5.1 Source of animals: Fresh tissue samples (lymph nodes, lungs with tuberculous lesions) were collected from postmortem hall (dairy farm Ludhiana ,Punjab),(n=25) suspected for bovine tuberculosis. Tissue impression smears were made from these tissues fixed with methanol (100%w/v) for further use. The tissue samples were collected in two containers separately, one in 10 % NBF for histopathology and frozen tissues in sterile container for PCR studies.
5.2 Clinical Specimen
A total of 25 tissue samples (lung and lymph node sections) from bovine tuberculosis suspected animals above 2 yrs of age at postmortem were routinely Acid-fast stained, formalin-fixed and paraffin embedded.
5.3 Cytological Smear preparation
Approximately 2 g of tissue from each sample (n= 25) was cut into small pieces and homogenized with 1.0 ml of sterile distilled water using a pestle and a mortar. The tissue homogenates (200 ml each) were decontaminated with 4% NaOH. Inoculated onto two slants of Lowenstein-Jensen (LJ) media with and without glycerol and incubated for 6–8 weeks at 370C.
5.4 For identification of Culture
Two loops full of tissue homogenate were smeared on glass slides. The smears were dried, heat fixed, stained with Ziehl-Neelsen (ZN) and examined for Acid Fast Bacilli (AFB).
5.5 PNA synthesis and labelling
Samples included in the study were identified by the MTBCcy3 Probe and MAVTAMRA hybridization assay (PNA Bios Probe, USA). Probes MTBCCy3 and MAVTAMRA, were used for specific detection of members of the M. tuberculosis complex and M. avium respectively, with the 16S rRNA Sequence database and the probe design program [18]. The probe sequences were customized from (PNA Bios USA). M. bovis and M. avium standard cultures were used for the standardization of PNA-FISH assay. Sequence of the probes is depicted the Table 2.
Table 2: PNA probe Sequences used for Assay [18].
Probe
|
Sequence (orientation)
|
Target species
|
MTBCCy3
|
TCC TGG TGC CCT ACG-Cy3 (3–5)
AGG ACC ACG GGA TGC (5-3)
|
M. bovis
(M. tuberculosis complex)
|
MAV
TAMRA
|
CTG GAG TTC TGC GTA-TAMRA (3_5)
GAC CTC AAG ACG CAT (5_3)
|
M. avium
|
5.6. Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) using PNA probes was performed on postmortem samples to demonstrate and identify Mycobacterium bovis and Mycobacterium avium. The procedure for the FISH technique was performed as per [13, 14] with slight modifications. Standard Culture-grown and fixed bacteria (20 µl) were spotted onto three-field microscope slides (Thermo scientific, India) and air dried. Then, the slides were dehydrated in 100% (v/v) methanol for 1 min and 100% (v/v) ethanol for 5 min, air dried again, and preheated to hybridization temperature. Slides with impression smears and tissue sections were prepared in similar pattern. Impression smears and tissue sections were applied with a 20 µl of Triton X 100 with a cover slip and kept for 20 minutes in dark chamber. The slides were then subjected to hybridization mixture containing 10% (w/v) dextran sulfate (Mp Biomedicals, India), 10 mM NaCl (Mp Biomedicals, India), 30 to 50% (v/v) formamide (Hi media India), 0.1% (w/v) sodium pyrophosphate (Mp Biomedicals, India),0.2% (w/v) polyvinyl pyrrolidone (Mp Biomedicals, India), 0.2% (wt/vol) Ficoll (Mp Biomedical, India), 5 mM disodium EDTA (Mp Biomedicals, India), 0.1% (vol/vol) Triton X-100 (Mp Biomedical, India), 50 mM Tris-HCl (pH 7.5), and a fluorescent probe(s) with a final concentration of 1 to 1.5 mol/liter were applied to each sample. Slides were incubated at a temperature optimized for each PNA probe (Table 3) in a preheated moisture chamber in the dark for 90 min. Coverslips were removed by submerging each slide in approximately 20 ml of prewarmed 5 mM Tris (pH 10), 15 mM NaCl (Mp Biomedical), and 0.1% (vol/vol) Triton X-100 (Mp Biomedical) (FISH wash buffer) in a water bath at 550C, following hybridization. The slides were then kept in water bath for 30 minutes.
After brief immersion in FISH wash buffer, the slides were washed with double distilled water, air dried and mounted with 1 drop of imaging mounting fluid (Vector Laboratories Inc., Burlingame, Calif.). Microscopic examinations were conducted using a fluorescence microscope (company). Unspecific hybridization of MTBCCy3 to M. bovis, was avoided by high-stringency hybridization conditions (55°C, 50% formamide). For impression smears and tissue sections hybridization conditions of formamide, were required at 40% and 50%. For M. bovis, the MTBCCy3 probe sequence, hybridization conditions (55°C, 40% and 50% formamide) were sufficient to prevent unspecific binding. For probe, MAVTAMRA (M. avium) unspecific binding was avoided by hybridization at 55°C and formamide concentration of 30%, (impression smears and tissue sections) respectively.
Immunohistochemical Studies
Detection of antibodies (ESAT-6 monoclonal and polyclonal, CFP-10 polyclonal) in tissues was done by immunohistochemical analysis. All tissue samples were separately collected and fixed in 10% Neutral Buffered Formalin and were further processed as per conventional methods [15]. Thick paraffin tissue sections were spread on Superfrost positively charged microscopic slides (Fisher Scientific, USA). Antigen retrieval was done in EZ antigen retrieval solutions using EZ-Retriever System (Bio Genex Laboratories Inc., California). After endogenous peroxidase and nonspecific protein blocking, the sections were incubated with standardized dilution of (ESAT-6 and CFP-10) antibodies in a humidified chamber at 4°C overnight. Secondary antibody conjugated with HRP (Vector Laboratories, USA) was added and incubated for 30 min at room temperature. Visualization of antigen antibody complex was performed using ImmPACT DAB Peroxidase Substrate Kit (Vector Laboratories, USA) followed by counterstaining with hematoxylin and AEC stain. AEC (3-Amino-9-Ethylcarbazole) is a widely used chromogen for immunohistochemical staining. AEC produces a red end product that is soluble in alcohol and must be used with an aqueous counterstain and mounting media.
Presence or absence of Mycobacterial antigens was evaluated by observing the stained cells showing positive reactivity (macrophages, giant cells, epithelioid cells) using light microscopy under oil immersion [16, 17].
5.7. PCR Primers:
The primer sequences for ESAT-6 were: Forward- 5'-GTACCAGGGTGTCCAGCAA AA-3' and Reverse 5'-CTGCAGCGCGTT GTTCAG-3' [12] giving a product size of 61 bp respectively was used for PCR amplification. All 4 samples found positive by PNA-FISH were further confirmed by esxA (ESAT-6) PCR. Tissue sample DNA was amplified by esxA (ESAT-6 PCR, for detection of M. bovis. Amplicons of 61 bp were considered positive for ESAT-6 PCR respectively.