Detection of Nontuberculous Mycobacterial Species from Tissue Samples of Cattle and Buffaloes by PCR and PRA (PCR-RFLP)

Non-tuberculous mycobacteria (NTM) are the ‘atypical mycobacteria’ belonging to species other than those in the Mycobacterium tuberculosis complex. Although most NTM are known to be saprophytic in nature, some NTM species are known to cause pulmonary infections (Griffith et al., 2007) due to which they are being recognized worldwide. These organisms are efficient in causing pulmonary disease, disseminated disease or localized lesions in both immuno-competent and immune-compromised hosts (animals as well as humans) (Jarzembowski and Young, 2008). NTM can activate non-specific immune response which leads to false positive reactions in tuberculin testing (Bouts et al., 2009; Kazda and Cook, 1988) and causes disease, lymphadenitis, soft tissue infections, skin infections and visceral or disseminated disease (Chan and Iseman, 2013). Pulmonary infections are most commonly caused by Mycobacterium avium complex (MAC), Mycobacterium kansasii and Mycobacterium abscessus. These infections are sometimes asymptomatic which does not always equate with active infection and are diagnosed with supportive radiographic and clinical findings (Johnson and Odell, 2014). NTM such as Mycobacterium chelonei, Mycobacterium fortuitum, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium thermoresistible cause disease including mastitis in cattle and cutaneous mycobacterial granuloma in cats and dogs. Lesions in respiratory tract and lymph nodes of cattle are produced by M. kansasii and can also be isolated from tissue samples of cattle giving a positive TST (tuberculin skin testing) (Waters et al., 2006; 2010).
 
Identification of NTM species can be done based on their phenotypic characteristics of biochemical testing, pigment production, growth characteristics and colonial morphology but these traditional methods are time-consuming (Witebsky and Kruczak-Filipov, 1996; Metchock et al., 1999). Thus, more advanced techniques for rapid identification of NTM such as commercial nucleic acid probes, 16S ribosomal DNA sequencing (Turenne et al., 2001), high-performance liquid chromatography (HPLC) (Butler and Guthertz, 2001) and PCR-restriction enzyme pattern analysis (PRA) (Telenti et al., 1993) methods have been developed.
 
Molecular detection of NTM by PCR based amplification of mycobacterial DNA with genus-specific primers has been used. The identification is done on the basis of comparison between nucleotide sequence and reference sequences. The most commonly used target gene is the gene coding 16S ribosomal RNA. In addition to this, many other genes have been targeted for this purpose (gene encoding 32kDa protein (Soini et al., 1994), the 65 kDa heat shock protein (Telenti et al., 1993) and the 16S-23S ribosomal RNA internal transcribed spacer (Roth et al., 1998).
The impact of NTM is both direct, causing more or less severe infections and loss of productivity, or indirect, by interfering with diagnosis and control of bovine tuberculosis and paratuberculosis. In this study, highly conserved 65 kDa heat shock protein was used for NTM detection by PRA as a target.

Lymph nodes, lungs tissue samples (n=30) suspected of bovine tuberculosis from cattle (n=20) and buffaloes (n=10) with gross lesions/anomaly (tubercle lesions, enlarged lymph nodes, abscess) and without any gross lesions/ anomaly were collected from postmortem hall GADVASU, Ludhiana. DNA from tissue samples as well as from standard cultures was extracted using NucleoSpin Tissue DNA extraction Kit (Machery-Nagel) as per the manufacturer’s protocol. Later, PCR and PRA (PCR-RFLP) was conducted on the extracted DNA for identification of presence of NTM in these samples.
 
Polymerase chain reaction
 
The extracted DNA was amplified using 5 sets of primers (Table 1).
 

 
PCR protocol
 
For the amplification, the reaction volume of 25 μl was made containing 12.5 μl of GoTaq® Green Master mix, 1 μl of forward primer (10 pmol/μl), 1 μl of reverse primers (10 pmol/μl), 2.5 μl of nuclease free water and 8 μl of DNA template along with the test sample DNA, known positive control DNA from standard cultures of M. kansasii (MTCC3058), M. smegmatis (MTCC6), M. vaccae (MTCC272), M. fortuitum subspp. fortuitum (MTCC929) and M. intracellulare (MTCC920) (IMTECH, Chandigarh) were also amplified.
 
Thermocycling conditions
 
The thermocycling conditions for M. kansasii M. smegmatis M. vaccae and M. intracellulare were same. Thermal cycling was performed in research thermal cycler and cycling conditions were as follows, initial denaturation at 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 1 minute, annealing of primers at 60°C for 1 minute and 62°C for 1 minute for M. fortuitum, extension at 72°C for 1 minute and final extension at 72°C for 10 minutes. The amplified PCR products were then run by agarose gel electrophoresis using 1.5 per cent agarose and visualized in Gel Documentation System (Alpha Innotech).
 
PCR-Restriction fragment length polymorphism analysis (PRA)
 
PRA uses the polymerase chain reaction to amplify the selected DNA regions (the internal transcribed spacer ITS regions) (Park et al., 2000). These PCR products are then digested by restriction enzymes and visualized on an agarose gel. This method was used to differentiate various species of NTM from samples collected.
 
Amplification of hsp65 gene
 
A highly conserved heat shock protein 65 portion gene of Mycobacteria was amplified using primer sequence forward (Tbll) 5¢- ACCAACGATGGTGTGTCCAT-3¢ and reverse (Tbl2) 5¢- CTTGTCGAACCGCATACCCT-3¢ of 439 bp (Schinnick, 1987) (Applied Biosystem). For the amplification, the reaction volume of 25 µl was made containing 12.5 µl of GoTaq® Green Master mix, 1 µl of forward primer (10 pmol/µl), 1 µl of reverse primers (10 pmol/µl), 2.5 µl of nuclease free water and 8 µl of DNA template. Along with the test sample DNA, known positive control DNA of M. kansasii (MTCC3058), M. smegmatis (MTCC6), M. vaccae (MTCC272), M. fortuitum subspp. fortuitum (MTCC929) and M. intracellulare (MTCC920) (IMTECH, Chandigarh) were also amplified. The reaction was subjected to 45 cycles of amplification which includes denaturation for 1 min at 94°C, annealing for 1 min at 56°C and extension for 1 min at 72°C and the final extension was done at 72°C for 10 min.
 
Restriction fragment length polymorphism (RFLP)
 
The amplified PCR product (439 bp) was then digested with two enzymes BstEII and HaeIII (promega). RFLP of the standard cultures of M. kansasii, M. smegmatis, M. vaccae, M. fortuitum and M. intracellulare was also done. For the digestion of PCR product with BstEII,  10 µl of PCR product was added directly to the mixture containing 1 µl (5 U) of enzyme, 2.5 µl of restriction buffer (5 x buffer B) and 11.5 µl of water the mixture was incubated at 60°C for 60 minutes. Similarly, for the digestion of PCR product with HaeIII, 10 µl of PCR product was added directly to the mixture containing 1 µl (5 U) of enzyme, 2.5 µl of restriction buffer (5 × buffer B) and 11.5 µl of water the mixture was incubated at 37°C for 60 minutes (Telenti et al., 1993). The enzymes and buffers were purchased from Promega.
 
Evaluation of restriction patterns
 
After the digestion, 4 µl of gel loading buffer (0.25% bromophenol blue, 40% sucrose in water) was added and 10 µl of the mixture was loaded onto a NuSieve 3:1 agarose gel (Lonza). A gene ruler DNA™ ladder plus 50 bp (Fermentas) was run along with the test samples. The gel was visualized in Gel Documentation system (AlphaImager 3400HP, AlphaInnotech). The size of the amplicon was determined by comparing it with the standard molecular weight marker. The results were interpreted as per the algorithm used by Telenti et al., (1993) and PRA site (http://app.chuv.ch/prasite/index.html).

PCR
 
In the present study 30 lymph nodes and lung tissue samples were subjected to PCR for detection of NTM. The size of the amplicon was determined by comparing it with the standard molecular weight marker. Amplicons of 152 bp, 628 bp, 172 bp and 450 bp and 500 bp were considered positive for M. kansasii, M. smegmatis, M. fortuitum and M. intracellulare and M. vaccae. Out of 30 samples 2 samples were positive for M. kansasii, one from tissues with gross lesions and one without any gross lession (Fig 1). In molecular detection of NTM 16S-23S internal transcribed spacer region is the most common genomic loci. In a cross-sectional study conducted by Hoza et al., (2016) a total of 744 sputum samples were collected from 372 TB suspects. They were detected by using various methods (16S rRNA and hsp65 gene sequencing). The prevalence of NTM was found to be 9.7% of the mycobacterial isolates. A similar study was done by Ghielmetti et al., (2018) in which M. kansasii was detected from tissue samples with and without having macroscopic lesions.
 

 
Differentiation of NTM by PRA
 
PCR-RFLP (PRA) is a rapid and reliable technique that gives the ability to identify different species of mycobacteria. In PRA method, 439 bp PCR product of hsp65 gene was amplified (in both standard culture DNA and samples) and digested with the BstEII and HaeIII restriction enzymes. The restriction patterns were analyzed for species identification as per Saifi et al., (2013). Similar study was conducted by Telenti et al., (1993) in which 65-kDa protein (hsp65 protein) was used.
 
PCR for presence of hsp65 gene
 
Among the clinical samples processed 3 out of 30 tissue
samples (10%) were positive for hsp 65 gene (Fig 3). The standard cultures (M. kansasii, M. smegmatis, M. fortuitum, M. vaccae, M. intracellulare) also showed the 439 bp band of hsp65 gene (Fig 2).
 

 

 
Restriction enzyme analysis of the hsp65 gene
 
The PCR product of hsp65 gene amplicon of standard cultures along with the samples was subjected to digestion with restriction enzyme using BstEIII and HaeIII (Fig 4). From 30 tissue samples, two were identified as M. kansasii (n=2) having the RFLP pattern as 245/220 bp when digested with BstEIII and 140/105/70 bp when digested with HaeIII (Fig 5) and one could not be identified as the RFLP pattern was different from other known patterns (Fig 6).
 

 

 
@figure6
 
M. kansasii is also known to have the potential to interfere with bTB diagnostics and in some cases it may interfere with bTB diagnosis giving false-positive reactions (Vordermeier et al., 2012). Although the infection caused by M. kansasii is rare and is often associated with respiratory tract and associated lymph nodes lesions, diagnosed at postmortem. Chang et al. (2002) treated 439-bp PCR product of 10 NTM with BstEII and HaeIII for identification of NTM to species level. Six different RFLP profiles were produced by digestion with BstEII and eight different RFLP profiles were produced by digestion with HaeIII. From this, 9 of 10 samples of NTM were identified to the species level. Six mycobacterial species were identified, including M. gordonae type I, M. gordonae type II, M. gastri, M. kansasii, two M. fortuitum subsp. 3rd variant, M. simiae, M. scrofulaceum and M. szuigai.
       
M. kansasii and M. persicum are known to have the potential to interfere with bTB diagnostics and, in some cases, to cause false-positive reactions leading to considerable economic losses (Vordermeier et al., 2007;et_al2006).
       
The hsp6 gene was chosen as the target for amplification because it’s highly conserved among all the Mycobacterium species as reported by Buchanan et al., (1987). Some other PCR based procedures capable of identifying multiple species of Mycobacteria have been classified by Plikaytis et al., (1992). The PCR-RFLP procedure by Telenti et al., (1993) is more reliable because of its ability to identify the more number of species without the need for probing or sequencing of the amplicons. Similar study by  Hafner et al., (2004) analysed the heat shock protein 65 (hsp65) gene restriction fragment length polymorphism (RFLP) patterns of some rarely isolated NTM for which patterns were not been published before (Mycobacterium bohemicum, Mycobacterium hassiacum, Mycobacterium heckeshornense, Mycobacterium monacense and Mycobacterium triplex). Also new hsp65-variants for Mycobacterium interjectum (type II), Mycobacterium mucogenicum (type V), Mycobacterium gordonae (type VIII) and Mycobacterium paraffinicum were described. Tortone et al., (2018) evaluated the usefulness of molecular methods, especially hsp65-PRA (PCR-Restriction Enzyme Analysis). For 56 NTM isolates recovered from 32 (42.1%) positive samples were used in the study and identification upto species level was done using hsp65-PRA.
 
Ong et al., (2010) also reported 5 novel restriction patterns, different from any of the patterns of the algorithm. Saifi et al., (2013) suggested that PRA is sensitive, specific and an effective assay for detection of nontuberculous mycobacterial species than other PCR based techniques and is capable of identification of large number of species without using any probe or amplicon sequencing. A study was conducted by Nour-Neamatollahie et al., (2017) in which (PRA) of the hsp65 gene was done on clinical samples (sputum, bronchial lavage, skin samples) of Tb suspected patients as a result of which majority of NTM were obtained along with M. bovis and M. tuberculosis. The most frequently detected Mycobacterium species were Mycobacterium kansasii, which was isolated in 5 (45.4%) out of 11 patients with NTM pulmonary disease.

NTM such as M. kansasi may cause infection in animals and PRA techniques was found to be a rapid tool for identification and differentiation of NTM upto species level.

The authors are thankful to DBT for providing the necessary funds under the scheme “Evaluation of diagnostic assays for quicker diagnosis of Mycobacterial infections in cattle and buffaloes” (BT/PR5776/MED/30/928/2012) and the Director of Research, GADVASU, Ludhiana for providing the necessary facilities.