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Mycobacterium paratuberculosis avium

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Mycobacterium paratuberculosis avium food pathogen of concern
41
Mycobacterium avium : A food borne pathogen of concern
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Page 1: Mycobacterium paratuberculosis avium

Mycobacterium avium : A food

borne pathogen of concern

Page 2: Mycobacterium paratuberculosis avium

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

Page 3: Mycobacterium paratuberculosis avium

Mycobacterium avium subspeciesparatuberculosis (MAP)

Gram positive rod (0.5 x 1.5 micron)

Acid fast

Facultative intracellular

Obligate parasitic pathogen

Requires iron for growth

Page 4: Mycobacterium paratuberculosis avium

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

Page 5: Mycobacterium paratuberculosis avium

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)

Page 6: Mycobacterium paratuberculosis avium

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

Page 7: Mycobacterium paratuberculosis avium

Disease

Digestive tract route of entrance of MAP

Multiplication of MAP in Intestinal mucosa

Phagocytosis by macrophages

Via lymph channels

Infiltration regional lymph node

Page 8: Mycobacterium paratuberculosis avium

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

Page 9: Mycobacterium paratuberculosis avium

Potential Human Exposure to MAP

Page 10: Mycobacterium paratuberculosis avium

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

Page 11: Mycobacterium paratuberculosis avium

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%

Page 12: Mycobacterium paratuberculosis avium

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)

Page 13: Mycobacterium paratuberculosis avium

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

Page 14: Mycobacterium paratuberculosis avium

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)

Page 15: Mycobacterium paratuberculosis avium

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

Page 16: Mycobacterium paratuberculosis avium

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)

Page 17: Mycobacterium paratuberculosis avium

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)

Page 18: Mycobacterium paratuberculosis avium

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)

Page 19: Mycobacterium paratuberculosis avium

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)

Page 20: Mycobacterium paratuberculosis avium

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)

Page 21: Mycobacterium paratuberculosis avium

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

Page 22: Mycobacterium paratuberculosis avium

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

Page 23: Mycobacterium paratuberculosis avium

Culture method

PCR based method

ELISA

Detection Mycobacterium avium subsp.

Paratuberculosis

Page 24: Mycobacterium paratuberculosis avium

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

Page 25: Mycobacterium paratuberculosis avium

Disadvantage

Long culture period – 4 - 12 week

Fastidious growth requirements

Contamination

Advantages

•Gold standard• Simple and widely used method

Page 26: Mycobacterium paratuberculosis avium

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)

Page 27: Mycobacterium paratuberculosis avium

Drawbacks

PCR inhibitors - present in fecal, milk, milkproduct samples

Cant differentiate between live and dead cell

Chances of cross amplification

Some protocols lack sensitivity

Page 28: Mycobacterium paratuberculosis avium

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)

Page 29: Mycobacterium paratuberculosis avium

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 %

Page 30: Mycobacterium paratuberculosis avium

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)

Page 31: Mycobacterium paratuberculosis avium

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

Page 32: Mycobacterium paratuberculosis avium

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

Page 33: Mycobacterium paratuberculosis avium

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)

Page 34: Mycobacterium paratuberculosis avium

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)

Page 35: Mycobacterium paratuberculosis avium

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

Page 36: Mycobacterium paratuberculosis avium

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

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