clark et al. 2006
USA 98 samples of retail cheese curds 5 0
Stephan et al. 2007
Switzerland 142 raw milk cheese samples (soft, semihard and hard)
4.2 0
MAP in Retail Dairy Products
MAP in Raw Meat Product
MAP isolated from GI tract and other organs of culled dairy animals.
(Antognoli et al, 2008)
Meat contaminated with MAP by
Dissemination of pathogen in tissue
Fecal contamination
Fleece contamination
Wool and skin
Redistribution during washing
Culture method
PCR based method
ELISA
Detection Mycobacterium avium subsp.
Paratuberculosis
MAP Detection-Culture Method
Media –Herrold’s EggYolk Medium (HEYM)
Antibiotics-
PANTA- Polymyxin B, Amphotericin B,
Nalidix Acid, Trimethoprin, Azocillin
VAN - Vancomycin, Amphoterin B, Nalidxic Acid
Additive- Mycobactin J
Decontamination of sample:
1) NaOH 2) HPC
Disadvantage
Long culture period – 4 - 12 week
Fastidious growth requirements
Contamination
Advantages
•Gold standard• Simple and widely used method
PCR Based Methods
IS 900 – for M. paratuberculosis
IS 901 – for M. avium
IS 1245 – for Mycobacterium avium complex
hsp X gene – putative heat shock protein
F57 – diagnostic probe for MAP
Real Time PCR-
IS 900 sequence – (Khare et al, 2004)
F57 – (Stephan et al, 2007)
Drawbacks
PCR inhibitors - present in fecal, milk, milkproduct samples
Cant differentiate between live and dead cell
Chances of cross amplification
Some protocols lack sensitivity
ELISA
ELISA tests based on:
IFN–Υ - Expression of IFN-Υ increases duringinfection
Protoplasmic antigen (PPA-3) – first used antigen
(Sweenay et al 1994)
Lipoarabinomannan polysaccharide antigen (LAM)
Advantages Disadvantage
Can performed similarly for all ruminants
Same test for milk and serum samples
Rapid and Low price
Early detection is not possible
Cross reactivity
False positive result in case of immunization
Sensitivity of ELISA
Subclinical Infected Animal – 15-57 % Clinically Infected Animals – 89 -95 %
Treatment
No drug approved
Expensive and unrewarding
Antibiotic therapy – No complete cure
Antibiotics used - Clofazimine or Isoniazidand either Rifabutin or Ethambutol
Treatment of goat affected with MAP
Streptomycin, Rifampicin, Levamisole(Das et al, 1992)
Vaccination
Heat killed or modified live preparation of M.paratuberculosis strain 18- reduces incidence
Provides partial protection
Decreases the No. of MAP shedding in feces
(Kormendy, 1994)
DisadvantagePositive antibody test, which may interfere with serological testing
Management
Over all cleanliness of farm
Manure handling
Care of new borne calf
Breed selection – jersey and Cuernsey more
susceptible
Routine check up – ELISA, PCR
MAP a human pathogen ?
Chron’s disease in human, a sever inflammatory enteritisinvolving the terminal ileum
Clinical symptoms of Crohn’s disease closely mimic thosefound in animals with Johne’s disease
M. paratuberculosis has been isolated from biopsy tissuesCrohn’s disease patients
Epidemiological evidence correlating exposure to M.paratuberculosis with incidence of Crohn’s disease is notreadily available (Stabel, 1997)
Probiotics and MAP
Recent study shows presence of MAP in pasteurized milkand other dairy products such as cheese, yoghurt, babyfoods
Map growth was inhibited (delayed) when supplementedwith supernatants from a number of Lb. paracasei isolates
When co-inoculated with probiotic strains in sterile milk for48 h (pH < 4.5) MAP could not be detected by culturemethod up to 50 days
In vitro inhibitory effect of some lactobacilli on MAP, maybe due to factors other than acid production.
(Donaghy et al,2005)
Conclusions
Economic losses of $1.5 billion/year
Pasteurized milk, cheese, other dairy products may
not be always free of MAP
Contaminated baby food with MAP expose children
and immuno-compromised people at high risk
Effectiveness of pasteurization affected by initial
concentration of MAP in raw milk
New technologies are required for the early detection
of infected animals
Identification and characterization of antigen protein
that are specific to MAP is necessary for improved
vaccine development
In the current state of knowledge, magnitude and
potential consequences of the presence of MAP in
dairy products on retail sale must not be ignored.
Conclusions