Detection of Mycobacterium avium subsp. paratuberculosis in bovine milk from the state of Pernambuco, Brazil

Abstract

The aim of this study was to detect the IS900 region of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine milk samples using real-time polymerase chain reaction (qPCR) and conventional PCR, and to study the agreement between these tests. A total of 121 bovine milk samples were collected from herds considered positive for MAP, from the State of Pernambuco, Brazil. MAP DNA was detected in 20 samples (16.5%) using conventional PCR and in 34 samples (28.1%) using qPCR. MAP DNA was detected in all of the 6 animal farms studied. Moderate agreement was found between qPCR and conventional PCR results, where the sensitivity and specificity of conventional PCR in relation to qPCR were 50% and 96.6%, respectively. Thus, the IS900 region of MAP was found in bovine milk samples from the State of Pernambuco. To the best of our knowledge, this is the first report of MAP DNA found in bovine milk in Northeast Brazil. We also demonstrated the qPCR technique is more sensitive than conventional PCR with respect to detection of MAP in milk samples.

Keywords:
Molecular biology; Diagnosis; Paratuberculosis; Milk

Introduction

Mycobacterium avium subsp. paratuberculosis (MAP) is a gram-positive, slow-growing bacillus of the family Mycobacteriaceae that possesses a cell wall rich in lipids, characteristic of this family. This microorganism is an intracellular pathogen, responsible for Johne's disease or paratuberculosis.11 Harris NB, Barletta RG. Mycobacterium avium subsp. paratuberculosis in veterinary medicine. Clin Microbiol Rev. 2001;14(3):489-512.,22 Manning EJB, Collins MT. Mycobacterium avium subsp. paratuberculosis: pathogen, pathogenesis and diagnosis. Rev Off Int Epizoot. 2001;20(1):133-150.

Paratuberculosis has been previously investigated in several studies in Brazil.33 Yamasaki EM, Tokarnia CH, Galvão A, et al. Aspectos clínicos patológicos e controle da paratuberculose em rebanho bovino leiteiro. Pesqui Vet Bras. 2010;30(11):921-932. Its occurrence in Pernambuco has been described in dairy cattle based on clinical signs, histopathology, and results from enzyme-linked immune sorbent assay (ELISA) serology (32.3% positive samples), isolation (50% positive samples), and polymerase chain reaction (PCR) studies.44 Mota RA, Pinheiro-Junior JW, Gomes MJP, et al. Paratuberculose em um rebanho leiteiro no Estado de Pernambuco, PE. Arq Inst Biol (São Paulo). 2007;74(2):73-79. Additionally, a prevalence rate of 2.7% (11/408) has been reported in the micro-region of Garanhuns, with 47.4% (9/19) outbreaks.55 Sá LM, Oliveira JMB, Santos GR, et al. Avaliação sorológica e de fatores de risco para a infecção por Mycobacterium avium subsp. paratuberculosis em rebanhos leiteiros da Microrregião de Garanhuns, Pernambuco. Pesqui Vet Bras. 2013;33(3):310-314.

The most common methods of MAP diagnosis in infected animals include isolation of bacteria from feces using selective culture media and antibody detection techniques such as ELISA. However, the greatest disadvantage of using culture media is the long incubation period, which can be as long as 16 weeks for a definitive diagnosis. ELISA can be performed within a few hours, although its sensitivity is estimated at only 45%, since antibodies to MAP may not be detectable in the initial stages of the infection.66 Collins MT. Diagnosis of paratuberculosis. Vet Clin North Am Food Anim Pract. 1996;12(2):357-371.,77 O’Mahony J, Hill C. Rapid real-time PCR assay for detection and quantitation of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Appl Environ Microbiol. 2004;70(8):4561-4568.

Molecular techniques to detect MAP, such as PCR, are rapid and qualitative in nature. Real-time PCR (qPCR) exhibits greater sensitivity than conventional PCR, and can determine the infective load in environmental samples, feces, milk, and cultures.77 O’Mahony J, Hill C. Rapid real-time PCR assay for detection and quantitation of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Appl Environ Microbiol. 2004;70(8):4561-4568.,88 Kawaji S, Taylor DL, Mori Y, Whittington RJ. Detection of Mycobacterium avium subsp. paratuberculosis in ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture. Vet Microbiol. 2007;125(1-2):36-48. One of the target genes used to detect MAP via PCR is the IS900 region, first described by Green et al.99 Green EP, Tizard MLV, Moss MT, et al. Sequence and characteristics of IS900, an insertion element identified in a human Crohn's disease isolate of Mycobacterium paratuberculosis. Nucleic Acids Res. 1989;17(22):9063-9073. and independently identified by Collins et al.1010 Collins DM, Gabric DM, De Lisle GW. Identification of a repetitive DNA sequence specific to Mycobacterium paratuberculosis. FEMS Microbiol Lett. 1989;51(1):175-178. The discovery of the IS900 region of MAP enables the diagnosis of paratuberculosis even in the initial stages of infection. The specificity and sensitivity of PCR have been enhanced up to the point of detecting 1 CFU of MAP in samples.1111 Vansnick E, Rijk P, Vercammen F, Geysen D, Rigouts L, Portaels F. Newly developed primers for the detection of Mycobacterium avium subspecies paratuberculosis. Vet Microbiol. 2004;100(3-4):197-204.

MAP has been detected in several animal products and byproducts. A study conducted in Switzerland detected the presence of the IS900 region in 19.7% (273/1384) milk samples collected from milk storage tanks.1212 Corti S, Stephan R. Detection of Mycobacterium avium subsp. paratuberculosis specific IS900 insertion sequences in bulk-tank milk samples obtained from different regions throughout Switzerland. BMC Microbiol. 2002;2:15. A study in Cyprus reported 63 (28.6%) positive out of a total of 220 milk samples from tanks, using real-time PCR for IS900 and F57.1313 Slana I, Liapi M, Moravkovaa M, Kralovaa A, Pavlika I. Mycobacterium avium subsp. paratuberculosis in cow bulk tank milk in Cyprus detected by culture and quantitative IS900 and F57 real-time PCR. Prev Vet Med. 2009;89(3-4):223-226.

The aim of this study was to detect the IS900 region of MAP in bovine milk samples from the State of Pernambuco (Brazil) using PCR and qPCR, and to investigate the agreement between these diagnostic tests.

Materials and methods

Sampling

In total, 121 bovine milk samples from the State of Pernambuco were collected from 6 dairy herds that already had a history of paratuberculosis. The animals were clinically healthy at the time of collection.

Sample collection and processing

Sample collection

The cow teats were cleaned with water and disinfected with 70% alcohol prior to collection of milk samples. The first 3 jets of milk were discarded. Subsequently, approximately 50 mL milk was pooled from the 4 mammary glands using sterilized and separate polypropylene tubes. The samples were stored in cool boxes containing recyclable ice and sent to the Laboratory of Bacteria in the Federal Rural University of Pernambuco for processing.

DNA extraction

DNA extraction was performed using 2 mL of each sample, which was centrifuged at 12,000 × g for 10 min, and the pellet was resuspended in 100 µL buffered saline solution with sterile phosphate (pH 7.2). A commercial kita a DNA Easy Blood and Tissues Kit®, Qiagen Biotechnology Brazil Ltda., São Paulo, Brazil. was used for extraction, following the manufacturer's instructions.

Polymerase chain reaction (PCR)

The extracted DNA was amplified in a final volume of 15 µL, containing the following: 5 µL genomic DNA; 0.5 µL each, of primers specific for IS900 at 20 pM (DF: 5′-GACGACTCGACCGCTAATTG-3′ and DR-1: 5′-CCGTAACCGTCATTGTCCAG-3′); 2.75 µL ultrapure Milli-Q water and 6.25 µL PCR kit mixture,b b MasterMix, Promega® Corp., Madison, WI. as per the manufacturer instructions. The DNA was amplified in a thermocyclerc c Bioer XP cycler®, Bioer Technology, Hangzhou, China. using the following conditions: initial denaturation at 96 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 1 min, annealing at 58 °C for 1 min and extension at 72 °C for 3 min, with a final extension at 72 °C for 10 min.1414 Taddei R, Barbieri I, Pacciarini ML. Mycobacterium porcinum strains isolated from bovine bulk milk: implications for Mycobacterium avium subsp. paratuberculosis detection by PCR and culture. Vet Microbiol. 2008;130(3-4):338-347. The amplified product of 99 bp, corresponding to MAP, was detected by electrophoresis in agarose gel (2%), followed by staining with blue green,d d Blue Green Loading Dye (LGC Biotecnologia, São Paulo, Brazil). and visualization under ultraviolet light. Images of the bands were captured.

Polymerase chain reaction in real time

The extracted DNA was amplified in a final volume of 25.0 µL containing: 5 µL genomic DNA; 1 µL each, of primers specific for IS900 at 10 pM (DF: 5′-GACGACTCGACCGCTAATTG-3′ and the DR-1: 5′-CCGTAACCGTCATTGTCCAG-3′), 5.5 µL ultrapure Milli-Q water, and 12.5 µL real-time PCR kit mixture,e e QuantiFast SYBR Green PCR Kit®, Qiagen Biotechnology Brazil Ltda., São Paulo, Brazil. according to the manufacturer instructions. The DNA was amplified in a thermocyclerf f Rotor-Gene Q thermocycler, Qiagen Biotechnology Brazil Ltda., São Paulo, Brazil. using the following conditions: initial denaturation at 95 °C for 5 min, followed by 40 cycles at 95 °C for 20 s and 60 °C for 30 s. The thermocycler software was used to monitor and interpret the results.

DNA from a MAP strain provided by the National Agricultural Laboratory in Minas Gerais identified as “Nakajima 1991” was used to standardize the qPCR. The melting curve consisted of 65 °C for 90 s for preparation, with a posterior gradual increase of 1 °C every 5 s from 60 °C to 95 °C. The peak of denaturation was at 82.9 °C. The number of copies was determined as previously described by Rodrígues-Lázaro et al.1515 Rodríguez-Lázaro D, D’Agostino M, Herrewegh A, Pla M, Cook N, Ikonomopoulos J. Real-time PCR-based methods for detection of Mycobacterium. Int J Food Microbiol. 2005;101(1):93-104. The copy number detected was divided by 15, which corresponds to the average number of IS900 elements in the MAP genome.1616 Kralik P, Slana I, Kralova A, Babak V, Whitlock RH, Pavlik I. Development of a predictive model for detection of Mycobacterium avium subsp. paratuberculosis in faeces by quantitative real time PCR. Vet Microbiol. 2011;149(1-2):133-138. The DNA of the MAP strain was 10-fold serially diluted and used to obtain the standard curve ranging from 2.27 × 107 to 22.6 MAP cells. The efficiency of the primers was 95.0% and the coefficient of linear correlation (R 2) was 0.99909, while the detection threshold (Ct) was between 9.58 and 30.08. The reactions were performed in duplicate. Finally, the number of copies in the positive field samples was quantified using the standard curve.

Sequencing of the samples and validation of conventional PCR

In order to validate the amplifications, one of the positives samples from the conventional PCR was purified using a commercial extraction kit,g g QIAquick Gel Extraction kit®, Qiagen Biotechnology Brazil Ltda., São Paulo, Brazil. as per the manufacturer instructions, before being sent for sequencing. Sequences with a Phred quality score above 20 were analyzed using the Basic Local Align Sequence Tool (BLAST; www.blast.ncbi.nlm.nih.gov/Blast.cgi).

Statistical analysis

The Kappa coefficient (K) was used to assess the agreement between tests. The qPCR was considered as a gold standard and the conventional interpretation of the K values adopted was as follows: 0.00-0.20 = weak; 0.21-0.40 = regular; 0.41-0.60 = moderate; 0.61-0.80 = good; and 0.81-1.00 = very good agreement; negative values were interpreted as 0.00.1717 Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics. 1977;33(1):159-174. Biostat softwareh h BioEstat version 5.0, Universidade Federal doPará, Pará, Brazil. was used to calculate the agreement, sensitivity, and specificity. The chi-squared test was used to determine associations between the tests, with a confidence interval of 95%.

Results

Of the 121 milk samples analyzed, the IS900 region of MAP was detected in 20 (16.5%) samples using conventional PCR and in 35 (28.1%) samples using qPCR. Milk samples contaminated with MAP were found in all six farms studied, ranging from 10.0 to 23.8% using conventional PCR and from 10.0 to 36.8% using qPCR (Table 1).

Table 1
Frequency of positive milk samples by animal farm using conventional PCR and qPCR with the mean value of MAP cells/mL per animal farm determined by qPCR.

The number of DNA copies, determined by qPCR in all positive samples exhibited a mean value of 160.59 MAP cells/mL, with minimum and maximum values of 12.2 and 1126.66 MAP cells/mL, respectively. The mean value of the quantifications per animal farm for the positive samples ranged from 35.97 to 368.15 MAP cells/mL (Table 1).

The agreement between conventional PCR and qPCR, analyzed by the Kappa coefficient, was 0.53, which is moderate (χ2 = 38.08; p < 0.000). The values for sensitivity and specificity of the conventional PCR were 50% and 96.6%, respectively.

The sequencing of the field sample exhibited a similarity of 98% with the DNA of M. avium subsp. paratuberculosis (MAP4, complete genome) deposited at the National Center for Biotechnology Information.

Discussion

This is the first record of the detection of MAP in bovine milk in Northeast Brazil. The presence of MAP in milk has been previously reported in Brazil by Carvalho et al.,1818 Carvalho IA, Silva A, Campos EB, Moreira MAS. Short communication: detection of Mycobacterium avium subspecies paratuberculosis by polymerase chain reaction in bovine milk in Brazil. J Dairy Sci. 2009;92(11):5408-5410. who found the IS900 region of DNA in 8/222 (3.6%) samples of raw bovine milk from Viçosa, Minas Gerais.

MAP has been detected previously in bovine milk in other countries. In Switzerland, DNA of IS900 was found in 273 (19.7%) of 1384 samples from milk tanks.1212 Corti S, Stephan R. Detection of Mycobacterium avium subsp. paratuberculosis specific IS900 insertion sequences in bulk-tank milk samples obtained from different regions throughout Switzerland. BMC Microbiol. 2002;2:15. Slana et al.1313 Slana I, Liapi M, Moravkovaa M, Kralovaa A, Pavlika I. Mycobacterium avium subsp. paratuberculosis in cow bulk tank milk in Cyprus detected by culture and quantitative IS900 and F57 real-time PCR. Prev Vet Med. 2009;89(3-4):223-226. collected a total of 220 milk samples from tanks in Cyprus and found 63 (28.6%) positive samples (IS900 and F57) using real-time PCR.

Paratuberculosis is considered uncommon in Brazil; however, outbreaks of the disease have been reported in 11 states of the Federation.33 Yamasaki EM, Tokarnia CH, Galvão A, et al. Aspectos clínicos patológicos e controle da paratuberculose em rebanho bovino leiteiro. Pesqui Vet Bras. 2010;30(11):921-932.,1919 Ristow P, Marassi CD, Rodrigues ABF, et al. Diagnosis of paratuberculosis in a dairy herd native to Brazil. Vet J. 2007;174(2):432-434. In Pernambuco, Mota et al.44 Mota RA, Pinheiro-Junior JW, Gomes MJP, et al. Paratuberculose em um rebanho leiteiro no Estado de Pernambuco, PE. Arq Inst Biol (São Paulo). 2007;74(2):73-79. reported the occurrence of paratuberculosis in a bovine dairy herd by isolating MAP from feces samples, with genetic confirmation of the IS900 region using PCR.

The high frequency of positive milk samples found in this study could be explained by the fact that the milk samples were collected from herds considered positive for paratuberculosis. It is important to note that the actual prevalence rate could be even higher, since not all infected animals eliminate the bacterium via milk.1212 Corti S, Stephan R. Detection of Mycobacterium avium subsp. paratuberculosis specific IS900 insertion sequences in bulk-tank milk samples obtained from different regions throughout Switzerland. BMC Microbiol. 2002;2:15.

Based on the health risks due to MAP and considering that this microorganism is implicated in Crohn's disease in humans, rapid and sensitive tests for detection of this bacterium in milk are urgently required.77 O’Mahony J, Hill C. Rapid real-time PCR assay for detection and quantitation of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Appl Environ Microbiol. 2004;70(8):4561-4568. In this study, the number of milk samples found positive via qPCR (34 samples) was higher than that via conventional PCR (20 samples). The qPCR technique is more efficient in detecting pathogenic bacteria and has several advantages over conventional PCR, including the potential to quantify the bacteria, the minimal risk of cross-contamination, as well as greater sensitivity and specificity that can be enhanced by the use of probes.2020 Mackay IM. Real-time PCR in the microbiology laboratory. Clin Microbiol Infect. 2004;10(3):190-212.

Sonawane and Tripathi2121 Sonawane GG, Tripathi BN. Comparison of a quantitative real-time polymerase chain reaction (qPCR) with conventional PCR, bacterial culture and ELISA for detection of Mycobacterium avium subsp. paratuberculosis infection in sheep showing pathology of Johne's disease. Springer Plus. 2013;2(1):45. reported that 2 groups of tissues (multibacillary and paucibacillary) were used to perform conventional PCR to detect the IS900 region of MAP. They recorded sensitivity levels of 82.6% and 13.3% for the multibacillary and paucibacillary groups, respectively. In the same study, qPCR recorded sensitivity levels of 100% in the detection of MAP for both groups.

Upon analysis of the agreement between nested PCR and qPCR in terms of detecting MAP, Fang et al.2222 Fang Y, Wu WH, Pepper JL, et al. Comparison of real-time, quantitative PCR with molecular beacons to nested PCR and culture methods for detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples. J Clin Microbiol. 2002;40(1):287-291. obtained a Kappa index of 0.85. This value, which is higher than that found in the present study, could be explained by the fact that nested PCR is more sensitive than conventional PCR, which was used in the present study for comparison with qPCR.2323 Moghadam MT, Sarv S, Moosakhani F, Badiie A. Detection of Mycobacterium avium subspecies paratuberculosis in milk and fecal samples in dairy cattle by PCR and nested-PCR. J Anim Vet Adv. 2010;9(24):3055-3061.

In the present study, 3 samples tested positive using conventional PCR and not by qPCR. The reason for this is unclear; however, a non-homogenous distribution and/or small quantity of MAP bacteria in the sample could be responsible for the result.2222 Fang Y, Wu WH, Pepper JL, et al. Comparison of real-time, quantitative PCR with molecular beacons to nested PCR and culture methods for detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples. J Clin Microbiol. 2002;40(1):287-291. Another possible reason is the presence of some inhibitors, such as calcium, in the extracts, which even in small quantities, could inhibit Taq and/or compete with magnesium, thus reducing the efficiency of qPCR.2424 Opel KL, Chung D, McCord BR. A study of PCR inhibition mechanisms using real time PCR. J Forensic Sci. 2010;55(1):25-33. This, however, does not refute the finding that the qPCR method reliably detected the presence of MAP DNA in a greater proportion when compared with conventional PCR.

Direct detection of viable MAP using cultures is considered the gold standard. However, MAP can be detected more quickly and with approximately 10 times higher sensitivity using qPCR.1313 Slana I, Liapi M, Moravkovaa M, Kralovaa A, Pavlika I. Mycobacterium avium subsp. paratuberculosis in cow bulk tank milk in Cyprus detected by culture and quantitative IS900 and F57 real-time PCR. Prev Vet Med. 2009;89(3-4):223-226.,2525 Slana I, Kralik P, Kralova A, Pavlik I. On-farm spread of Mycobacterium avium subsp. paratuberculosis in raw milk studied by IS900 and F57 competitive real time quantitative PCR and culture examination. Int J Food Microbiol. 2008;128(2):250-0257. This discrepancy between the methods could be explained by the low concentration of MAP in the sample, since qPCR is a more sensitive detection technique than culturing, from both, feces and milk.2626 Pinedo PJ, Rae DO, Williams JE, Donovan GA, Melendez P, Buergelt CD. Association among results of serum ELISA, faecal culture and nested PCR on milk, blood and faeces for the detection of paratuberculosis in dairy cows. Transbound Emerg Dis. 2008;55(2):125-133.

Considering that bovines with sub-clinical infections can eliminate very low quantities of MAP via milk,2727 Sweeney RW, Whitlock RH, Rosenberger AE. Mycobacterium paratuberculosis cultured from milk and supramammary lymph nodes of infected asymptomatic cows. J Clin Microbiol. 1992;30(1):166-171. it is essential to use adequate and sensitive diagnostic tests to detect this bacterium. The sensitivity of qPCR, in terms of detecting MAP in milk samples, has been reported to range from 1 to 100 CFU/mL milk.2828 Slana I, Paolicchi F, Janstova B, Navratilova P, Pavlik I. Detection methods for Mycobacterium avium subsp. paratuberculosis in milk and milk products: a review. Vet Med (Praha). 2008;53(6):283-306. Additionally, determination of MAP bacterial load in milk samples is also possible with qPCR. Conventional PCR as well as culture methods are not quantitative and culture methods also cannot assess this factor due to the decontamination process of the samples, which decreases the quantity of MAP in the sample.77 O’Mahony J, Hill C. Rapid real-time PCR assay for detection and quantitation of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Appl Environ Microbiol. 2004;70(8):4561-4568.

Raw bovine milk has received considerable attention, as it is known to be an important factor in the transmission of MAP from cow to calf. A study has shown that milk and the colostrum may be risk factors for transmission of MAP infection.2929 Nielsen SS, Bjerre H, Toft N. Colostrum and milk as risk factors for infection with Mycobacterium avium subspecie paratuberculosis in dairy cattle. J Dairy Sci. 2008;91(12):4610-4615.

MAP still remains to be implicated in Crohn's disease, and if humans can be infected by MAP, there are several forms of transmission, including transmission between humans. However, since animals are the main source of these bacteria, transmission from animals to humans is more likely,3030 Collins MT. Mycobacterium paratuberculosis: a potential food-borne pathogen?. J Dairy Sci. 1997;80(12):3445-3448. which can occur via food. Viable MAP have been recovered from pasteurized milk samples in Brazil3131 Carvalho IA, Pietralonga PAG, Schwarz DGG. Short communication: recovery of viable Mycobacterium avium subspecies paratuberculosis from retail pasteurized whole milk in Brazil. J Dairy Sci. 2012;95(12):6946-6948. and other countries as well.3232 Ellingson JLE, Anderson JL, Koziczkowski JJ, et al. Detection of viable Mycobacterium avium subsp. paratuberculosis in retail pasteurized whole milk by two culture methods and PCR. J Food Prot. 2005;68(5):966-972. The presence of MAP in milk products such as cheese has been confirmed in Greece and the Czech Republic.3333 Ikonomopoulos J, Pavlik I, Bartos M, et al. Detection of Mycobacterium avium subsp. paratuberculosis in retail cheeses from Greece and the Czech Republic. Appl Environ Microbiol. 2005;71(12):8934-8936.

Conclusion

In this study, the IS900 region of MAP was detected in bovine milk samples obtained from the State of Pernambuco, and to our knowledge, this is the first such report of MAP DNA in bovine milk in Northeast Brazil. We also demonstrated that the qPCR technique is more sensitive than conventional PCR in terms of detecting MAP in milk samples.

Ethical approval

The Ethics Committee on Animal Use of the Federal Rural University of Pernambuco approved the ethical procedures of this study under license No. 034/2013.

  • a
    DNA Easy Blood and Tissues Kit®, Qiagen Biotechnology Brazil Ltda., São Paulo, Brazil.
  • b
    MasterMix, Promega® Corp., Madison, WI.
  • c
    Bioer XP cycler®, Bioer Technology, Hangzhou, China.
  • d
    Blue Green Loading Dye (LGC Biotecnologia, São Paulo, Brazil).
  • e
    QuantiFast SYBR Green PCR Kit®, Qiagen Biotechnology Brazil Ltda., São Paulo, Brazil.
  • f
    Rotor-Gene Q thermocycler, Qiagen Biotechnology Brazil Ltda., São Paulo, Brazil.
  • g
    QIAquick Gel Extraction kit®, Qiagen Biotechnology Brazil Ltda., São Paulo, Brazil.
  • h
    BioEstat version 5.0, Universidade Federal doPará, Pará, Brazil.

References

  • 1
    Harris NB, Barletta RG. Mycobacterium avium subsp. paratuberculosis in veterinary medicine. Clin Microbiol Rev. 2001;14(3):489-512.
  • 2
    Manning EJB, Collins MT. Mycobacterium avium subsp. paratuberculosis: pathogen, pathogenesis and diagnosis. Rev Off Int Epizoot 2001;20(1):133-150.
  • 3
    Yamasaki EM, Tokarnia CH, Galvão A, et al. Aspectos clínicos patológicos e controle da paratuberculose em rebanho bovino leiteiro. Pesqui Vet Bras. 2010;30(11):921-932.
  • 4
    Mota RA, Pinheiro-Junior JW, Gomes MJP, et al. Paratuberculose em um rebanho leiteiro no Estado de Pernambuco, PE. Arq Inst Biol (São Paulo). 2007;74(2):73-79.
  • 5
    Sá LM, Oliveira JMB, Santos GR, et al. Avaliação sorológica e de fatores de risco para a infecção por Mycobacterium avium subsp. paratuberculosis em rebanhos leiteiros da Microrregião de Garanhuns, Pernambuco. Pesqui Vet Bras 2013;33(3):310-314.
  • 6
    Collins MT. Diagnosis of paratuberculosis. Vet Clin North Am Food Anim Pract. 1996;12(2):357-371.
  • 7
    O’Mahony J, Hill C. Rapid real-time PCR assay for detection and quantitation of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Appl Environ Microbiol 2004;70(8):4561-4568.
  • 8
    Kawaji S, Taylor DL, Mori Y, Whittington RJ. Detection of Mycobacterium avium subsp. paratuberculosis in ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture. Vet Microbiol 2007;125(1-2):36-48.
  • 9
    Green EP, Tizard MLV, Moss MT, et al. Sequence and characteristics of IS900, an insertion element identified in a human Crohn's disease isolate of Mycobacterium paratuberculosis. Nucleic Acids Res. 1989;17(22):9063-9073.
  • 10
    Collins DM, Gabric DM, De Lisle GW. Identification of a repetitive DNA sequence specific to Mycobacterium paratuberculosis. FEMS Microbiol Lett 1989;51(1):175-178.
  • 11
    Vansnick E, Rijk P, Vercammen F, Geysen D, Rigouts L, Portaels F. Newly developed primers for the detection of Mycobacterium avium subspecies paratuberculosis. Vet Microbiol 2004;100(3-4):197-204.
  • 12
    Corti S, Stephan R. Detection of Mycobacterium avium subsp. paratuberculosis specific IS900 insertion sequences in bulk-tank milk samples obtained from different regions throughout Switzerland. BMC Microbiol. 2002;2:15.
  • 13
    Slana I, Liapi M, Moravkovaa M, Kralovaa A, Pavlika I. Mycobacterium avium subsp. paratuberculosis in cow bulk tank milk in Cyprus detected by culture and quantitative IS900 and F57 real-time PCR. Prev Vet Med. 2009;89(3-4):223-226.
  • 14
    Taddei R, Barbieri I, Pacciarini ML. Mycobacterium porcinum strains isolated from bovine bulk milk: implications for Mycobacterium avium subsp. paratuberculosis detection by PCR and culture. Vet Microbiol 2008;130(3-4):338-347.
  • 15
    Rodríguez-Lázaro D, D’Agostino M, Herrewegh A, Pla M, Cook N, Ikonomopoulos J. Real-time PCR-based methods for detection of Mycobacterium. Int J Food Microbiol 2005;101(1):93-104.
  • 16
    Kralik P, Slana I, Kralova A, Babak V, Whitlock RH, Pavlik I. Development of a predictive model for detection of Mycobacterium avium subsp. paratuberculosis in faeces by quantitative real time PCR. Vet Microbiol 2011;149(1-2):133-138.
  • 17
    Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics 1977;33(1):159-174.
  • 18
    Carvalho IA, Silva A, Campos EB, Moreira MAS. Short communication: detection of Mycobacterium avium subspecies paratuberculosis by polymerase chain reaction in bovine milk in Brazil. J Dairy Sci 2009;92(11):5408-5410.
  • 19
    Ristow P, Marassi CD, Rodrigues ABF, et al. Diagnosis of paratuberculosis in a dairy herd native to Brazil. Vet J. 2007;174(2):432-434.
  • 20
    Mackay IM. Real-time PCR in the microbiology laboratory. Clin Microbiol Infect 2004;10(3):190-212.
  • 21
    Sonawane GG, Tripathi BN. Comparison of a quantitative real-time polymerase chain reaction (qPCR) with conventional PCR, bacterial culture and ELISA for detection of Mycobacterium avium subsp. paratuberculosis infection in sheep showing pathology of Johne's disease. Springer Plus 2013;2(1):45.
  • 22
    Fang Y, Wu WH, Pepper JL, et al. Comparison of real-time, quantitative PCR with molecular beacons to nested PCR and culture methods for detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples. J Clin Microbiol. 2002;40(1):287-291.
  • 23
    Moghadam MT, Sarv S, Moosakhani F, Badiie A. Detection of Mycobacterium avium subspecies paratuberculosis in milk and fecal samples in dairy cattle by PCR and nested-PCR. J Anim Vet Adv 2010;9(24):3055-3061.
  • 24
    Opel KL, Chung D, McCord BR. A study of PCR inhibition mechanisms using real time PCR. J Forensic Sci 2010;55(1):25-33.
  • 25
    Slana I, Kralik P, Kralova A, Pavlik I. On-farm spread of Mycobacterium avium subsp. paratuberculosis in raw milk studied by IS900 and F57 competitive real time quantitative PCR and culture examination. Int J Food Microbiol. 2008;128(2):250-0257.
  • 26
    Pinedo PJ, Rae DO, Williams JE, Donovan GA, Melendez P, Buergelt CD. Association among results of serum ELISA, faecal culture and nested PCR on milk, blood and faeces for the detection of paratuberculosis in dairy cows. Transbound Emerg Dis 2008;55(2):125-133.
  • 27
    Sweeney RW, Whitlock RH, Rosenberger AE. Mycobacterium paratuberculosis cultured from milk and supramammary lymph nodes of infected asymptomatic cows. J Clin Microbiol. 1992;30(1):166-171.
  • 28
    Slana I, Paolicchi F, Janstova B, Navratilova P, Pavlik I. Detection methods for Mycobacterium avium subsp. paratuberculosis in milk and milk products: a review. Vet Med (Praha) 2008;53(6):283-306.
  • 29
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Publication Dates

  • Publication in this collection
    Jan-Mar 2017

History

  • Received
    14 Mar 2015
  • Accepted
    25 Apr 2016
Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
E-mail: bjm@sbmicrobiologia.org.br