Rapid detection of M. bovis and M. avium in cytological smears and tissue sections by PNA-FISH

Background: Bovine Tuberculosis is globally the paramount cause of death from single pathogen in cattle and other species. Rapid and explicit identication of mycobacteria is essential to hold back tuberculosis in bovines. We performed a uorescence Peptide nucleic acid uorescence in situ hybridization (PNA-FISH) procedure for specic detection of Mycobacterium bovis and Mycobacterium avium in bovine was optimized on cytological smears and tissue sections of bovines suspected for bovine tuberculosis. Results: PNA-FISH was performed on lung and lymph node tissues impression smears. The probes were standardized for standard bovine mycobacterial cultures at 50% formamide concentration for M.bovis and 30% formamide concentration for M.avium. All the cytological smears were positive from M.bovis probe (MTBCcy3) which was standardized at hybridization conditions of (55 o C and 40% formamide) concentrations. Results revealed four out of twenty ve were positive in tissue sections with a bright red uorescence with cy3 lter (MTBC probe). No results were seen with (MAV TAMRA ) probe for M.avium which was standardized at hybridization conditions of (55 o C and 30% formamide) respectively. No uorescence was seen in control tissue sections .In addition, results were juxtaposed to other commonly used detection methods like IHC and PCR by targeting esxA gene. None of the sample was found positive for M. avium. Conclusion:


Background
Infections caused by Mycobacterium spp. are often associated with worst clinical outcome including death. Bovine Tuberculosis (bTB) is caused by Mycobacterium bovis,that belongs to the Mycobacterium tuberculosis complex (MTC) which mainly infects cattle [1,2]. Use of conventional methods for detection of mycobacteria can be done by ZN staining as well as Immunohistochemical staining. But these methods suffer from several disadvantages including lower sensitivity. Lately, conventional methods such as acid-fast staining, phenotypic differentiation have been augumented by nucleic acid probes and ampli cation-based methods which is rapid and sensitive as well substantially reducing the time to diagnosis [3]. In recent years, molecular methods based on target ampli cation have become available for identi cation of Mycobacterium species in clinical specimen.Visualization of Mycobacteria by speci c techniques, e.g., by uorescence in situ hybridization (FISH), would be a great help in directly identifying bacteria in clinical samples. Peptide nucleic acids (PNAs) are pseudo-peptides with DNAbinding capability. These were rst reported in the early 1990s in connection with a series of attempts to design nucleotide analogues capable of hybridizing, in a sequence-speci c fashion, to DNA and RNA [4,5]. The relative hydrophobic character of PNA as compared to DNA and RNA might be particularly useful in diagnostic applications where access of detection probes to their molecular target will depend upon e cient diffusion of the probe through a hydrophobic environment, such as a cell wall, under su ciently mild conditions for the morphology of the cell to be preserved [6]. The in situ hybridization procedure is relatively simple, requires only minimal equipment, and permits morphologic evaluation of positive signals. The aim of our study was to detect M. bovis and M. avium in cytological smears and tissue sections in bovine tuberculosis by PNA-FISH.

Results
In the present study, lung and lymph node tissue samples suspected for bovine TB (n=25) were subjected to Ziehl Neelsen staining. 19 out of 25 samples were positive by ZN staining. The maximum percent positivity of 76% was found in samples with the presence of 15-20 acid fast bacilli per eld (Fig.1).In this study, 4 out of 25 were positive by PNA-FISH. The probes were standardized for standard bovine Mycobacterial cultures at 50% formamide concentration for M. bovis and 40% formamide concentration for M. avium. All the cytological smears ( Tissue impression smear) were positive from M. bovis probe (MTBCcy3) which was standardized at hybridization conditions of (55 o C and 40% formamide)  (Fig.11). The results are given in (Table   1) respectively.

Discussion
The discovery of PNA has raised a number of novel possibilities relating to molecular diagnostics. We have shown the potential of labeled PNA oligomers as a powerful means of identifying Mycobacteria spp. directly in cytological tissue and impression smears of lungs and lymph node by PNA-FISH. The method presented here provides a combination of the high speci city offered by molecular techniques and the simplicity of direct microscopy [7]. Although detection of Mycobacteria with oligonucleotide probes is di cult since probe penetration is hampered by mycolic acid in mycobacterial cell walls. The development of PNA probes that enter mycobacteria without further pretreatment was, hence, a breakthrough [8]. Labeling of probes with TAMRA or Cy3 resulted in advanced signal intensity and succeeded in direct FISH detection of mycobacteria in tissue sections and cytological smears. In addition, uorescent labeled PNA probes represent an economical way to identify mycobacterial cultures isolated from clinical specimens [9]. All Four of the isolates obtained in this study were identi ed unequivocally as M. bovis. Assuming a time to result of about 3 hours for a FISH procedure (including xation, hybridization, and microscopy) and considering its low cost, FISH is a suitable method for fast identi cation of isolated mycobacterial species. Another precedence of FISH is that no biosafety level 3 research area is essential. It provides a top to bottom identi cation of microorganisms at different taxonomical levels without the need to determine traditional phenotypic characteristics, extract and amplify DNA, or to sequence. Laboratory methods used for diagnosis are conventional, mainly based on acid-fast staining, microscopy which is low in sensitivity and identi cation of mycobacteria causing the disease, so the interpretations of results by conventional methods are highly subjective and prone to errors [10]. Histopathology is considered a reliable tool for diagnosis of bovine TB but cannot differentiate between mycobacterial species, therefore in the present study the histopathological lesions were used for screening the cases for bovine TB and they were later subjected to PNA-FISH to detect M. bovis and M. avium organisms. Further the samples positive from PNA-FISH were con rmed by IHC and PCR. These tests aided in proving that the results from PNA -FISH were promising and authentic and there were no cross reactions with other mycobacteria. PNA-FISH is less time consuming, a 3-4 hr process time is required to obtain the results whereas other techniques are more laborious. Thus, the results of the present study suggested that IHC and molecular methods like PCR are required for con rmation of M.
bovis. The advantage of IHC is that it is robust and can even detect fragmented tubercle bacilli [11]. In our study, PNA FISH procedure was used to identify and visualize mycobacteria in clinical specimens and directly within the tissue sections, and was shown to be a fast and appropriate tool for research and diagnostic purposes. In the present study, acid fast bacilli were observed in nineteen out of twenty ve cases, detected by ZN staining technique. The microscopic examination of an Acid fast stain is simple and fast, and a positive AFB stain is the rst sign of possible TB. However, staining can yield below par predictive values for TB in clinical settings in which NTM is frequently isolated, because it does not allow differentiation of MTB from NTM [12].

Conclusion
It was concluded from the study that among conventional and molecular diagnostic methods, PNA-FISH can be used in cytological impression smears and tissue sections. It is less time consuming in diagnosis of bTB in post mortem cases than PCR. The tissue samples were collected in two containers separately, one in 10 % NBF for histopathology and frozen tissues in sterile container for PCR studies.

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-xed and para n embedded.

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 37 0 C.

For identi cation of Culture
Two loops full of tissue homogenate were smeared on glass slides. The smears were dried, heat xed, stained with Ziehl-Neelsen (ZN) and examined for Acid Fast Bacilli (AFB).

PNA synthesis and labelling
Samples included in the study were identi ed by the MTBCcy3 Probe and MAV TAMRA hybridization assay (PNA Bios Probe, USA). Probes MTBCCy3 and MAVTAMRA, were used for speci c 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.

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 modi cations. Standard Culture-grown and xed bacteria (20 µl) were spotted onto three-eld microscope slides (Thermo scienti c, 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 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 uid (Vector Laboratories Inc., Burlingame, Calif. unspeci c 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 xed in 10% Neutral Buffered Formalin and were further processed as per conventional methods [15]. Thick para n tissue sections were spread on Superfrost positively charged microscopic slides (Fisher Scienti c, USA). Antigen retrieval was done in EZ antigen retrieval solutions using EZ-Retriever System (Bio Genex Laboratories Inc., California). After endogenous peroxidase and nonspeci c protein blocking, the sections were incubated with standardized dilution of (ESAT-6 and CFP-10) antibodies in a humidi ed 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].
Tissue sample DNA was ampli ed by esxA (ESAT-6 PCR, for detection of M. bovis. Amplicons of 61 bp were considered positive for ESAT-6 PCR respectively.

Declarations
Ethics Approval and Consent to participate We are thankful to the ethical committee for approving the use of animals and the approval of IAEC/CPCSEA is obtained vide reference no. IAEC/2015/26/013.The current study was approved by the Institutional Ethics Committee, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana (Approval number -IAEC/2015/26-013). The study was conducted from January 2017 to June 2019. ] Consent for publication.

Not applicable
Availability of data and material The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.