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Research Article
 

Identification of Mycobacterium tuberculosis Complex by Culture and Duplex Polymerase Chain Reaction in Bovines



D. Neeraja, B.M. Veeregowda, M. Sobha Rani, D. Rathnamma, H.D. Narayanaswamy, M.D. Venkatesha, G. Leena, R. Apsana, S.H. Somshekhar, M. Saminathan, K. Dhama and S. Chakraborty
 
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ABSTRACT

Bovine tuberculosis (bTB) is an economically important zoonotic disease (can spread to human through inhalation or ingestion) caused by Mycobacterium bovis which belongs to Mycobacterium tuberculosis complex (MTC). Control and eradication of infection is difficult even in organized dairy farms. So combinations of tests like culturing and nucleic acid-based diagnostics are used for the isolation and identification of mycobacterial infections in cattle. Even though, there are many advances in diagnosis of bovine TB infection in cattle but till now, isolation identification of the etiological agent from clinical samples stands as a definitive and gold standard test. Nucleic acid based methods like Polymerase Chain Reaction (PCR) which have advantages of speed, sensitivity and specificity can be used for diagnosis of tuberculosis along with isolation. In the present study, isolation of Mycobacterium tuberculosis complex organisms was attempted from nasal swabs and milk of cattle using Lowenstein-Jensen (LJ) media without glycerol. Cattle which were positive for tuberculosis either by skin test or gamma interferon test were selected. Two of the twelve nasal swabs and none of the seven milk samples showed typical mycobacterial colonies on LJ media after 8 weeks of incubation. Ziehl-Neelsen staining of colonies showed slender, rod shaped acid fast organisms suggestive of Mycobacterium. Deoxy ribo nucleic acid (DNA) was extracted by boiling method and amplified by duplex PCR for 245 and 500 bp amplicons specific for MTC (IS6110) and M. bovis (RvD1Rv2031c), respectively. Electrophoresis revealed 245 bp product but not 500 bp which confirmed the identity and relatedness of the isolated mycobacterium to Mycobacterium tuberculosis complex.

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D. Neeraja, B.M. Veeregowda, M. Sobha Rani, D. Rathnamma, H.D. Narayanaswamy, M.D. Venkatesha, G. Leena, R. Apsana, S.H. Somshekhar, M. Saminathan, K. Dhama and S. Chakraborty, 2014. Identification of Mycobacterium tuberculosis Complex by Culture and Duplex Polymerase Chain Reaction in Bovines. Asian Journal of Animal and Veterinary Advances, 9: 506-512.

DOI: 10.3923/ajava.2014.506.512

URL: https://scialert.net/abstract/?doi=ajava.2014.506.512
 
Received: April 08, 2014; Accepted: June 15, 2014; Published: July 26, 2014



INTRODUCTION

Bovine tuberculosis (bTB) is an economically important zoonotic disease caused by Mycobacterium bovis which belongs to Mycobacterium tuberculosis complex. The complex also includes Mycobacterium bovis, M. tuberculosis, M. africanum, M. microti, M. canetti and M. caprae (Cousins et al., 2003; Good and Duignan, 2011; Atkins and Robinson, 2013). The disease can spread to humans, typically by the inhalation of aerosols or the ingestion of unpasteurized milk (Phillips et al., 2003; Evans et al., 2007; Verhagen et al., 2011). Office International des Epizooties (OIE) classified bTB as a list B transmissible disease of public health importance and is of high significance to the international trade of animals and animal products (OIE, 1999) and presently is a listed disease. Bovine tuberculosis occurs in almost every country of the world and is of major importance in dairy cattle due to high morbidity and loss of production as infected animals lose 10-25% of their productive efficiency (Cousins, 2001). It also has high economic impact on animal industry causing annual loss of three billion dollars worldwide affecting 50 million cattle (Cousins, 2001; Challu, 2007). Prevalence of bTB in Indian cattle and buffaloes was 1.6-16 and 3-25%, respectively (Challu, 2007). High prevalence of tuberculosis was detected in some South Indian states like Karnataka (30-35%) and Tamil Nadu (34.58%) (Mukherjee, 2006).

Control and eradication of infection is difficult even in organized dairy farms for sensitive, specific and reliable diagnostic tests for the detection of pre-clinical infections. Accurate diagnosis of disease in live animals is of paramount importance for an effective disease control and eradication program. So combinations of tests like culturing and nucleic acid-based diagnostics are used for the isolation and identification of mycobacterial infections in cattle. Even though, there are many advances in diagnosis of bovine TB infection in cattle but till now, isolation and identification of the etiological agent from clinical samples stands as a definitive and gold standard test. However isolation is slow, cumbersome and requires atleast 8 weeks (Rohonczy et al., 1996; Collins, 2011; Figueiredo et al., 2012).

The Ziehl-Neelsen acid fast staining is a simple and rapid method but cannot detect the species and lacks specificity and sensitivity (Barouni et al., 2004). Nucleic acid based methods like Polymerase Chain Reaction (PCR) which have advantages of speed, sensitivity and specificity can be used for diagnosis of tuberculosis along with isolation (Lermo et al., 2010; Collins, 2011; Gan et al., 2013). IS6110 and RvD1Rv2031c sequences are detected in M. tuberculosis complex and M. bovis, respectively which yield 245 and 500 bp fragment on amplification in a single tube reaction.

MATERIALS AND METHODS

Culturing
Samples: Ante mortem samples like milk samples (n = 7) and nasal swabs (n = 12) were collected from twelve cattle which were positive for skin or gamma interferon test.

Cultural isolation: The samples for isolation of mycobacteria were processed by decontaminating with 4% NaOH (modified Petroff’s method). Nasal swabs were agitated in 1 mL of Phosphate Buffered Saline (PBS) and 5 mL of milk taken in to sterile centrifuge tubes separately. Double volume of sterile NaOH (4%) was added to the sample, mixed and kept on shaker for 10-15 min, centrifuged at 3,000x g for 15 min and supernatant was discarded. Sterile distilled water was added up to neck of the bottle, mixed thoroughly and centrifuged at 3000x g for 15 min. The supernatant was discarded and about 50 μL of sediment was inoculated in to Lowenstein-Jensen (LJ) medium without glycerol and incubated at 37°C for 8 weeks in CO2 incubator (Srivastava et al., 2008).

Duplex-Polymerase Chain Reaction (PCR): The DNA was extracted from colonies on LJ slants by boiling method (Hosek et al., 2006). A loop full of inoculum was added to 30 μL of nuclease free water and boiled at 95°C for 15 min and kept at -20°C for 10 min. It was then re-centrifuged for 15 min at 13,000 rpm and 5-10 μL supernatant was used as a template. Duplex PCR was performed in a reaction mix (50 μL) containing 5 μL of 10X PCR buffer (Invitrogen®), 200 μM dNTP, 2.5 U of recombinant Taq polymerase (Invitrogen®), 0.2 μM of each primer (Figueiredo et al., 2009) -JB21(5’-TCG TCC GCT GAT GCA AGT GC-3’) and JB22 (5’-CGT CCG CTG ACC TCA AGA AAG-3’) for amplifying RvD1Rv2031c genes specific to M. bovis and INS1 (5’-CGT GAG GGC ATC GAG GTG GC-3’) and INS2 (5’-GCG TAG GCG TCG GTG ACA AA-3’) for amplifying IS6110 gene specific to M. tuberculosis complex, 2.0 mM MgCl2 and 5 μL of purified DNA template. Amplification was carried out in a Palm cyclerTM (Corbett Research, Australia) with the cycling conditions of 94°C for 5 min, followed by 30 cycles of 1 min at 94°C, 1 min at 68 and 72°C with a final extension at 72°C for 7 min. PCR products were checked by electrophoresis on 1.75% agarose gel containing ethidium bromide (10 μg mL-1) (Figueiredo et al., 2009).

RESULTS AND DISCUSSION

Two of the twelve nasal swabs and none of the milk samples showed characteristic colonies suggestive of Mycobacterium spp. (Fig. 1) after 8th week of incubation at 37°C on LJ medium without glycerol. Ziehl-Neelsen staining of colonies showed slender, rod shaped acid fast organisms (Fig. 2) suggestive of Mycobacterium spp.

Image for - Identification of Mycobacterium tuberculosis Complex by Culture and Duplex Polymerase Chain Reaction in Bovines
Fig. 1:Lowenstein Jensen's medium with mycobacterial colonies (8 weeks old)

Image for - Identification of Mycobacterium tuberculosis Complex by Culture and Duplex Polymerase Chain Reaction in Bovines
Fig. 2:Ziehl-Neelsen staining technique showing acid fast organisms from 8 week old colonies (1000X)

Image for - Identification of Mycobacterium tuberculosis Complex by Culture and Duplex Polymerase Chain Reaction in Bovines
Fig. 3:
Agarose gel electrophoresis of duplex PCR amplified products of IS6110 (245 bp specific for M. tuberculosis complex) and RvD1Rv2031c genes (500 bp specific for M. bovis), Lane M:100 bp ladder, P: Positive control, 1, 2, 3 and 4: Sample numbers and N: Negative control

In duplex PCR, the two isolates produced specific amplicons of 245 bp for IS6110 gene specific for M. tuberculosis complex (Fig. 3) but not 500 bp amplicon which is specific for M. bovis.

Culturing, though considered as definitive test, is limited to post mortem samples than ante mortem samples due to intermittent shedding of the organism. After 8 weeks of inoculation, isolation could be made from only two nasal swabs (16.67%) and none from milk samples. The results are in agreement with Figueiredo et al. (2012) who opined that nasal mucus samples are better than milk samples because in milk, mycobacterium could be killed by the action of macrophages and it is reported that only 5% of tuberculin reactors would excrete mycobacterium in milk (Palmer and Waters, 2006). However, Srivastava et al. (2008) in a study isolated both M. bovis (6/40; 15%) and M. tuberculosis (4/14; 28.5%) from the milk samples.

Definitive diagnosis of tuberculosis is carried out by isolation, biochemical identification and species level differentiation to MTC by culturing from post mortem samples. However, isolation from milk, nasal swabs and other such ante mortem samples is difficult because of varied factors. Those include, (a) Animals positive for Cell Mediated Immunity (CMI) based tests may not excrete organisms always in milk and nasal secretions because of intermittent shedding, (b) Clinical samples with less mycobacterial load might lose the bacteria while sample preparation (decontamination and antibiotic treatment), there is a loss of 5-10% viable bacteria (Stavri et al., 2003) and decontaminants like 4% NaOH are more toxic compared to others (Corner et al., 2012), (c) Mycobacterium being fastidious and slow growing in nature (Adams, 2001) requires at least six weeks for visible growth, (d) There are reports that only about 5% of Tuberculin-Reacting Cattle (TRC) can eliminate M. bovis. Because of all these factors, isolation from milk and other ante mortem samples became difficult and it is, therefore, restricted to post mortem samples.

M. bovis isolation from nasal swabs indicated that aerosol transmission of disease to humans and other animals is evident and isolation of Mycobacterium from healthy cattle indicate sub-clinical infection/latent infection in the herd (Srivastava et al., 2008). Further, isolation is laborious, time consuming, less sensitive and more specific method. Presence of some positive cultures is sufficient to declare the outbreak of tuberculosis in the herd (Figueiredo et al., 2010).

Duplex PCR was applied to two isolates obtained from nasal swabs to differentiate MTC and M. bovis in single tube reaction. Two isolates were confirmed as MTC as they produced 245 bp size amplicon but not as M. bovis, since it did not yield 500 bp amplicon. The results are not in agreement with Figueiredo et al. (2010) who could differentiate M. bovis from MTC in single-step multiplex PCR and Figueiredo et al. (2009) conducted multiplex PCR for the identification of M. bovis isolates having 100% concordance with the conventional culturing and biochemical reactions.

The isolates obtained in this study were confirmed as MTC but not M. bovis. There are chances that it may be M. bovis, since 13.3% of M. bovis failed to produce 500 bp fragment in multiplex PCR (mPCR) as reported by Figueiredo et al. (2010) and they may be M. caprae also, as it is naturally infecting the cattle. Alvarez et al. (2012) confirmed the presence of bTB in 42 herds by isolation of M. bovis/M. caprae. Good and Duignan (2011) reported bTB as a chronic bacterial disease caused by M. bovis and M. caprae, both cause infectious disease in cattle and the diagnostic tests used for detection of disease caused by M. caprae is not considered to be substantially different from that caused by M. bovis.

CONCLUSION

Confirmation of the identity and relatedness of the isolated mycobacterium to Mycobacterium Tuberculosis Complex (MTC) can be done exclusively based on molecular diagnostic tools like the duplex PCR which can be applied for differentiation of MTC and M. bovis in single tube reaction to further strengthens the diagnosis.

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