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 Table of Contents  
FULL LENGTH ARTICLE
Year : 2016  |  Volume : 5  |  Issue : 3  |  Page : 269-272

Detection of Mycobacterium bovis in artisanal cheese in the state of Pernambuco, Brazil


1 Federal Rural University of Pernambuco, Dois Irmãos, Recife, Pernambuco, Brazil
2 Department of Immunology, Aggeu Magalhães Research Center, Oswaldo Cruz Foundation, Cidade Universitária, Recife, Pernambuco, Brazil
3 Academic Unit of Garanhuns, Federal Rural University of Pernambuco, Garanhuns, Pernambuco, Brazil
4 National Agricultural Laboratory of Pernambuco, Ministry of Agriculture, Livestock and Food Supply of , Dois Irmãos, Recife, Pernambuco, Brazil

Date of Web Publication13-Feb-2017

Correspondence Address:
José W. Pinheiro Junior
Federal Rural University of Pernambuco, Rua Dom Manuel de Medeiros, s/n, Dois Irmãos, CEP 52171-900 Recife, Pernambuco
Brazil
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Source of Support: None, Conflict of Interest: None


DOI: 10.1016/j.ijmyco.2016.04.007

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  Abstract 


Objective/background: The present study was aimed at detecting Mycobacterium bovis in artisanal cheese using real-time quantitative polymerase chain reaction.
Methods: One hundred seven cheese samples (250 g) were purchased in 107 commercial establishments including neighborhood grocery stores, bakeries, and open-air markets from 19 municipalities of the state of Pernambuco, Brazil. Ten grams of each cheese sample were macerated with sterile saline solution in a sterile bag and DNA was extracted from 20 mg of the macerated material using the Wizard SV Genomic DNA Purification System. The quantitative polymerase chain reaction amplified a fragment corresponding to the region of difference 4 of M. bovis.
Results: Of the 107 samples analyzed, three (2.8%) were positive for M. bovis and their identities were confirmed by sequencing. This is perhaps the first report of the presence of M. bovis in artisanal cheese in the state of Pernambuco, Brazil.
Conclusion: The results of the present study highlight the need for improving sanitary measures during the production of artisanal cheese to prevent zoonotic tuberculosis in humans, resulting from the consumption of food contaminated with M. bovis.

Keywords: Cheese, Food safety, PCR, Tuberculosis


How to cite this article:
Cezar RD, Lucena-Silva N, Borges JM, Santana VL, Junior JW. Detection of Mycobacterium bovis in artisanal cheese in the state of Pernambuco, Brazil. Int J Mycobacteriol 2016;5:269-72

How to cite this URL:
Cezar RD, Lucena-Silva N, Borges JM, Santana VL, Junior JW. Detection of Mycobacterium bovis in artisanal cheese in the state of Pernambuco, Brazil. Int J Mycobacteriol [serial online] 2016 [cited 2019 Oct 19];5:269-72. Available from: http://www.ijmyco.org/text.asp?2016/5/3/269/200063




  Introduction Top


Artisanal cheeses are produced with raw milk in small farms around the world [1]. The production of artisanal cheese often does not comply with the hygienic–sanitary requirements demanded by official agencies, especially regarding to the possibility of being contaminated with pathogens due to the use of raw milk from unhealthy dairy cows and lack of hygiene during processing [2].

Mycobacterium bovis is the etiological agent of bovine tuberculosis and can cause human tuberculosis, in which clinical symptoms are indistinguishable from those caused by Mycobacterium tuberculosis. Consumption of milk and its derived products contaminated with M. bovis represent the main infection route of zoonotic tuberculosis for humans [3],[4].

The presence of the bacillus in cheese and its association with human tuberculosis has already been reported [5],[6],[7]. In Brazil, studies have demonstrated the presence of M. bovis in raw cattle milk [8],[9].

Considering the importance of artisanal cheese in Brazil and its participation in the transmission chain of zoonotic tuberculosis to humans, the aim of the present study was to detect M. bovis in artisanal cheeses commercialized in the state of Pernambuco, Brazil, using molecular techniques.


  Material and methods Top


Sampling

One hundred and seven samples of artisanal cheese “coalho” type were purchased in 107 commercial establishments including neighborhood grocery stores, bakeries, and open-air markets located in 19 municipalities of the Garanhuns microregion, state of Pernambuco, Brazil, as follows: Angelim (six samples), Bom Conselho (six samples), Brejão (six samples), Caetés (six samples), Calçado (seven samples), Canhotinho (six samples), Correntes (six samples), Garanhuns (six samples), Iati (four samples), Jucati (six samples), Jupi (eight samples), Jurema (five samples), Lagoa do Ouro (six samples), Lajedo (six samples), Palmerina (three samples) Paranatama (five samples), Saloá (six samples), São João (six samples), and Terezinha (three samples).

The cheese samples were sent to the Garanhuns Laboratories Center, located in the Garanhuns Academic Unit of the Federal Rural University of Pernambuco in an isothermal box containing reusable ice.

DNA extraction

The cheese samples (250 g) were partitioned and 10 g of cheese were macerated with 20 mL of 0.9% sterile saline solution in a sterile bag. DNA extractions were performed with 20 mg of the macerated material using the Wizard SV Genomic DNA Purification System (Promega. Promega Corporation, USA, 2012) following the manufacturer's instructions.

Positive control

The M. bovis American Type Culture Collection (ATCC) 19274 strain was provided by the Oswaldo Cruz Foundation (Fundação Osvaldo Cruz, Rio de Janeiro, Brazil) and it was used for the construction of a plasmid harboring the target sequence, which was the positive control in the molecular tests. The genomic DNA of M. bovis ATCC 19274 strain was extracted and the fragment corresponding to the region of difference 4 (RD4) was amplified with the specific primers reported by Sales et al. [10]. The target fragment was cloned using  Escherichia More Details coli XL1 blue strain and TA cloning kit (Invitrogen. Invitrogen Corporation, Califórnia, 2006) according to the manufacturer's instructions.

Real-time polymerase chain reaction

Quantitative real-time polymerase chain reaction (qPCR) was performed using the same primer set used for the amplification of the RD4 fragment of the M. bovis ATCC 19274 strain in the presence of TaqMan Universal PCR Master Mix (Applied Biosystems, USA, 2010) and DNA sample. The reaction solution (25 μL) consisted of 2.0 μL of DNA and 12.5 μL of the master mix with 1.0 μL of each primer (5pmol), 0.5 μL of probe (5pmol), and 8-μL water. The amplification conditions were 95 °C for 15 min (denaturation) followed by 40 cycles of 94 °C for 15 s and 60 °C for 60 s. The qPCR was performed in an ABI 7500 Real-Time PCR (Applied Biosystems, USA, 2010) system set for absolute quantification. In all PCR runs, standard curves were obtained using plasmid DNA encompassing the mycobacteria RD4 sequence as positive control. The positive control was prepared in triplicate by serial dilution of 10× plasmid DNA from 200 ng (quantification cycle = 11.8) to 0.0002 ng (quantification cycle = 32.2). The standard curve slope was −3.40 and R = 0.999 with 97% efficiency.

Sequencing

The commercial kit ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction v3.1 (Applied Biosystems, USA, 2009) was used for DNA sequencing following the manufacturer's recommendations. The RD4 fragments were sequenced using the Sanger method and the reaction products were analyzed in the ABI 3500xL Genetic Analyzer (Applied Biosystems, USA, 2009).

All the sequences obtained in the present study were compared with the RD4 fragment of the reference genome (88bp; GenBank Access number BX248339.1) using the software Blast-N (http://www.ncbi.nlm.nih.gov) and MEGA6 [11].


  Results and discussion Top


This is the first report on the occurrence of M. bovis in artisanal cheese commercialized in the Garanhuns microregion, a dairy region of the state of Pernambuco, northeastern Brazil.

Of the 107 samples analyzed in the present study, three (2.8%) were positive for M. bovis and their identities were confirmed by sequencing. The positive samples were from two neighborhood grocery stores of the municipality of Correntes (one out of six) and Lagoa do Ouro (one out of six), and an open-air market located in Bom Conselho (one out of six; [Table 1]).
Table 1: Results of quantitative polymerase chain reaction of cheese samples collected fromcattle of the microregion of Garanhuns, state of Pernambuco, Brazil, 2014.

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Few studies in Brazil have evaluated the presence of M. bovis in cheese. In the present study, M. bovis DNA was present in 2.8% of the samples analyzed, which is lower than the 10% positivity found in samples of the same type of cheese collected in the state of Piauí, northeastern Brazil [12]. Our study and the one of Silva et al. [12] shows the risk of human infection by M. bovis through the consumption of cheese, yogurt, and milk without heat treatment, as 41% of milk is illegally produced in Brazil [10]. Melo et al. [13] evaluated the presence of M. bovis in 147 cheese samples confiscated from travelers coming from 21 countries to Brazil and found M. bovis genetic material in 17.5% of the samples, showing that cheese may contribute to the spread of bacteria to different countries.

More than 200 samples of fresh cheese from the USA were analyzed and only 0.5% of them were positive for M. bovis [1],[5]. However, the consumption of Mexican fresh cheese accounts for 90% of cases of zoonotic tuberculosis affecting persons of Hispanic ethnicity living in the USA [14]. A study by Rodwell et al. [15] compared spoligotyping by M. bovis strains isolated from humans in the USA with strains from cows raised in Mexico and found that 91% of the human M. bovis isolates from the USA had spoligotypes identical to those found in Mexican cattle. Of 95 samples of fresh cheese from the state of Hidalgo, Mexico, 6.31% (six from 95) were positive for the bovine bacillus and four samples (4.21%) were from regions with enzootic bovine tuberculosis. A positive correlation between cases of zoonotic tuberculosis and the consumption of cheese made from raw milk in endemic areas for bovine tuberculosis has been previously described [16].

In 2001, one third of all cases of pediatric tuberculosis diagnosed in San Diego, California, USA were caused by M. bovis, and the consumption of dairy products was associated with positive tuberculin skin test in children [17]. In a study performed in Brazil, of 189 patients with tuberculosis, only three (1.59%) were diagnosed with M. bovis; however, the consumption of cheese made with raw milk was associated with two of the cases of zoonotic tuberculosis [7].

Although in Brazil a National Program for Control and Eradication of  Brucellosis More Details and Tuberculosis was established in 2001 [18], tuberculosis is still present in several states [19] with an average prevalence of 3.3% [20]. The Garanhuns microregion, where the present study was performed, is enzootic for bovine tuberculosis [21],[22].

The consumption of raw milk and its by-products are the main infection route associated with human cases of tuberculosis caused by M. bovis, especially in cases of extrapulmonary tuberculosis [5],[7],[20]. It is believed that M. bovis is responsible for approximately 4000 of the 100,000 cases of human tuberculosis diagnosed in Brazil [23].

The manufacturing process of artisanal cheese, which uses raw milk, does not eliminate the tuberculosis bacillus. Experimental studies have shown that the bacillus can persists for up to 305 days depending on the type of cheese [24].

Milk pasteurization and use of pasteurized milk in cheese production dramatically reduces the incidence of zoonotic tuberculosis [25]. Due to the risk that dairy products made with raw milk may pose to public health, it is important to study cases of human tuberculosis caused by M. bovis in order to provide better assistance to patients as well as to improve the diagnosis, control, and prevention measures of tuberculosis in cattle herds.

Detection of M. bovis in samples of artisanal cheese intended for human consumption may pose a risk to public health, showing that measures to prevent and control tuberculosis in cattle herds should be improved. Cheese quality must also be monitored, which can be achieved using molecular techniques. Artisanal cheese should be consumed with caution.

Ethical considerations

The Ethics Committee on Animal Use of The Federal Rural University of Pernambuco provided scientific and ethical clearance for the present study (reference number 23082.004671/2013, license number 028/2013).


  Conflicts of interest Top


The authors declare that they have no competing interests.



 
  References Top

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    Tables

  [Table 1]


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