|Year : 2014 | Volume
| Issue : 2 | Page : 115-121
A polymerase chain reaction and enzyme linked immunosorbent assay based approach for diagnosis and differentiation between vaccinated and infected cattle with Mycobacterium bovis
Mohamed Sabry1, Ahmed Elkerdasy2
1 Department of Bacteriology, Mycology and Immunology, College of Veterinary Medicine, Sadat University, Egypt
2 Department of Biochemistry, College of Veterinary Medicine, Sadat University, Egypt; Department of Biomedical Science, College of Pharmacy, Shaqra University, Al-Dawadmi, Saudi Arabia
|Date of Submission||03-Oct-2013|
|Date of Decision||21-Oct-2013|
|Date of Acceptance||29-Oct-2013|
|Date of Web Publication||20-Mar-2014|
Department of Biochemistry, College of Veterinary Medicine, Sadat University, Egypt; Department of Biomedical Science, College of Pharmacy, Shaqra University, Al-Dawadmi, Saudi Arabia
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: In most African and Arabic countries tuberculosis (TB) causes great economic losses in bovine species and constitutes serious zoonotic problem. As the traditional diagnostic method delay the research because of low sensitivity and specificity, a rapid method of diagnosis is of outmost importance. Aim: The study was designed to evaluate the two rapid diagnostic methods of TB in cattle, further to differentiate between infected and bacillus Calmette-Guerin (BCG) vaccinated animals. Materials and Methods: Intradermal tuberculin test was applied to 300 cattle. Of these cattle, 15 cattle were vaccinated from cattle negative to tuberculin test with BCG. Blood samples were taken for lymphocyte separation to apply polymerase chain reaction (PCR) upon and for serum preparation for the enzyme-linked immunosorbent assay (ELISA) application, this blood collected from 65 cattle classified into three groups, viz. positive tuberculin test (35 animals), negative tuberculin test (15 animals), and vaccinated cow with BCG (15 animals). From blood samples lymphocytes were separated and the isolated lymphocytes were subjected to PCR and serum for ELISA application. Blood samples, specimens from lymph nodes and specific tissues were taken for PCR and for cultivation and isolation of Mycobacterium bovis. Results and Conclusions: The results of this study revealed that PCR can be used as rapid efficient and accurate diagnostic test in detection of ruminant TB. Moreover, cattle's ELISA reading showed higher sensitivity in positive tuberculin animals. However, the differentiations between vaccinated and infected animals not clear by using a single antigen only.
Keywords: Enzyme-linked immunosorbent assay, polymerase chain reaction, tuberculosis
|How to cite this article:|
Sabry M, Elkerdasy A. A polymerase chain reaction and enzyme linked immunosorbent assay based approach for diagnosis and differentiation between vaccinated and infected cattle with Mycobacterium bovis. J Pharm Bioall Sci 2014;6:115-21
|How to cite this URL:|
Sabry M, Elkerdasy A. A polymerase chain reaction and enzyme linked immunosorbent assay based approach for diagnosis and differentiation between vaccinated and infected cattle with Mycobacterium bovis. J Pharm Bioall Sci [serial online] 2014 [cited 2019 Dec 11];6:115-21. Available from: http://www.jpbsonline.org/text.asp?2014/6/2/115/126584
Tuberculosis (TB) in humans and animals continues to cause major health problems on a global scale. Human TB accounts for 8 million cases of clinical disease and 3 million deaths annually and is predominantly caused by Mycobacterium tuberculosis. Bovine TB is a major cause of economic loss and represents a significant zoonotic infection.  Mycobacterium bovis is the etiological agent of bovine TB and is closely related to M. tuberculosis within the TB complex. The prevalence of tuberculin test was 0.96% in the examined farms in Egypt during the period from November 2010 until December 2011 and M. bovis isolation represented 66.7% of infected cases while the prevalence of tuberculin among human patients was 8.3% and the M. tuberculosis isolation was 83.3% from tuberculin positive human patients  Although higher prevalence in cattle farms 2.46-4.6% was recorded. , M. bovis diagnosed mainly by the usage of old methods as culturing, staining and phenotypic traits which take more times (2 months at least), therefore many researchers directed their work for reduction of this time utilizing more recent, rapid and sensitive diagnostic methods. Several serological tests are being developed and there is also the possibility that they can be adapted to detect mycobacterial antibody in bulk milk tank samples for herd testing.  The low sensitivity of enzyme-linked immunosorbent assay (ELISA) limits its usefulness as a diagnostic tool for bovine tuberculosis (BTB) eradication campaigns  but utilization of combinations of strain-specific mycobacterial antigens like MTB40, ESAT6, CFP10, MBP70, Rv3870 and Rv1580c could resolve both specificity and sensitivity.  Due to dysgonic and very slow growth, the identification of M. bovis by conventional biochemical methods is cumbersome and time-consuming. Direct use of polymerase chain reaction (PCR) on biological samples enables diagnosis to be reached within 48 h.  The PCR amplification of the M. tuberculosis deoxyribonucleic acid (DNA) is a rapid, reliable method for diagnosis.  So far, the only currently available vaccine for M. bovis is bacillus Calmette-Guerin (BCG). BCG is an attenuated strain of M. bovis strain that isolated from a cow with tuberculous mastitis in France prepared by serial passage on glycerinated bile potato medium between 1908 and 1919. The efficacy of BCG was determined by the skin test and the plasma level of interferon gamma (INF-γ). Development of tests able to differentiate between infected and vaccinated animals is under research, alongside efforts to identify vaccine candidates for BT. Differential diagnosis can be achieved through the use of defined proteins that are present in M. bovis but not in BCG, such as ESAT-6 and CFP-10. These antigens have been used to identify M. bovis-infected cattle and differentiate them from those cattle exposed to other mycobacterial species including Mycobacterium avium and BCG. Using these antigens in combination with purified protein derivatives from M. bovis (PPD-B) and M. avium (PPD-A) improve the specificity of diagnosis.  And also using IFN-γ and PCR with Internal Spacer 1081 will be beneficial in this differentiation . There is a great need for the development of sensitive diagnostic test which can distinguish M. bovis infected cattle from vaccinated and those infected with non tuberculous mycobacteria.
The experiment was done in a private farm of Friesian cattle in Menoufia governorate in Egypt after the owner consent using 300 animals (age ranges from 2 to 4 year, females and they appeared apparently healthy) during the routine farm program.
| Materials and Methods|| |
Avian tuberculin PPD-A (avian PPD 25,000 IU/ml was purchased from Assure quality limited, National Center for Biosecurity and Infectious Disease - Walaceville-New Zealand). Bovine tuberculin (Mammalian PPD tuberculin: 2 mg/ml) was obtained from bacterial diagnostic products department, Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo, Egypt. ELISA coating MBP70 (KVKNTIAATSFAAAGLAALAVAVSPPAA) manufactured by (GENOSPHERE BIOTECHNOLOGIES France: www.genosphere-biotech.com--info@.genosphere-biotech.com and with PPD-B was from New Zealand, Rabbit anti-bovine labeled with horseradish peroxidase (HRP) was purchased from Labor Diagnostik Leipzig - Germany. PCR master mix was from Promega Corporation, Madison, WI. QIAGEN spin-column genomic DNA isolation kit was purchased from QIAGEN (QIAGEN Inc., Lane Valencia, CA 91355, USA).
Agarose A was from Bio Basic INC. Konrad Cres, Markham Ontario.
| Experimental Design|| |
The test was carried out according to the method previously described by Lesslie et al.  Ordinary intradermally tuberculin test was applied to 300 cattle. Briefly, Shaving two sites on one side of the neck (12 cm apart) and recording the initial skin thickness, 0.1 ml of avian tuberculin PPD-A (avian PPD 25,000 IU/ml) was injected intradermally into the upper site and an equivalent dose of bovine tuberculin (Mammalian PPD tuberculin: 2 mg/ml) was injected into the lower site of the neck. 72 h post injection the skin - fold thickness at each site was measured again and recorded. The results were interpreted based on standard interpretation (i.e. after 72 h). Bovine - positive reactors and avian positive reactors were obtained using the following formulae: ([Bov72-Bov0]-[Av72-Av0]) and ([Av72-Av0]-[Bov72-Bov0]) respectively, Bov0 and Av0 indicated skin-fold thickness before injection of tuberculin, Bov72 and Av72 indicated skin-fold after injection of tuberculin with 72 h. Subsequently, the negative cows to the tuberculin test (15 animals) were vaccinated with BCG. Application of tuberculin on vaccinated animals was performed after 4 months from BCG vaccination to 15 animals under vaccination study according to previous work. , The differences in thickness between bovine and avian antigens were interpreted as follows
- Skin folds Thickness < 3 mm is negative.
- Skin fold Thickness =3-4 mm is doubtful.
- Skin fold Thickness ≥ 5 mm is positive.
How about the thickness of 4 mm?
After tuberculin application, animals were divided into 3 groups
First group: It contains 35 animals positive to tuberculin, slaughtered and examined for the presence of a lesion.
Second group: It contains 15 animals negative to tuberculin and considered as control.
Third group: It contains 15 animals negative to tuberculin, were vaccinated by BCG.
Blood samples were collected from each of the 65 cattle from the three groups (35 cows positive to tuberculin test, 15 cows negative tuberculin test; control and 15 cows vaccinated with BCG). About 10 ml of blood obtained from the jugular vein puncture in sterile McCartney tube was collected from each cow to prepare serum. Other samples of 37.5 ml blood collected from each animal and mixed with 12.5 ml acid citrate dextrose as anti-coagulant for separation of lymphocytes.  Animal's positive reactors for tuberculin were slaughtered (according to Egyptian law) and subjected to post-mortem (PM). The following lymph nodes were collected (i) mandibular, (ii) cranial and caudal mediastinal, (iii) left and right bronchial, (v) hepatic and (vi) mesenteric lymph nodes. Furthermore organ tissues samples were collected from seven lobes of the two lungs, liver, spleen and intestine. These organs were sliced finely and examined grossly for lesions. When gross lesions suggestive of BT were found in any of the examined tissues, the animal was classified as visible lesion (VL). Animals in which lesions were not found were classified as non-visible lesion. These samples were used for both DNA extraction to PCR analysis and also for cultivation and isolation of M. bovis.
The test was carried out according to the method described by Amadori et al. The coating MBP70 (KVKNTIAATSFAAAGLAALAVAVSPPAA) with PPD-B. Flat-bottom maxy-Sorb Ni-NTA His Sorb micro titer plates with 100 μl of purified antigens (0.50 μg/well) in carbonate bicarbonate buffer 0.1 M, pH 9.6 added in all wells except 4 wells (2 wells as a positive control and 2 wells as a negative control), the plates were then kept overnight at 4°C. The plates were washed three times with phosphate buffered saline (PBS) containing 0.05% Tween 20 and over coated with 0.5% skimmed milk for 1 h at 37°C, to each well 100 μl of sera diluted to 1/16 in dilution buffer (PBS/tween 20/1% BSA) was added. The plates were then incubated at room temperature for 1 h at 37°C. Each plate contained 2 wells of negative sera run in the same dilutions to calculate the cut-off value. The Plates were then decanted, washed 3 times with washing buffer. To each well, 100 μl of rabbit anti-bovine labeled with HRP was added and plates then incubated for 30 min at room temperature. The plates were washed 3 times with washing buffer. 100 μl of freshly prepared 3, 3′, 5, 5′-tetra methylbenzidine substrate buffer was added to each well and the plates were then incubated in dark for 1 h at room temperature. To each well, 100 μl of stop solution was added. The optical density was measured at 450 nm using ELX800ELISA reader. A serum dilution was considered positive if it yielded a mean OD of each group equal to/or greater than the cut-off value. 
Tissue samples from lymph nodes showing VL and those showing no VLs were processed for isolation of mycobacteria  and cultured on Lowenstein Jensen media with or without pyruvate  All cultures were evaluated for colony growth on a weekly basis up to 10 weeks and these colonies were confirmed to be positive by Ziehl Neelsen staining to detect acid fast mycobacteria.
DNA extraction from tissues, bacterial culture and collected lymphocytes
DNA was extracted from the tissue and culture was performed using QIAamp DNA Mini Kit (Quiagen) and Quiagen spin column genomic DNA isolation kit according to the manufacturer instructions.
PCR was directly applied upon extracted DNA from lymph nodes, organ tissues, lymphocytes (obtained from VL and NVL tuberculin positive cattle) and isolated cultures. It was also applied on lymphocytes from vaccinated cattle. PCR was conducted in a final volume of 25 μl consisting of 1 μl DNA, 1 μl of 10 picomolar of each primer (BW-6, 5'- CGA CAC CGA GCA GCT TCT GGC TG-3'and BW-7, 5'- GTC GGCACC ACG CTG GCT AGT G-3'- ) and 12.5 μl PCR master mix. The final volume was brought to 25 μl using sterilized, nuclease-free de-ionized water. PCR was carried out using a PeX 0.5 thermal Cycler with the cycle sequence of denaturing at 94°C for 5 min for one cycle, followed by 35 cycles each of which consisted of denaturing at 94°C for 1 min, annealing at 68°C and extension at 72°C for 1 min with additional cycle as a final extension at 72°C for 5 min. PCR products (306-bp region of the multicopy insertion sequence IS-1081  were electrophoresed on 1% agarose A gel in TE (Tris-ethylenediaminetetraacetic acid) buffer at 100 V for 30 min with ethidium bromide staining. PCR products were visualized under ultra violet light and photographed.
PCR primer and program
a. Primers (BW-6 [5' CGA CAC CGA GCA GCT TCT GGC TG 3'] and BW-7 [5'GTC GGC ACC ACG CTG GCT AGT G 3']) aimed at the 306-bp region of the multicopy insertion sequence IS-1081  were used to amplify mycobacterial DNA in tissue, culture and blood leukocyte samples.
b. Amplification parameters included an initial denaturation at 94°C for 5 min followed by 35 cycles each of denaturation at 94°C for 1 min, annealing at 68°C for 1.5 min, and extension at72°C for 2 min. The extension step in the 35 th cycle was held for 10 min before the samples were shifted to 4°C for storage.
| Results|| |
At the beginning of this study, tuberculin screening test was conducted on all (three hundred) animals under the study. Thus intradremal inoculation of avian tuberculin and bovine tuberculin in the neck was performed. After 72 h post injection the skin fold thickness was measured. The results showed that 35 of the 300 tested animals (11.7%) were positive to tuberculin test while 256 animals (88.3%) of these tested animals were not reactive to tuberculin test [Table 1].
|Table 1: Comparison between tuberculin positive and negative animals using (PM examination, culture and AFB (acid fast bacilli staining) and application of PCR using IS 1081 on tissue and obtained culture|
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PM examination and culturing
Examination of the tuberculin positive animals confirmed the development of macroscopic lesions of TB in many tissues and organs such as different lymph nodes, liver, lung, spleen, intestine as indicated in materials and methods section. Furthermore the presence of acid fast bacilli was confirmed in culture isolated from all cattle showing gross lesion (100%) and detected by Ziehl Neelsen stain of the acid fast tubercle bacilli as indicated by staining with Ziehl Neelsen stain from this lesion. This result is indicated in [Table 1] which shows also that the presence of M. bovis in all tuberculin positive animals was confirmed by PCR. The BCG vaccination was applied on 15 cattle from the tuberculin negative cases animals. Four month later, tuberculin test was performed to these vaccinated animals. The results showed that all vaccinated animals (100%) were tuberculin positive with mean skin thickness of 11 mm although it was less than the infected cases whose mean skin thickness was 16 mm [Table 2].
|Table 2: Comparison between tuberculin positive, vaccinated and tuberculin negative cattle using skin thickness, ELISA (dependent on MBP70 antigen) and application of PCR using IS 1081 on lymphocytes|
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PCR analysis and ELISA assay
To test the validity of PCR analysis and ELISA assay in diagnosis of TB and in differentiation between TB-infected and BCG-vaccinated cattle, PCR was conducted on tissue samples from the lesions, lymph nodes from all tuberculin positive animals and isolated culture. Also PCR was conducted on lymohcytes of tuberculin positive, tuberculin negative, and vaccinated cows. As shown in [Table 2], PCR analysis using IS1081 could detect M. bovis in lymphocyte samples from 13 of 35 (37.14%) tuberculin positive animals while neither in vaccinated nor tuberculin negative animals, M. bovis could be detected. This is also clear in [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5] and [Figure 6] which show a band of the expected size (300 pb) is detected in tissue samples and isolated cultures from tuberculin positive animals. These results indicate that PCR could be used as a diagnostic tool to differentiate between BCG-vaccinated and TB-infected cattle. In respect to ELISA, using MPB70 antigen for application of ELISA, could not discriminate between TB infected and BCG-vaccinated cattle where all tuberculin positive (35/35, 100%), vaccinated (15/15, 100%) were positive to ELISA.
|Figure 1: Lanes 1-11 are positive, but lane 12 −ve control and lane 13 +ve control|
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|Figure 2: Lanes 1-3, 6 and 8-11 are positive, but lane 12 −ve control and lane 13 +ve control|
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|Figure 3: Lanes 2-5, 8-10 are positive, but lane 12 −ve control and lane 13 +ve control|
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|Figure 4: Lanes 2-10 are positive, but lane 12 −ve control and lane 13 +ve control|
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|Figure 5: Lanes from 5 to 10 are positive, but lane 12 −ve control and lane 13 +ve control|
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|Figure 6: Lanes 2, 3 are positive, also lane 6-10 are positive, but lane 12 −ve control and lane 13 +ve control|
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Moreover, 20% (3/15) of tuberculin negative cows were positive to ELISA [Table 2]. This result indicates that using ELISA based on single antigen is less specific and sensitive in diagnosis and differentiate between BCG-vaccinated and TB-infected cattle.
As shown in [Table 1] it was clear after comparing tuberculin positive and negative animals using PM examination, culture and AFB (acid fast bacilli staining) and application of PCR using IS-1081 on tissue and obtained culture, the percentage of tuberculin positive cattle was 35/300 (11.7%). This percentage is nearly similar to that obtained in the previous work  in which it was found that 10% of herds were positive for bovine TB in intensive rearing herds and this result also showed agreement with the similar work where it was reported that the prevalence of BTB in Nili Ravi buffaloes varied from 5.48% to 12.72%.  The differences in the percentage of infection may be due to the hygienic condition of breeding and/or the locality and the period of flock testing. All the tuberculin positive cattle were slaughtered and were examined in the slaughter house. All of them were expressing VLs 100% (35/35), and these obtained results were in agreement with the previous work  in which infected 5 calves (Friesian cross castrated male calves of 6 months of age were obtained from bovine TB-free herds) with M. bovis, all expressed gross pathology 100%. Considering mycobacterial isolation it was noted that all VL expressing cattle was positive to culture 100% (35/35) and this result coincides with the result obtained in literature , where researchers isolated M. bovis from all VL expressing animals.  The obtained isolates mainly were tested by staining with Ziehl Neelsen stain and acid fastness was confirmed. Using PCR analysis of tissue samples from slaughtered animals the presence of M. bovis was confirmed with 100% sensitivity and specificity and that mainly agrees with the similar result elucidated by a group of researchers.  Hence the results indicated that the PCR method used in the present work is quicker, reproductive, and reliable for the study of slow-growing mycobacteria, particularly in cultures where the small number of bacilli hinders identification by classical methods.
The BCG vaccine is an attenuated strain of M. bovis, has been widely used for control of human TB despite controversy over its protective efficacy.  In cattle, BCG has been used in a series of trials; with various degrees of protection against M. bovis.  Moreover, BCG is utilized due to its immunostimulant effect. However, major constraint in the use of attenuated mycobacterial vaccines such as BCG is that vaccination of humans or cattle interferes with detection of TB by means of the tuberculin skin test and that is clear from [Table 2]. After comparing infected, vaccinated and tuberculin negative cattle using tuberculin test, we found that all vaccinated animals 100% (15/15) were tuberculin positive with mean skin thickness 11 mm although lower than the infected cases where mean skin thickness was found to be 16 mm. This result is almost similar to the published result by a group of researchers.  They tried to prove that after vaccination with BCG, vaccinated cattle gave positive tuberculin test with increased skin thickness and also showed that these vaccinated cattle produced moderate IFN-γ responses to PPD-B but very weak responses to the recombinant antigens. Also comparing these cattle under study by application of ELISA using MPB70 antigen, similar results for infected 100% (35/35) and vaccinated 100% (15/15) were obtained and this result mainly confirmed by a published result  where it was stated that MBP70 was a unique product of M. bovis and it was also found in M. tuberculosis and BCG but was expressed at a low level in some BCG strains. The presence of some tuberculin negative cattle with percentage of 20% (3/15) mainly confirmed by another published result.  Hence cross-reactive responses elicited by exposure to non tuberculous mycobacteria often confound the interpretation of ante mortem tests for M. bovis infection of cattle, so the use of recombinant or native M. bovis proteins such as MPB59, MPB64, MPB70 and MPB83 must enhance test specificity.  These proteins however, are difficult to synthesize and/or purify might lack sensitivity when used individually. , Therefore cocktails of antigens will be required to achieve broad population coverage. After application of PCR on the separated lymphocytes from infected cattle there was found that 37.14% (13/35) were positive with IS 1081 sequence primer and that nearly similar to results obtained from another published work.  Furthermore nearly similar published result could be found where a group of scientists utilized blood as extrapulmonary sample for testing it with PCR to detect mycobacterial DNA.  The other finding also indicated that 36.3% of human cases were positive to mycobacterial infection. Nearly similar to our data, another group of researchers reported 39% positive cases for mycobacterial infection using PCR upon human blood samples.  Although not all tested cases gave positive PCR results and that may be because of difficulty in recording signs of dissemination and bacteremia in tested cattle, when compared with human cases as these signs can be recorded, the developed PCR appears to have a potential to discover mycobacterial DNA in blood of tuberculin positive. It is field applicable, cost effective and has a turnaround time of 8 h for individual blood testing. Considering the BCG vaccinated and tuberculin negative cattle they gave negative PCR results after testing the lymphocytes separated from them by PCR. Hence the application of PCR with specific primers on blood or on lymphocytes could be used in discriminating infected animals from vaccinated and tuberculin negative cattle.
Hence the development of tests which can distinguish between infected with M. tuberculosis or M. bovis; atypical mycobacterial infection and BCG vaccinated cattle could greatly assist in the diagnosis of early infection as well as enhance the use of TB vaccines on a wider scale.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]