1. Bharathy.S., L.Gunaseelan., K.Prabu
UTILITY OF MILK AS A NON INVASIVE SAMPLE FOR CONFIRMATION OF Mycobacterium avium subspecies paratuberculosis (MAP) EXCRETORS IN GOATS
Bharathy.S., L.Gunaseelan., K.Prabu By
Dr.Bharathy.S (O174)
SS18.
M.V.Sc Scholar
Department of Veterinary Public Health and Epidemiology,
Madras Veterinary College

2. INTRODUCTION Crohn’s disease???? Johne’s disease (JD)
Mycobacterium avium subsp.paratuberculosis (MAP) causative for
Crohn’s disease????
Johne’s disease (JD)
Chronic infectious enteritis
Chronic granulomatous, incurable ileocolitis
Severe abdominal pain, diarrhea, bleeding, bowel
obstruction
Progressive weight loss and profuse diarrhea

3. MAP and Crohn’s disease
MAP Excretion and Transmission
Sheds intermittently by subclinically and clinically infected animals.
Feces and milk and colostrum.
Faecal–oral route transmission.
Subclinical shedders- act as a source for new infections.
(Chiodini et al, 1984 and Streeter et al,1995)
MAP and Crohn’s disease
The pathological changes in intestines of Crohn’s disease is similar to Johne’s disease in cattle.
Chiodini et al.,1984 isolated MAP from terminal ileum of three patients with Crohn’s disease.
MAP has been successfully cultured from breast milk of lactating Crohn’s disease patients (Naser et al.,2000b)

4. MAP excretion in milk Objectives
Pinedo et al.,2008 isolated MAP in milk 45% from clinically affected animals and 22% in colostrum and 8% in milk of subclinical cases.
Giese and Aherens., 2000 detected MAP in 6 milk samples out of 11 infected animals.
Grant et al.,2001 reported MAP DNA in raw goat’s milk 1.1% in the UK.
Nebbia et al.,2006 recovered MAP DNA intermittently in milk samples from 13 out of 29 asymptomatic sheep and goat.
In India, MAP isolated from milk and feces of infected goat (Singh and Vihan 2004)
Objectives
To detect MAP by IS900 PCR in goat milk samples.
To describe the excretion pattern of MAP in milk of the known JD positive goats with public health significance.
To describe the utility of goat milk for diagnostic sample for MAP detection in small ruminant flocks.

5. Materials and methods Sampling and Handling of milk samples:
A total of 15 Goat Milk samples were collected from known JD positive cases (confirmed by Acid fast staining and fecal IS900 PCR)
8 samples from Kattupakkam farm, PGRIAS
7 samples from Madras Veterinary College Clinics.
5-10mL were collected in a sterile tube from the 2 quarters by hand milking.
Samples were refrigerated at 4˚C until analysis.

6. DNA Extraction from milk sample
5 mL of milk samples in sterile tube
Centrifuge at 3000 rpm for 15 minutes.
Discard supernatant and cream fat layer, restore the pellet
Wash the pellet 3 times with PBS
Centrifuge 1000rpm for 10 minutes.
DNA extraction from pellet by commercial kit method

7. IS900 Polymerase chain reaction
IS900 PCR was performed (Pillai and Jayarao., 2002)
Forward primer: 5’-CCGCTAATTGAGAGATGCGATTGG-3’
Reverse primer: 5’AATCAA CTCCAGCAGCAGCGCGGCCTCG-3
Cycling Condition:
Events
Temperature
(˚C)
Time
(minutes)
Initial denaturation 94 3
Denaturation 1
Annealing 55
Extension 72
Final extension 7
Sequencing of PCR products:
Seven positive PCR products were sequenced to confirm the identity of the 229 bp amplified fragment.
Automated sequencing was carried out with an DNA Sequence Analyser
Nucleotide sequences were processed by BLAST analysis

8. RESULTS All the 15 goat milk samples were shown IS900 PCR positivity.
The Percentage of positivity for IS900 PCR for milk samples were 100%.
BLAST analysis revealed that the nucleotide sequence of the 229-bp fragment amplified by the IS900 PCR assay was identical to the known IS900 of MAP.
L1 L L L M L L L7 L L9 L10
229bp
Mycobacterium avium subsp.paratuberculosis specific amplicons (229 bp) using IS900 specific primers in goat milk samples. M:100 bp ladder; Lane 3: Negative control; Lane 4: Positive control; Lane1,2,6,7,8,9,10 : tested DNA samples

9. DISCUSSION
In our study all the 15 milk samples from positive case for JD shown positive result by IS900 PCR, explains about the excretory pattern of MAP in milk.
MAP excretion therefore through milk established the utility of a non invasive sample to identify MAP excretors in JD flocks.
It also indicates the potential hazard of MAP infection to humans consuming raw goat milk

10. CONCLUSION There is the risk of organism is being shed into the milk.
Kids can become infected by nursing from, or being bottle-fed milk from, an infected doe.
People considering consumption of raw goat milk has some medicinal value, so results in potential hazard for humans .
The detection of MAP directly from milk by IS900 PCR could become a valuable diagnostic or screening test for MAP excertors in goats.
So there is a need to minimize the potential threat of this organism as a zoonotic agent of Crohn’s disease??????

11. Prohibit the use of milk derived from animals with clinical Johne’s
Control measures and
Awareness .
Further optimization of pasteurization time and temperature
Reduce public health risk by means of strengthening collaboration.

12. Team worked and helped for this study
Thank You
Team worked and helped for this study
Dr.L.Gunaseelan
Professor and Head
VPH & E
MVC, Chennai
Dr.C.Sreekumar
Professor, Post Graduate Research Institute in Animal Sciences, Kattupakkam
Dr.K.Porteen
Assistant Professor
VPH & E
MVC,Chennai