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البقرة البقرة سو'رة سو'رة ( (3232))

العظيم الله العظيم صدق الله صدق

Mce4A gene is a specific gene for detection of M.bovis in

raw milk and some milk products

Assiut UniversityFaculty of Vet. Medicine Dep. of Food. Hygiene

بوفز الميكوباكتيريم عن للكشف متخصص Mce4Aجين

االلبان منتجات وبعض الخام اللبن فى

Presented by Presented by

Saad El-din, N.; Saad El-din, N.;

Nagah, M. S.; Nagah, M. S.;

Nasr,E. A.; Nasr,E. A.;

Nahed, M. Wahba Nahed, M. Wahba

and Walaa, M. Elsherifand Walaa, M. Elsherif

TuberculosisTuberculosis is a chronic disease of many species and man is a chronic disease of many species and man caused by Mycobacteria species.caused by Mycobacteria species.

Found in the spines of Found in the spines of Egyptian mummies Egyptian mummies from (3000-2400 BC).from (3000-2400 BC).

Ibn SinaIbn Sina identify pulmonary tuberculosis in his book identify pulmonary tuberculosis in his book The The CanonCanon of Medicine.of Medicine.

Robert KochRobert Koch in in March 24, 1882 March 24, 1882 identified The bacillus identified The bacillus causing tuberculosis and received the causing tuberculosis and received the Nobel Prize Nobel Prize in in medicine for his discovery .medicine for his discovery .

According to the World Health Organization (WHO 2009) According to the World Health Organization (WHO 2009)

2 billion 2 billion people, people, 1/31/3 the world's population, the world's population, infectedinfected by TB. by TB. 2 million 2 million people people diedie from the from the disease worldwide.disease worldwide.

AnnuallyAnnually, , 8 million 8 million people become illpeople become ill with tuberculosis.with tuberculosis.

Between 2000 and Between 2000 and 20202020, nearly one, nearly one billion people will be newly infected. billion people will be newly infected.

The economic losses caused by the disease in animals

Infect over 50 million cattle world-wide resulting Infect over 50 million cattle world-wide resulting in economic losses of approximately $ 3 billion.in economic losses of approximately $ 3 billion.

Reduction of 10-20% in milk production.Reduction of 10-20% in milk production. Reduction of 10-20% in weight Reduction of 10-20% in weight .. Infertility, Infertility, ⇩⇩10-25% of the reproductive efficiency.10-25% of the reproductive efficiency. Condemnation of meat.Condemnation of meat.

Significant risk of M. Bovis

Consumption of un-pasteurized milk and dairy products

Aerosols

Tuberculous animals and their carcasses

The current study aimed to:

1. Isolation and identification of Mycobacteria spp. in milk and some milk

products )Kareish cheese, Butter and locally manufactured yoghurt(

2. Using Mce4A gene in PCR for confirmation of Mycobacterial isolates.

Collection of samples

Milk fromtuberculin positive reactors

tuberculin negative reactors

Locally manufactured

yoghurt Kareish cheese

Cooking butter

50

50

50

50

50

50

Marketable milk

2- Tuberculin test

Bovine TB Eradication Program2222

InjectInjectBovine PPD tuberculin Bovine PPD tuberculin

(here)(here)

Inject Inject AviumAviumPPD tuberculin PPD tuberculin (here)(here)

Bovine TB Eradication Program2424

Bovine TB Eradication Program2121

Skin ThicknessSkin Thickness

Measure and Measure and record record

skin thicknessskin thickness

measured measured -- before before injection and isinjection and isrecordedrecorded

The samples were prepared according to the technique as recommended by (A. P. H. A., 1992).

*culture of Mycobacteria

Take sediment Add HCL 6%

Then incubate at 37 oC for 30 min.

To destroy M.O. other than acid fast bacilliCentrifuge at 3000

r.p.m. for 15 min. to remove acid

* Neutralization method 4% sterile NaOH sol.

culture on

Lowenstein Jensen medium

1.1. Morphological characters (Z.N. Stain) .Morphological characters (Z.N. Stain) .2.2. Rate of growth.Rate of growth.3.3. Effect of light on pigment production.Effect of light on pigment production.4.4. Growth at different temperature degrees.Growth at different temperature degrees.5.5. Biochemical tests.Biochemical tests.

Identification of the recovered Mycobacteria

~Primer 1 (Sense):~Primer 1 (Sense):5´-5´-CAC-CTT-CCT-CAT-CCC-CTC-CAC-CTT-CCT-CAT-CCC-CTC-3´3´

~ Primer 2 (Antisense):~ Primer 2 (Antisense):5´-5´-GAT-GAG-GGA-TTG-GAA-CAA-C GAT-GAG-GGA-TTG-GAA-CAA-C -3´-3´

Initial denaturing (95Initial denaturing (95ooC/3 min)C/3 min) )40 cycles()40 cycles( denaturingdenaturing 9595ooC/1 minC/1 min

annealingannealing 56 56 ooC/45 secC/45 sec

extensionextension 7272ooC/1 minC/1 min

final final extensionextension

7272ooC/10 C/10 minmin

14

6

2

0

2

4

6

8

10

12

14

%

Milk from tuberculinpositive reactors

Milk from tuberculinnegative reactors

Marketable milk

Milk from tuberculinpositive reactors Milk from tuberculinnegative reactorsMarketable milk

Incidence of Acid Fast Bacilli in milk samples examined microscopically using Ziehl-Neelsen stain.

AFB in milk

Incidence of Mycobacteria isolated from milk samples using conventional culture method.

4

2 2

0 0 0

0

0.5

1

1.5

2

2.5

3

3.5

4

%

Milk from tuberculinpositive reactors

Milk from tuberculinnegative reactors

Marketable milk

pyruvatedglycerinated

Incidence of Acid Fast Bacilli in some milk products samples examined microscopically using Ziehl-Neelsen stain.

8

10

4

0

1

2

3

4

5

6

7

8

9

10

%

locally manufacturedyoghurt

Kareish cheese Cooking butter

locally manufactured yoghurt

Kareish cheese

Cooking butter

Incidence of Mycobacteria isolated from some milk products samples using conventional culture method.

4

0

2 2 2 2

0

0.5

1

1.5

2

2.5

3

3.5

4

%

locally manufacturedyoghurt

Kareish cheese Cooking butter

pyruvatedglycerinated

Identification of Mycobacteria isolated from milk and some milk products samples.

4

2 2

0 0 0

2 2 2 2 2 2

0

0.5

1

1.5

2

2.5

3

3.5

4

%

Milk fromtuberculin

positivereactors

Milk fromtuberculinnegativereactors

Marketablemilk

locallymanufactured

yoghurt

Kareish cheese Cooking butter

M.bovis

MOTT

Results of PCR testingResults of PCR testing

700 pb

Conclusion and Suggestions

On the basis of the results achieved in the present study, it could be

concluded that Mycobacteria spp. including M. bovis and MOTT were

detected in raw milk, locally manufactured yoghurt, Kareish cheese and cooking butter in

different percentages.

Identification of isolated M. bovis using Mce4A gene indicated positive result

Therefore, the aforementioned data proved that great attention must be paid to the

problems of these pathogens in our foods.

So, many steps taken to protect the milk supply from contamination with

Mycobacteria spp. including M. bovis are:

Suggestions

1-Test and slaughter policy and retesting of cattle herds at an interval of 2 months

Suggestions

2- Using mce4A gene in PCR reaction for detection of local M. bovis isolates was advised since it

proved to be specific for contamination of M. bovis infection.

3- Efficient pasteurization for milk and milk products and Preventing the manipulation of

raw milk and milk products.

Thank You

للسادة شكرا

الحضور