This document presents a study on the prevalence of Mycobacterium avium subspecies paratuberculosis (MAP) infection in cows. The objectives were to determine the prevalence of MAP in cow fecal samples using microscopy and molecular characterization using PCR. Samples were collected from 32 cows near GLA University in Mathura. Microscopy found MAP bacilli in 10 samples (31.25% prevalence). DNA was isolated from samples and tested with IS900 PCR, finding 2 positive samples (6.25% prevalence). The study concludes MAP infection affects over 30% of cows by microscopy and 6% by PCR, indicating control measures are needed to prevent spread between animals and potentially to humans.

1.  Presented by -
 Name: Neetu Yadav
 B.Sc Hons Biotechnology
 Roll No.: 194300028
 Supervisor
 Dr. Kundan kumar chaubey
 Assistant Professor- Research
 Department of Biotechnology
 GLA University, Mathura
Prevalence of Mycobacterium avium subspecies
paratuberculosis infection in cows and it’s
morphological and molecular characterization

2. OBJECTIVES
1- PREVALENCE OF MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS
(MAP) IN FECAL SAMPLE OF COWS.
2- MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF MAP USING
MICROSCOPY AND PCR.
 Materials required
 Glasswares
 Plastic waves
 Eqipments
 Oligonucliotides
 Miscellaneous items
 Methods
 ZN Staining
 Microscopy
 DNA Isolation
 IS900 PCR
 Agarose Gel
Electrophoresis
 Indigenous ELISA for
MAP infection.

3. ZIEHL-NEELSEN STAINING
Cleaning of slide
Smear preparation
1% Carbol fushin
25% Sulphuric acid
0.1% Methylene blue
Dry and Observation
ZN STAINING AND MICROSCOPY OF MILK SAMPLE

4. SAMPLE COLLECTION AND PROCESSING
Sample Collection
Gaushala near by GLA University, Mathura- 32 Cow Sample

5. PROCESS :

6. - RESULTS -
- Microscopy (ZN staining) -
Cow (n=32)
Positive Samples - 10 (31.25%)
MAP bacilli seen in fecal
smears under the microscope

7. DNA ISOLATION
500 µl milk sample +100 µl milk lysis buffer
Incubation at room temperature for 15 min.
Add 100 µl 0f 24% SDS,10 min incubation at room temperature
Add 20 µl of proteinase K and incubation at 56°C for 2 hours
Add 100 µl of 5M NaCl and 64 µl CTAB-NaCl; incubate at 65°C for 30 minutes
Add equal volume of phenol : Chloroform : Isoamyl alcohol (25:24:1)

8. CONTINUED………
Centrifuge at 10000 rpm for 15 minutes at 4°C
Transfer the aqueous phase to sterilized eppendorf tube
Precipitate the DNA by adding 0.6 volume of chilled iso-propanol
Keep tube at -20° C for overnight
Pellet the DNA by centrifuging at 10000 rpm for 20 min at 4°C
Discard the supernatant and wash the pellet with 1 ml 70 % ethanol
Resuspended the pellet the 30 µl 1X TE Buffer. Store the DNA at -20°C

9. DNA ISOLATION AND PCR
- IS900 PCR -
413 bp
Lane M: 1.0 kb DNA ladder or Marker (#SM0243,
Fermentas)
Lane 1: IS900 PCR product from DNA of MAP ‘S
5’ Indian Bison type (Positive Control)
Lane 2 and 4: IS900 PCR product from DNA
harvested from cow fecal sample showing positive
results.
Lane 2, 3 and 4: Sample negative in PCR
1 2 3 4
Genomic DNA from cow feces
DNA Bands Visualization
Cow (n=32)
Positive Samples - 2
(6.25%)

10. DISCUSSIONS AND CONCLUSION
 Study reported 31.25% and 6.25% bio-load of MAP infection
using microscopy (ZN staining) and IS900 PCR, respectively.
 This may help to estimate the actual status of the disease in
cow which help in control the disease to spread to other
animals.
 Consumption of milk products and sharing habitat with
infected cows spread the infection to humans.
 Vaccine based control may be effective on spread of the
infection to other domestic animals in addition to humans.

11. Thank You