About the district satna
Satna district is surrounded by five districts of the state of Madhya Pradesh namely, Rewa, Shahdol, Umaria, Katni and Panna and one district of Uttar Pradesh state called as Banda (
https://satna.nic.in/en/map-of-district/ Accessed on 12
th June 2022. It has an area of 7,502 square kilometers with 2,125 villages. Its population is over 2 million (2,228,619) (
https://satna.nic.in/en/ Accessed on 21
st May 2023) that have literacy rates of 65.12% slightly below the country’s 65.38% and above the state’s 64.11%. The district Satna has seven tehsils namely; Raghuraj Nagar, Rampur Baghelan, Nagod, Unchehra, Amarpatan, Ram Nagar, and Maihar. It has a population density of 249 per square kilometer above the state (196) but below the country (324). Most of the agricultural laborers are women (
https://cdn.s3waas.gov.in/s38e6b42f1644ecb1327dc03ab345e618b/uploads/2016/04/2018041238.pdf Accessed on 12
th June 2022). The location of Satna in the Madhya Pradesh state and India is depicted in Fig 1 using assistance of Google Maps 2022 (
https://satna.nic.in/en/map-of-district/and https://en.wikipedia.org/wiki/Satna_district#/media/File:MP_Satna_district_map.svg Accessed on 12
th June 2022).
Livestock, collection and transportation of samples
In the present study, a representative randomly selected 26 fecal samples from two different dairy farms Rajouri dairy (adult Murrah buffalo) and Kushwaha dairy (adult cattle Sahiwal and Jersey breeds) and 50 milk samples from a Private farm located at Satna district in Madhya Pradesh were collected for investigating on Paratuberculosis or Johne’s disease infection. The animals were healthy but weak in bodily conditions. These samples were collected in February in the year 2019. After collection, these samples were then transported and stored in the deep freezer (-40°C) to the Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Nanaji Deshmukh Veterinary Science University, Dr. Ambedkar Nagar-Mhow, Indore, Madhya Pradesh, India. These samples were further transported at cold chain conditions to the Microbiology Laboratory, Animal Health Division, Central Institute for Research on Goats (CIRG), Makhdoom-Farah, Mathura, Uttar Pradesh, India. Fecal and milk samples were tested for the presence of MAP infection by different tests during the year 2019-2021. Processed fecal and milk samples were examined by microscopical and molecular methods for the presence of
Mycobacterium avium subspecies
paratuberculosis and gene-specific amplification from extracted genome from the organisms, respectively.
Processing of fecal samples and microbial staining of fecal and milk smears
Individual fecal sample, 2 g was taken into sterile mortar and pestle and crushed well after adding 10 ml Phosphate Buffer Saline (PBS) solution. This mixture was poured in a 15 ml centrifuge tube and it was kept for centrifugation for 45 minutes at 4,500 revolutions per minute (rpm). The resulting supernatant was discarded and from the middle layer, a fecal smear was prepared on a glass slide. The smear was further examined after acid-fast staining for the presence of acid-fast bacilli. Milk smears were also prepared on glass slides and examined microscopically as above.
Isolation of deoxyribonucleic acid (DNA) from feces and milk samples
2 g of fecal material was taken in a sterile mortar containing 4 ml of sterile PBS solution. A pestle homogenized the fecal material. The resulting homogenate was transferred to a 15 ml centrifuge tube containing 8 ml of sterile PBS. The tube was centrifuged at 3,500 revolutions per minute for 45 minutes to concentrate bacilli. The tube was kept undisturbed for 1 hour at room temperature. The supernatant was discarded and the semisolid middle layer was collected with a sterilized swab and transferred to 300 ml of PBS in an Eppendorf® tube. The mixture was suspended to heat at 95°C for 10 minutes. A volume of 40 ml of Lysozyme (20 mg/ml) was added and the tube was incubated at 37°C for 2 hours with continuous shaking. Then, 20 ml of Proteinase K (10 mg/ml) and 50 ml of 10% Sodium Dodecyl Sulfate (SDS) were added to the mixture and heated at 56°C for 2 hours with continuous shaking. After the addition of 64 ml Cetyltrimethylammonium bromide-Sodium Chloride (CTAB-NaCl) solution in the mixture, it was heated at 65°C for 30 minutes. Equal volume of Chloroform-Isoamyl alcohol (24:1) was added to the tube and on mixing properly it was centrifuged at 10,000 rpm for 20 minutes at 4°C. The aqueous layer (upper layer) was transferred to the fresh 1.5 ml Eppendorf® tube. On addition 0.6 volume of isopropanol to the aqueous layer, it was mixed by gentle inversion and DNA precipitation was allowed at -20°C for overnight. Then, the tube was centrifuged at 10,000 rpm for 20 minutes at 4°C and discarded the supernatant. The resulting pellet was washed with 1 ml of 70% ethanol by centrifugation at 10,000 rpm for 10 minutes at 4°C. The supernatant was discarded and air dried the pellet. The pellet was resuspended in 30 ml of Tris Ethylenediaminetetraacetic acid (TE) buffer and stored at 4°C overnight to dissolve the DNA. It was further stored at -20°C for long-term usage. Nucleic acid extraction from milk samples was performed as per
Matoli et al., (2018).
IS900 polymerase chain reaction (PCR)
Primers sequences used were specific to MAP (IS
900 P90/91) (Forward primer: 5'-GAA GGG TGT TCG GGGCCGTCG CTT AGG-3' and Reverse primer: 5'-GGC GTT GAG GTC GATCGC CCA CGT GAC-3')
(Millar et al., 1995; Millar et al., 1996; Singh et al., 2010). A reaction master mix (with deoxyribonucleotide triphosphates, dNTPs; Taq DNA polymerase; assay buffer, Magnesium Chloride, MgCl
2 and loading dye) was used to perform the test. A total reaction volume of 25 ml containing 2.5 ml of a test DNA sample and 0.5 ml of each primer. Positive (DNA from native Bison type S 5 strain of MAP) and negative controls were also kept. Thermocycler chain reaction conditions were initial denaturation at 94°C for 3 minutes and subsequently for the next 30 cycles (denaturation at 94°C for 30 seconds, annealing at 63°C for 15 seconds and extension at 72°C for 1 minute) and final extension at 72°C for 10 minutes and at the end of the reaction it was kept at 4°C. The PCR product (413 bp) was analyzed on a 1% agarose gel in 1X Tris Borate Ethylenediaminetetraacetic acid (TBE) buffer containing 0.5 mg/ml of Ethidium Bromide at 80 V for 1 hour. IS
900 PCR product from MAP S 5 Indian Bison type DNA as positive control and 1.5 kb DNA ladder or Marker (Fermentas) were also run.
IS1311 PCR amplification for genotyping of MAP
A 25 ml reaction volume consisted of 12.5 ml master mix (Genei, Bengaluru), 5 ml template, 0.5 m of each of primers (Forward primer (M56): 5'-GCG TGA GGC TCT GTG GTG AA-3' and Reverse primer (M119): 5'-ATG ACG ACC GCT TGG GAG AC-3';
Sevilla et al., 2005) and remaining amount of nuclease-free water. Thermocycler chain reaction conditions were initial denaturation at 94°C for 5 minutes and subsequently for the next 37 cycles denaturation at 94°C for 30 seconds, annealing at 62°C for 30 seconds and extension at 72°C for 1 minute and final extension at 72°C for 10 minutes and at the end of the reaction it was kept at 4°C. No template control and a positive control from reference strain, were included in each PCR amplification. The DNA band of 608 bp was considered PCR positive after separation in 1% agaros
e gel stained with Ethidium Bromide. The PCR products (608 bp) were digested using HinfI/MseI enzymes and patterns were compared as Cattle type/Sheep type/India Bison type
(Whittington et al., 2001). 
