Research Article - (2019) Volume 7, Issue 5

Direct DNA Modified CTAB Preparation from Nasal Exudate in Live M. bovis Infected Cattle in Mexico Provide with a Valuable Assay Extrapolated to Humans TB Diagnostic Test
Sanchez-Garza JJ1, Garcia-Roque BM1,2, Mayorquin-Luna GA3, Cepeda Hortensia4, Vázquez AJ3, Favela-Hernández JM5 and Guerrero GG1*
 
1Academic Unit of Biological Sciences, Laboratory of Immunobiology, Autonomous University of Zacatecas, Zacatecas, Zac, Mexico
2Academic Unit of Chemical Sciences, Program in Chemistry Pharmacobiology, Autonomous University of Zacatecas, Zacatecas, Zac, Mexico
3National Polytechnic Institute, Zacatecas, Zac, Mexico
4Antonio Narro Agrarian University, Torreón Unit, Coahuila, Mexico
5Avicena Institute, Torreón, Coahuila, Mexico
 
*Correspondence: Guerrero GG, Academic Unit of Biological Sciences, Laboratory of Immunobiology, Autonomous University of Zacatecas, Zacatecas, Zac, Mexico, Tel: +52 492 1564376, Email: ,

Received: 21-Jun-2019 Published: 25-Sep-2019, DOI: 10.35248/2329-891X.19.7.325

Abstract

Detection, identification, and differentiation of members of the MTB complex rely on specificity, sensitivity, and accuracy of the methods that have been developed since the decades of the ’90s. Despite this, still, in endemic areas of developing countries tuberculin field test as well as conventional techniques (histopathology and bacteriology) are performed due primarily to the costs and availability. Therefore, it is an urgent need to have a routine assay to boost field test (false positive and negative tests) in live cows while avoiding the unnecessary sacrifice of animals. To this end, in the present work, we designed a dual experimental strategy that can be used as a routine assay for the M. bovis or M. tuberculosis detection through PCR mediated amplification of RD’s. DNA can be prepared from fast-growing colonies (7 to 8 days) or from homogenized tissue, nasal exudate and purification mediated cetyl-trimethylammonium bromide (CTAB) cationic buffer. The method was extraplated to positive TB positive nasal/oral human exudate.with similar results. Collectively these findings indicate that this strategy represent a valuable tool for TBb epidemiological survey and research.

Keywords

M. bovis; M. tuberculosis; Cetyl-trimethyl-ammonium bromide (CTAB); PCR; DNA

Introduction

Bovine tuberculosis (TBb) is a zoonotic, chronic disease of cattle caused by Mycobacterium bovis, a member of the M. tuberculosis complex that represents a serious health problem [1,2]. In accordance with a recent report [3], it has been estimated that more than 50 million cows are infected worldwide with M. bovis leading to $3 billion annually losses in Agriculture [3]. The problem is aggravated by the number of asymptomatic infected animals that have not been notified, diagnosed leading to an underestimation of TBb in endemic regions [4,5]. Despite extensive efforts to control and eradicate the disease in endemic regions, still there an urgent need to make available faster test from live cows, since most of the diagnostic test rely; either in the field tests (caudal fold, comparative cervical and simple cervical) and/or confirmatory assays (negative or positive) (bacteriological and histopathological) in slaughtered cattle [6-8].

From the annals of the literature toward the development of molecular detection methods in TBb, a test that relies in the rRNA determination was found but not further studies were made [9]. Instead, in the last decades, M. bovis molecular detection based on different types of PCR (nested, multiplex, real-time) have been developed with promising results [10,11-16]

For example, Liébana et al. [10] performed a simple detection of M. bovis, directly from samples of bovine tissue by means of a PCR technique, but the test was not able to distinguish between the different members of the M. tuberculosis complex [10] In addition, PCR test was not as sensitive as the culture and it did not always detect those samples that contained a small number of organisms, which could due to the difficulty of extracting DNA from the isolates (tissue) of infected animals [17]. Moreover, the sensitivity of PCR can be affected by inhibitors in clinical samples, inhibitors reagents derived from the extraction method or the yield of DNA [18] or procedures for bacteria lysis and DNA extraction involve time-consuming steps and costly which is not always available in developed countries Amaro et al. [19], or a lack of specificity and sensitivity because the region to be amplified is not the optimal [18-20] Amaro et al. [19]. In contrast, Lázaro et al. [21] and Cepeda and Vázquez. [22], respectively, showed that RD4 amplification could lead to higher specificity and sensitivity to distinguish between M. bovis from the rest of the Mycobacteria that belong to the complex of M. tuberculosis (from 20/20, 100% sensible and specific) [20] and M. bovis in the nasal exudate of TST+ cows. To note is that DNA preparation for PCR amplification was undertaken with a cationic buffer, the cetyl trimethyl ammonium bromide (CTAB) [22].

Interestingly, Duarte de Silva et al. [15] were able to detect M. bovis directly in bovine milk and blood samples using real-time PCR. However, the milk sample that tested positive for M. bovis did not belong to any of the animals that had positive blood samples whereas the presence of proteins and milk fats that may have altered the extraction of M. bovis DNA. In a more recent study, it was compared the analytical performance of a multiplex real-time PCR assay for the detection of M. bovis, M. californicum and M. bovigenitalium in culture versus samples of bovine milk, semen, and nasal exudate. Samples were also seeded on the appropriate solid growing medium and except for the nasal exudate; colonies were observed [23]. The authors concluded that the PCR technique has a better analytical performance than the traditional culture for the detection of mycobacteria in nasal exudates of bovines [24]. To note is the several advancements for the improvement of detection, differentiation of the members of the MTB complex [25-31], each other have provided with a key point to take in the account either for TBb or human tuberculosis [25-31].

In the light of all these data from the literature, we pursued to design an experimental strategy that can be used as routine assay for the M. bovis or M. tuberculosis detection through PCR mediated amplification of Regions of Difference (RD’s) DNA can be prepared from fast-growing colonies (7 to 8 days) or from homogenized tissue, nasal exudate, and purification mediated cetyl-trimethyl-ammonium bromide (CTAB) cationic buffer. This has been extrapolated nasal/oral human exudate. diagnostic with similar results. Collectively these findings provide a valuable assay for TBb epidemiological survey and research.

Materials and Methods

Animals

Cattle were selected based on the inclusion criteria and according to the NOM-031-ZOO-15. Negative and positive bovines to the Tuberculin Skin Test (TST) for live studies: TST (-) to the caudal anus and the comparative cervical test while TST (±) to either of the field tests but negative for bacteriological and histopathological exams, considered as a reactor. TST (+) was considered positive either to field tests as well as for bacteriological and histopathological exams was considered as positive.

Samples collection

Tissues: A piece of tissue samples of slaughtered cattle were collected in PBS or borate buffer, homogenized and processed as described below.

Exudate: The exudate is collected by introducing a sterile swab into the nostril of the immobilized animal. Thereafter, a nasal swab is placed and homogenized in 5 ml of phosphate buffer (PBS) and maintained on ice. The swab is removed and the suspension is centrifuged at 10,000 rpm/4ºC. The supernatant is discarded and the pellet is recovered, which is resuspended in TE buffer and stored at -30°C until processing.

Culture middlebrook medium

Solid Middlebrook (7H10) medium dissolved in 900 ml of sterile MQ water, with vigorous stirring. Subsequently, 5 ml of glycerol is added and it is homogenized with agitation. The medium is sterilized and OADC, (dextrose, catalase, and albumin) (Gibco, Co) PANTA and THF (2- thiophenocarboxylizocidhydrazide) (2 mg/ml) (Sigma, Aldrich, Co were added and Petri dishes were prepared [32].

Direct DNA preparation using cationic cethyl-tri-methylammonium bromide (CTAB) from tissue or exudate

The extraction of DNA from the tissue sample is carried out from tissue homogenate (milteny homogenizer) in TE buffer. Incubate for 2 hours at a temperature of 37°C with 50 μl of lysozyme (10 mg/ml). Time to which 75 μl of 10% SDS are added, and mixed by inversion. 50 μl Proteinase K (1 mg/ml) is added, with gentle shaking and incubated at 65°C for 20 minutes [22,33]. Subsequently, 100 μl of 5 M NaCl is added and mixed gently by inversion. 100 μl of pre-warmed CTAB/ NaCl buffer is added, and shake in the vortex until the sample is white incubating at 65°C for 10 minutes. The separation of the organic and aqueous phases is carried out by the addition of 750 μl of chloroform/isoamyl alcohol (24:1) and centrifugation at 12,000 rpm/5 min/room temperature. DNA is precipitated from the aqueous phase with 750 μl of isopropanol, incubation for 30 min at -20°C. Centrifuge at 12,000 rpm/5min. The pellet is recovered, washed with 1 ml of 70% ethanol and finally resuspended in 50 μl of DEPEC water and stored at -30°C until use.

DNA preparation extraction using ethyl-tri-methylammonium bromide (CTAB) and filter mediated purification

The direct DNA extraction method using the CTAB extraction reagent (materials and methods) [23] yielded a DNA of varying concentration. To optimize this parameter, the aqueous phase that is usually added isopropanol to precipitate the DNA, which was done was transferred to a column of the Qiagen kit (indirect method) and centrifuged at 10,000 rpm for 30 seconds. The DNA bound to the membrane of the column was washed with the AW1 buffer and the AW2 buffer, respectively, and centrifuged each time at 10,000 rpm for 20 seconds. In a third wash, 50 μl of the elution buffer was added and incubated at room temperature/1 min, and again centrifugation at 10,000 rpm. This step was repeated until obtaining a final volume of 100 μl, which was stored until its use [32].

Exudate DNA extraction

the nasal swabs were removed from the tubes containing PBS and these were centrifuged to subsequently eliminate the liquid phase, while the pellet was homogenized in 400 μl of TE buffer and subsequently incubated at 37°C with 50 μl of lysozyme at a concentration of 10 mg/ml). Purification of DNA was made as above described [22,33].

PCR amplification of the RD4 (M. bovis) or RD5 (M. tuberculosis) DNA fragment purified by the cethyl-trimethyl- ammonium bromide (CTAB) modified method

The amplification reaction of a M. bovis DNA fragment was carried out, following the instructions of the manufacturer (Qiagen protocol). Briefly, to each reaction mixture, 0.25 μl of the forward, M. bovis RD4 region forward (FAACGCGACGACCTCATATTC) and 0.25 μl of the reverse (R- AAGGCGAACAGATTCAGCAT). M. tuberculosis RD1-RD5 region: forward, F (GGTTTTGGGTCTGACGAC); reverse, R (CCGAGAGGGGACGGAAAC) [21,22,34-36] were added and incubated at temperature environment for 15 minutes, then add 25 μl of the master mix or master mix it contains, the enzyme Hot Star Taq polymerase, dNTPs (dATP, dCTP, dGTP, dTTP), MgCl2 and KCl. Mix gently and add 16.5 μl of DEPEC wáter. Finally, 8 μl of the DNA sample (~ 0.7-1 μg/ml) are added. They are homogenized gently. For the amplification of a DNA fragment, the one recommended by the manufacturer (QIAGEN, Co) was followed. The analysis of the PCR products was carried out on a 1% agarose gel in TAE buffer and syber safe staining. The gel is prepared by weighing 0.30 g of agarose and dissolved in 30 ml of the buffer with heating. Allow to warm, and add 1 μl of syber Safe (Invitrogen, Co). The DNA samples are prepared by mixing a given volume with 4 μl of loading buffer. The gel is run in TAE buffer in a 90 volt electrophoresis chamber. The DNA bands are visualized in a photodocument (Chemidoc) (Bio-Rad).

Results

A protocol designed for the molecular detection of Mycobacterium bovis from live cows

Despite the huge amount of methodologies for the molecular detection of members of the MTB complex, still, there is a percentage of variability and sensitivity [12] in the data depending on the experimental settings used for that purpose. Therefore, we aimed to design a simple protocol to be used as a routine assay for the extensive epidemiological survey as well as for research. To this end, we collected nasal swabs from TST (+) cattle and TST (-). From this, we followed two mains steps for DNA preparation (Figure 1). Nasal swabs were also seeded as described in Figure 1 and the suspension in buffer was used for DNA extraction. As positive controls, we used M. bovis BCG and M. bovis AN5 [35,36] and RD1 primers (present only in AN5). In either case, it was obtained a band of around 7 kb (the amplicon) (Figure 2, lanes 1 and 7). No product was detected without DNA (Figure 1, lane 2); with only primers (Figure 1, lane 3); with DNA from E. coli (Figure 1, lane 6). RD4 amplification was obtained for M. bovis BCG (Figure 1, lane 4); for AN5 (Figure 1, lane 8). RD1 amplification for AN5 (Figure 1, lane 9) but not for M. bovis BCG (Figure 1, lane 5).

tropical-diseases-experimental-protocol

Figure 1: A experimental protocol designed for the study. A simple and rapid method was designed as a routine assay for M. bovis or M. tuberculosis detection either direct from the simple collected or from seeding in solid Middlebroth culture médium supplemented with THF as described in material and methods.

tropical-diseases-tissue-nasal-exudate

Figure 2: Direct detection from tissue or nasal exudate from M. bovis infected cattle from Mexico. Lymph nodes (A) or nasal exudate (B) were collected after field tuberculin tests. DNA was extracted using a
modified CTAB cationic buffer (this work) as described in materials and methods An amplicon of around 7 kb was obtained. The methods was reproducible in all cases. RD4 PCR amplification was made in
either case, a fragment of around 400 bp was obtained (Figure 2A, lanes 1-6) and/or (Figure 2B, lanes 1-6).

RD4 amplification from CTAB DNA prepared through direct sampling (nasal exudate) from in live cattle in Mexico

Next, DNA was prepared from nasal swabs using CTAB cationic detergent modified (this work) (materials and methods) (Figure 2B, lanes 4-7) which depending on the system, can eliminate genomic DNA or other undesirable components that affect the yield and integrity of DNA (Figure 2 A-B). This methodology was applied also to tissues (lymph nodes) (with similar results (Figure 2A, lanes 1-3).

The DNA obtained in either case, tissue (Figure 2A, lanes 1-3) and/or nasal exudate (Figure 2B, lanes 4-6) fragment of fragment of 400 bp corresponding to RD4 of M. bovis genome, [21,35,36]. As negative controls from TST (-) cows (Figure3C, lane 1). Neither amplicon was obtained (Figure 3C), nor RD4 PCR product (Figure 3C, lane 1).

tropical-diseases-molecular-detection

Figure 3: The validation of the designed methodology for molecular detection of M. bovis was extrapolated to human nasal/oral exudate (2 independent individuals). The DNA preparation (amplicon of around
7 kb) was made using the CTAB modified protocol (this work) (A, lanes 1-4) as well as the filter column kit (3C, lanes 1-3). RD4 amplification (B, lanes 1-4 nasal/oral, respectively for 2 individual) and D, amplification of the RD4 region from DNA of colonies of AN5 (lane 1) and from nasal/oral of one individual is shown (lanes 1-2).

Seeding of nasal exudate yielded fast-growing colonies of M. bovis isolated from live cattle in Mexico

To confirm the above results (Figures 3 and 4), nasal exudate and lymph nodes (retropharyngeal and mesenteric) from live cattle (TST+) and slaughtered cattle respectively were seeded on solid growing medium as described in Materials and Methods, In either case, 7-8 days post-seeding colonies were obtained. DNA preparation from these colonies was made using a filter column as indicated by the manufacturer. A thin band of the amplicon (around 7 kb) was obtained from tissue (Figure 2A, lanes 1-4) or nasal exudate (Figure 2A, lanes 5-7) [25,35-36]. RD4 amplification of M. bovis DNA obtained from nasal exudate or from tissue was made and one fragment of around 400 bp was obtained in either case, tissue (Figure 2B, lanes 1-4) or exudate (Figure 2B, lanes 5-7).

tropical-diseases-colony-Mycobacterium

Figure 4: DNA (amplicon of around 7 kb) from a colony of Mycobacterium bovis BCG or reference M. bovis strain AN5 grown in soil Middlebroth culture médium supplemented with THF (AN5) (lane 4)
or without (BCG) (lane 1) as described in material and methods and amplified for RD4 region (AN5, lane 5) and BCG (lane 2) or RD1 (AN5, lane 6).

Amplification of the RD4 region of M. bovis DNA obtained from human nasal/oral exudate of infected individuals

To extrapolate these findings from infected live or slaughtered cattle (in the case of the tissues), we applied the same methodology. DNA preparation from nasal/oral exudate respectively using the above-described methodologies (CTAB cationic buffer) or filter columns yielded a band of an amplicon of around 7 kb (Figure 5A, lanes 1-4; 5B, lanes1-3). A fragment of around 400 bp was obtained for M. bovis (Figure 5B, lanes 1-4; 4D, lanes 1-3). Furthermore, DNA either from bovine or human were tested with M. tuberculosis primers yielding two bands (in preparation).

tropical-diseases-nasal-exudate

Figure 5: Tissues (Lymph node) or nasal exudate were seeded and DNA was prepared from colonies. RD4 amplification yielded also a fragment of 400 bp.

Discussion and Conclusion

In the present work, we are reporting an experimental strategy to be used as a routine assay from live cows for the detection of Mycobacterium bovis or M. tuberculosis RD’s. The validation was made in human nasal/oral exudate.

Detection, identification, and differentiation of members of the MTBC complex rely on specificity, sensitivity, and accuracy of the methods that have been developed since the decades of the ’90s. Despite this, still in endemic areas developed countries tuberculin field test, as well as conventional techniques (histopathology and bacteriology), is performed due primarily to the costs and availability [25-31].

Therefore, it is an urgent need for endemic regions to have a routine assay to boost field test (false positive and negative tests) in live cows [37]. Moreover, the development of optimal PCR methods that can boost field tuberculin tests (false positive/false negative) of live cows while avoiding the unnecessary sacrifice of animals a molecular method should fulfill some basic requirements such as the high-quality DNA, the regions to be amplified and primers design [8,16,22,24-31,37-39]. M. bovis detection either from cattle or humans samples has moderate sensitivity, mainly attributed to the difficult for DNA extraction (bacterial lysis, long manipulation steps) or quality of the sample collection [17,25-28]. Nowadays, there are a several molecular methods for the detection of M. bovis that goes from simple PCR to PCR in real-time using either milk, semen, urine or nasal exudate [16,23,24-31], some of them fill one or two of the basic requirements mentioned above, but not all. In some of them, the results should be corroborated throughout histopathology and bacteriology, which would imply, time, and costs; and therefore, it can not be used extensively in epidemiological surveys [37-42]. Thus, in the present work, we aimed to design develop an assay that can be used as routine assay for the M. bovis or M. tuberculosis detection that involves DNA extraction prepared from fast-growing colonies (7 to 8 days) (Figures 3 and 4) or a direct from nasal exudate using cetyl-trimethylammonium bromide (CTAB) cationic buffer improved the quality of DNAs because denatures proteins, solubilize cell wall and lipid proteins [26] and PCR mediated molecular detection [25-31,38,39,43,44]. Furthermore, we observed complementation between the direct method for DNA preparation from exudate nasal/oral and PCR RD ’ s J Trop Dis, Vol.7 Iss.5 No:1000325 5 amplification either from M. bovis or M. tuberculosis (in human MTb in preparation) with exudate seeding, colonies growth and RD’s PCR amplification method [15,17,25] (Figures 3, 4 and 5B), To note is that tissues samples can be obtained only from slaughtered cattle, representing thus a hindrance to perform more extensive screening studies. Thus, herein we are reporting that through this improved nasal exudate M. bovis detection protocol, it is possible to avoid the unnecessary sacrifice of animals, and strengthen tuberculin test, offering thus an advantage and a promising alternative that can be extrapolated to humans, for TB epidemiological surveys.

Conflict of Interest Statement

The authors declare no conflict of interest.

Acknowledgment

The authors are grateful for the financial support of SNI CONACYT, SAGARPA-PIDETEC 2015, PRODEP, and PERFIL-PRODEP. We are also grateful with Dr. Myriam Bobadilla (INCMSZ; Mexico) for providing AN5 strain, with MVZ Sergio Vazquez from CEFOP, Calera, Zacatecas, Mexico for cattle sample collection.

REFERENCES

Citation: Sanchez-Garza JJ, Garcia-Roque BM, Mayorquin-Luna GA, Hortensia C, Vázquez AJ, Favela-Hernández JM, et al. (2019) Direct DNA Modified CTAB Preparation from Nasal Exudate in Live M. bovis Infected Cattle in Mexico Provide with a Valuable Assay Extrapolated to Humans TB Diagnostic Test. J Trop Dis 7:325. doi: 10.35248/2329-891X.19.7.325

Copyright: © 2019 Sanchez-Garza JJ, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.