Isolation and identification of Mycobacterium bovis in milk from cows in northeastern Brazil

Isolamento e identificação de Mycobacterium bovis em leite de vacas no Nordeste do Brasil

Joelson Marcolino Ramos Marcos Bryan Heinemann José Soares Ferreira Neto Antônio Francisco de Souza Filho Nicolás Céspedes Cárdenas Clebert José Alves Sérgio Santos de Azevedo About the authors

ABSTRACT:

Milk samples from 16 cows that tested positive on the tuberculin test in the state of Paraíba, northeastern Brazil, were used for mycobacteria isolation and identification. Mycobacteria were isolated from five (31.25%) of the 16 milk samples; three samples were classified asM. bovis, and two as belonging to theMycobacterium genus. This is probably the first study of isolation and identification ofM. bovis in milk from cows in Northeastern Brazil, which suggests that humans are at risk of contamination by ingestion.

Key words:
Mycobacterium bovis; isolation; milk; PCR; cattle

RESUMO:

Amostras de leite de 16 vacas positivas no teste da tuberculinização no Estado da Paraíba, Nordeste do Brasil, foram utilizadas para isolamento e identificação de micobactérias. Foram isoladas micobactérias em cinco (31,25%) das 16 amostras de leite: três amostras foram classificadas comoM. bovis e duas como pertencentes ao gênero Mycobacterium. De acordo com nosso conhecimento, este é o primeiro estudo de isolamento e identificação de M. bovis em leite de vacas no Nordeste do Brasil, o que sugere que os seres humanos estão em risco de contaminação por ingestão.

Palavras-chave:
Mycobacterium bovis; isolamento; leite; PCR; bovinos.

Bovine tuberculosis is a chronic, zoonotic disease caused by Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex. The disease is responsible for economic losses due to reduced reproductive efficiency, decreased milk production and decreased weight gain, carcass condemnation, and restrictions on international trade of animals and animal products (BRASIL, 2006BRASIL - MINISTÉRIO DA AGRICULTURA, PECUÁRIA E ABASTECIMENTO. Programa Nacional de Controle e Erradicação da Brucelose e da Tuberculose Animal (PNCEBT). Brasília: MAPA/SDA/DAS, 2006. 188p.).

While most cases of tuberculosis in humans are caused by M. tuberculosis, it is estimated that about 3.1% of the cases of human tuberculosis in the world are caused by M. bovis (EL-SAYED et al., 2016EL-SAYED, A. et al. Molecular epidemiology of Mycobacterium bovis in humans and cattle. Zoonoses and Public Health, v. 63, n. 4, p. 251-264, 2016. Available from: http://onlinelibrary.wiley.com/doi/10.1111/zph.12242/abstract;jsessionid=0BD56CCA865867B220F15F41ACCAA9AE.f02t01 doi: 10.1111/zph.12242>. Accessed: Mar. 20, 2016. doi: 10.1111/zph.12242.
http://onlinelibrary.wiley.com/doi/10.11...
). However, infection in humans is generally not confirmed by isolation and identification of the agent, making it impossible to identify the possible source of infection. In addition, the human diseases caused by M. tuberculosis and M. bovis are indistinguishable using clinical, radiological and pathological methods (ROCHA et al., 2011ROCHA, A. et al. Genotyping did not evidence any contribution of Mycobacterium bovis to human tuberculosis in Brazil. Tuberculosis (Edinburgh), v. 91, n. 1, p. 14-21, 2011. Available from: http://www.tuberculosisjournal.com/article/S1472-9792(10)00129-0/fulltext>. Accessed: Jan. 20, 2016. doi: 10.1016/j.tube.2010.10.003.
http://www.tuberculosisjournal.com/artic...
). Transmission of M. bovis to humans occurs through consumption of raw milk and dairy products, or through contact with secretions from fistulated abscesses.

In the state of Paraíba, official data indicated the occurrence of 1,015 cases of human tuberculosis in 2014 (BRASIL, 2015BRASIL - MINISTÉRIO DA SAÚDE. SECRETÁRIA DE VIGILÂNCIA EM SAÚDE. DEPARTAMENTO DE VIGILÂNCIA EPIDEMIOLÓGICA. Sistema de Informação de Agravos de Notificação - Sinan, 2015. Available from: http://portalsaude.saude.gov.br/images/pdf/2015/setembro/24/Casos-novos-tuberculose-1990-2014-base-jun-2015.pdf>. Accessed: Jan. 19, 2016.
http://portalsaude.saude.gov.br/images/p...
). In areas where human and bovine tuberculosis coexist, the differentiation between M. bovis and M. tuberculosis is important for monitoring the spread of M. bovis among cattle and from cattle to humans. Thus, the objective of this study was to isolate and identify M. bovis in milk from cows with a positive diagnosis identified using Single Intradermal Comparative Cervical Tuberculin (SI CCT) test in the state of Paraíba, Northeastern Brazil.

SICCT test was performed by veterinarians of the Official Veterinary Service of the Paraíba State following the strategies of the National Program for Control and Eradication of Bovine Brucellosis and Tuberculosis (PNCEBT); an animal was considered positive to the SICCT test when the 72-hour reaction to bovine tuberculin (Bov) was greater than the reaction to avian tuberculin (Av) by more than 4mm (BRASIL, 2006). All SICCT-positive animals were euthanized according to PNCEBT technical regulation.

Milk samples from 16 mixed-breed cows with average age of 6.4 years that tested positive on the SICCT test from seven different properties located in the municipalities of Cacimba de Areia, Patos, Piancó and São Mamede were used (Figure 1). Animals did not show any clinical signs of tuberculosis, chronic mastitis or local lymphadenopathy. Properties were labeled A (Coordinates: 07º13'28.9"S 37º56'21.7"W), B (07º08'41.7"S 37º07'16.7"W), C (06º56'41.7"S 37º10'22.6"W), D (06º58'37.3"S 37º16'38.7"W), E (06º57'53.5"S 37º20'32.2"W), F (06º58'36.0"S 37º17'21.9"W) and G (07º00'24.3"S 37º16'15.6"W). Milk was collected directly from the teats of the animals before milking, following all methods of antisepsis. An average of 15mL of milk per animal was collected, utilizing sterile and previously identified vials. These were sent to the laboratory in isothermal boxes with ice.

Figure 1
Geographical distribution of selected properties according to municipalities. Detail shows the State of Paraíba, Brazil.

For the isolation of Mycobacterium spp., a 5mL aliquot of milk was used, centrifuged at 3000rpm for 20 minutes. In this way, two phases of milk were obtained, fat and sediment, which were packaged in different vials and both decontaminated using the Petroff method (KANTOR, 1979KANTOR, I.N. Bacteriológia de la tuberculosis humana y animal. Buenos Aires: Centro Panamericano de Zoonosis, Organización Panamericana de la Salud, 1979. 63p. ), inoculated in duplicate in Stonebrink and Lowenstein-Jensen media, and incubated at 37°C for 90 days. Colonies suggestive of mycobacteria were stained using the Ziehl Neelsen method; DNA was extracted using thermolysis (MAZARS et al., 2001MAZARS, E. et al. High-resolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology. Proceedings of the National Academy Sciences of the United States of America, v. 98, n. 4, p. 1901-1906, 2001. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC29354/>. Accessed: Jan. 20, 2016. doi: 10.1073/pnas.98.4.1901.
http://www.ncbi.nlm.nih.gov/pmc/articles...
). Samples identified as Acid Fast Bacilli (AFB) in Ziehl Neelsen were subjected to molecular identification.

Identification of Mycobacterium species and differentiation of the M. tuberculosis complex, M. avium complex, M. intracellurare complex, and other Mycobacterium spp. was done using the TB Multiplex PCR (WILTON & COUSINS, 1992WILTON, S.; COUSINS, D. Detection and identification of multiple mycobacterial pathogens by DNA amplification in a single tube. PCR Methods and Applications, v. 1, n. 4, p. 269-273, 1992. Available from: http://genome.cshlp.org/content/1/4/269.long>. Accessed: Jan. 20, 2016
http://genome.cshlp.org/content/1/4/269....
). For this, the primers used were: MYCGEN-F (G1) (5'-AGAGTTTGATCCTGGCTCAG-3') and MYCGEN-R (G2) (5'-TGCACACAGGCCACAAGGGA-3') - associated with genus; TB-1F (5'-GAACAATCCGGAGTTGACAA-3') and TB-1R (5'-AGCACGCTGTCAATCATGTA-3') - related to the M. tuberculosis complex; MYCAV-R (5'-ACCAGAAGACATGCGTCTTG-3') - related to M. avium ; and MYCINT-F (5'CCTTTAGGCGCATGTCTTTA-3') - related to M. intracellulare. Positive controls used were the M. bovis strain AN5 and M. tuberculosis strain H37Rv for the M. tuberculosis complex. The 50μL reactions were performed containing reaction buffer dNTP (1.25mM each), 20pmol of each oligonucleotide, 50mM KCl, 10mM Tris-HCl (pH 8.3), 1.5mM MgCl2, 10pmol μL-1 of primers, 1.25 units of TAQ polymerase (1.0μL) and 5μL of the DNA under study. Amplification cycles used were the following: once at 94°C for 10 minutes, 61°C for 2 minutes, and 72°C for 3 minutes; 33 times at 94°C for 30 seconds, 61°C for 2 minutes, and 72°C for 3 minutes; once at 94°C for 30 seconds, 61°C for 2 minutes, and 72°C for 10 minutes. Samples containing an amplification of 1030bp were identified as belonging to the Mycobacterium genus, and samples with 372bp fragments were considered members of the M. tuberculosis complex (WILTON & COUSINS, 1992WILTON, S.; COUSINS, D. Detection and identification of multiple mycobacterial pathogens by DNA amplification in a single tube. PCR Methods and Applications, v. 1, n. 4, p. 269-273, 1992. Available from: http://genome.cshlp.org/content/1/4/269.long>. Accessed: Jan. 20, 2016
http://genome.cshlp.org/content/1/4/269....
).

All DNA samples with consistent amplification for M. tuberculosis complex using the TB Multiplex PCR were amplified with the following primers: RD4 (RD4-1 5'-ATG TGC GAG CTG AGC GAT G-3'; RD4-2 5'-TGT ACT ATG CTG ACC CAT GCG-3'; and RD4-3 5'-AAA GGA GCA CCA TCG TCC AC-3') for identification of M. bovis (WARREN et al., 2006WARREN, R.M. et al. Differentiation of Mycobacterium tuberculosis complex by PCR amplification of genomic regions of difference. International Journal of Tuberculosis and Lung Disease, v10, n. 7, p. 818-822, 2006. Available from: http://docserver.ingentaconnect.com/deliver/connect/iuatld/10273719/v10n7/s19.pdf?expires=1461095505&id=86722049&titleid=3764&accname=Guest+User&checksum=69A041568F1DEA686ED74EF48B6DE8B5>. Accessed: Feb. 02, 2016.
http://docserver.ingentaconnect.com/deli...
). 25μL reactions were conducted containing the reaction buffer dNTPs (1.25mM each), 20pmol of each oligonucleotide, 50mM KCl, 10mM Tris-HCl (pH 8.3), 1.5mM MgCl2, primers, 1.25 units of TAQ polymerase, and 5μL of the genomic DNA being studied. Positive controls used were the M. bovis AN5 strain and M. tuberculosis strain H37Rv. PCR cycles used were: once at 95°C for 15 minutes, 45 times at 94°C for 1 minute, 62°C for 1 minute, and 72°C for 1 min, and once at 72°C for 10 minutes. Sizes of the PCR products indicate that the region studied is absent or present in the respective members of the M. tuberculosis complex. Thus, the 268bp fragment resulting from amplification is consistent with the RD4 absent in the sample, characteristic of the M. bovis species.

Mycobacteria were isolated from five (31.25%) of the 16 milk samples; three samples were classified as M. bovis, and two as belonging to the Mycobacterium genus. This is the first study of isolation and identification of M. bovis in milk from cows in Northeastern Brazil. Several studies have been conducted in other states in order to isolate and identify mycobacteria in milk from cows. In São Paulo, PARDO et al. (2001PARDO, R.B. et al. Isolation of Mycobacterium spp. in milk from cows suspected or positive to tuberculosis. Brazilian Journal of Veterinary Research and Animal Science, v. 38, n. 6, p. 284-287, 2001. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1413-95962001000600007>. Accessed: Feb. 16, 2016. doi: 10.1590/S1413-95962001000600007.
http://www.scielo.br/scielo.php?script=s...
) isolated M. bovis in one sample of raw milk; LEITE et al. (2003LEITE, C.Q.F. et al. Isolation and identification of mycobacteria from livestock specimens and milk obtained in Brazil. Memórias do Instituto Oswaldo Cruz, v. 98, n. 3, p. 319-323, 2003. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762003000300005> Acessed: Jan. 20, 2016. doi: 10.1590/S0074-02762003000300005.
http://www.scielo.br/scielo.php?script=s...
), using milk samples from the states of São Paulo, Paraná, Santa Catarina, Goiás and Pará, isolated M. bovis in one sample of raw milk. FRANCO et al. (2013FRANCO, M.M.J. et al. Occurrence of mycobacteria in bovine milk samples from both individual and collective bulk tanks at farms and informal markets in the southeast region of Sao Paulo, Brazil. BMC Veterinary Research, v. 9, p. 746-6148, 2013. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3650655/>. Accessed: Jan. 20, 2016. doi: 10.1186/1746-6148-9-85.
http://www.ncbi.nlm.nih.gov/pmc/articles...
), in the southeast region of São Paulo, isolated M. bovis from one milk sample from an individual cooling tank.

Milk and its derivatives, when contaminated and unpasteurized, present a significant risk of tuberculosis transmission to humans. It is estimated that around 50% or more of milk produced in Brazil is unpasteurized (LEITE et al., 2003LEITE, C.Q.F. et al. Isolation and identification of mycobacteria from livestock specimens and milk obtained in Brazil. Memórias do Instituto Oswaldo Cruz, v. 98, n. 3, p. 319-323, 2003. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762003000300005> Acessed: Jan. 20, 2016. doi: 10.1590/S0074-02762003000300005.
http://www.scielo.br/scielo.php?script=s...
), attributing serious risks of transmission of various diseases to humans, including tuberculosis caused by M. bovis. It is worth highlighting that it is common in northeastern Brazil to consume raw milk.

An important aspect was the isolation and identification of Mycobacterium spp. in two samples, probably nontuberculous environmental mycobacteria (NTM), which are present in the environment and can be transmitted to animals and humans (FRANCO et al., 2013FRANCO, M.M.J. et al. Occurrence of mycobacteria in bovine milk samples from both individual and collective bulk tanks at farms and informal markets in the southeast region of Sao Paulo, Brazil. BMC Veterinary Research, v. 9, p. 746-6148, 2013. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3650655/>. Accessed: Jan. 20, 2016. doi: 10.1186/1746-6148-9-85.
http://www.ncbi.nlm.nih.gov/pmc/articles...
) through inhalation or ingestion, resulting in permanent or temporary colonization of the respiratory and digestive tracts (PRIMM et al., 2004PRIMM, T.P. et al. Health impacts of environmental mycobacteria. Clinical Microbiology Reviews, v. 17, n. 1, p. 98-106, 2004. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC321467/>. Accessed: Jan. 12, 2016. doi: 10.1128/CMR.17.1.98-106.2004.
http://www.ncbi.nlm.nih.gov/pmc/articles...
). The increasing number of individuals infected with HIV predisposes an increase in the number of cases of emerging and re-emerging diseases, especially those caused by opportunistic agents, such as Mycobacterium spp. In this context, non-pasteurized milk infected with mycobacteria poses a serious risk to individuals with HIV (FRANCO et al., 2013FRANCO, M.M.J. et al. Occurrence of mycobacteria in bovine milk samples from both individual and collective bulk tanks at farms and informal markets in the southeast region of Sao Paulo, Brazil. BMC Veterinary Research, v. 9, p. 746-6148, 2013. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3650655/>. Accessed: Jan. 20, 2016. doi: 10.1186/1746-6148-9-85.
http://www.ncbi.nlm.nih.gov/pmc/articles...
).

The presence of M. bovis in milk from cows in the state of Paraíba in Northeastern Brazil suggests that humans are at risk of contamination by ingestion. This reinforces the need for optimization of milk and dairy products quality programs, enhancing sanitary inspections of these products, and conducting studies for isolating and identifying mycobacteria in milk from other northeastern states

ACKNOWLEDGMENTS

The authors would like to thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) by financial support under the code 302131/2012-4

BIOETHICS AND BIOSSECURITY COMMITTEE APPROVAL

This study was approved by the institutional Committee for Ethics in the Use of Animals (Universidade Federal de Campina Grande – UFCG) (protocol 25-2012).

REFERENCES:

Publication Dates

  • Publication in this collection
    Dec 2016
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