Non-tuberculous mycobacteria (NTM) are opportunistic human pathogens of increasing public health significance (Falkinham, Reference Falkinham2009). Human to human transmission of NTM is rare, so it is important to understand the environmental sources associated with human infection (Primm et al. Reference Primm, Lucero and Falkinham2004). One of the most common NTM responsible for human illness is Mycobacterium avium Complex (MAC) which involves a group of organisms including M. avium (M. avium subspecies avium (MAA), M. avium subspecies hominissuis (MAH), M. avium subspecies paratuberculosis (MAP)) and Mycobacterium intracellulare (Rubin & Rahal, Reference Rubin and Rahal1994).
One of the most common clinical consequences of MAC is cervical lymphadenitis, which is infection of the cervical and mandibular lymph nodes, typically affecting children aged between 6 months to 2 years (Kaevska et al. Reference Kaevska, Slana, Kralik, Reischl, Orosova, Holcikova and Pavlik2011). This coincides with the period of time that teeth are erupting, and as such contaminated food and water is often considered a likely source of infection (Grant et al. Reference Grant, Pope, O'Riordan, Ball and Rowe2000). MAP has also been linked to Crohn's disease, an incurable inflammatory bowel disease in humans and is also the causative agent of chronic enteritis in ruminants known as Johne's disease (Naser et al. Reference Naser, Sagramsingh, Naser and Thanigachalam2014). As such, there have been numerous studies detecting MAP in pasteurised milk from Brazil, India, Czech Republic, USA, Argentina, UK, Iran, Ireland and the United Kingdom (Millar et al. Reference Millar, Ford, Sanderson, Withey, Tizard, Doran and Hermon-Taylor1996; Grant et al. Reference Grant, Ball and Rowe2002; Paolicchi et al. Reference Paolicchi, Zumarraga, Gioffre, Zamorano, Morsella, Verna, Cataldi, Alito and Romano2003; O'Reilly et al. Reference O'Reilly, O'Connor, Anderson, Harvey, Grant, Donaghy, Rowe and O'Mahony2004; Ayele et al. Reference Ayele, Svastova, Roubal, Bartos and Pavlik2005; Ellingson et al. Reference Ellingson, Anderson, Koziczkowski, Radcliff, Sloan, Allen and Sullivan2005; Shankar et al. Reference Shankar, Singh, Singh, Singh, Sohal and Greenstein2010; Anzabi & Hanifian, Reference Anzabi and Hanifian2012; Carvalho et al. Reference Carvalho, Pietralonga, Schwarz, Faria and Moreira2012). MAC has also been detected from raw milk from Brazil (Bezerra et al. Reference Bezerra, dos Reis, Rodrigues, Cenci, Cerva and Mayer2015) and Tanzania (Mdegela et al. Reference Mdegela, Karimuribo, Kusiluka, Kabula, Manjurano, Kapaga and Kambarage2005) and M. intracellulare specifically has also been detected from raw milk in Brazil (Franco et al. Reference Franco, Paes, Ribeiro, de Figueiredo Pantoja, Santos, Miyata, Leite, Motta and Listoni2013).
This is the first study in Australia to use real time PCR (qPCR) to investigate the presence of MAC in commercial milk. MAC was chosen instead of specifically looking at MAP due to the public health significance of the other MAC organisms (Falkinham, Reference Falkinham2009; Kaevska et al. Reference Kaevska, Slana, Kralik, Reischl, Orosova, Holcikova and Pavlik2011). The chosen method for detection in this study was qPCR, due to the slow growth rate of MAC on culture medium and the need for further identification of mycobacterial colony (Mendoza et al. Reference Mendoza, Lana and Díaz-Rubio2009).
Materials and methods
Milk samples
A total of 180 samples of Australian commercially available pasteurised milk were tested. Specifically, three times during the year the same 20 types of commercially available pasteurised milk, from 14 different brands, with different expiry dates, were selected. The different milk brands were produced from South Australia, New South Wales, Victoria or Western Australia. In triplicate, 30 ml of milk was collected aseptically from each milk carton for DNA extraction.
Incubated milk samples
An additional 14 milk samples, one from each brand of commercially available pasteurised milk were also selected. From each, 30 ml of milk was aseptically collected for DNA extraction. The milk cartons were then sealed and incubated at 37 °C for 3 weeks. After 3 weeks an additional 30 ml of milk was aseptically collected from each milk carton for DNA extraction. This was done to give any potential MAC present the right conditions to replicate to a higher concentration to facilitate detection.
Positive control
To ensure the DNA extraction method was suitable for MAC detection in milk a spiked milk sample was tested. MAC culture was grown up on R2A Agar (Thermo Fisher Scientific, Adelaide, Australia) for 3 weeks at 37 °C. Cells were collected aseptically using a sterile loop and suspended in phosphate buffered saline (PBS: ThermoFisher Scientific, Victoria, Australia). Cells were then spun down (600 g) and resuspended in PBS to achieve an OD600 of 1 which is equivalent to approximately 107 CFU/ml. This was confirmed by spread plating dilutions on R2A Agar and incubating at 37 °C for 3 weeks prior to counting colonies. 3 µl of this suspension was then aseptically added to 29·97 ml of milk (to give a final concentration of 104 MAC CFU/ml milk) prior to DNA extraction.
DNA extraction
DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals Australasia, Sydney, NSW, Australia) following manufacturer's instructions giving a final volume of 100 µl of DNA extract. The soil extraction kit was chosen as it has been demonstrated to effectively remove PCR inhibitors (Dineen et al. Reference Dineen, Aranda, Anders and Robertson2010).
Real time PCR (qPCR)
MAC qPCR was then performed in triplicate as previously described (Whiley et al. Reference Whiley, Keegan, Fallowfield and Bentham2014). The primers used were MACF primer (5′-CCCTGAGACAACACTCGGTC-3′) and MACR primer (5′-ATTACACATTTCGATGAACGC-3′ (Park et al. Reference Park, Jang, Kim, Chung, Chang, Park and Song2000). Briefly, the 25 µl reaction volume contained 1 × PCR buffer (Invitrogen), 2·5 mm MgCl2 (Invitrogen), 2·5 mm SYTO9 fluorescent dye (Invitrogen), 0·2 mm deoxynucleoside triphosphate mix (Invitrogen), IU platinum Taq DNA polymerase (Invitrogen), 0·3 µm MACF primer, 0·3 µm MACR primer and 5 µl of template DNA. The cycling conditions included an initial hold at 95 °C for 5 min, followed by 45 cycles consisting of 94 °C for 15 s, 50 °C for 30 s, and 72 °C for 20 s (Whiley et al. Reference Whiley, Keegan, Fallowfield and Bentham2014).
All qPCR reactions were carried out in a RotorGene 3000 (Corbett Research, Sydney, Australia) with data acquisition at 72 °C on the 6-carboxyfluorescein channel (excitation at 470 nm, detection at 510 nm) at a gain of 5. Melt curve data was also acquired on this channel at gains of 2 and 5 using a ramping rate of 1 °C/60 s from 75 to 95 °C. Each qPCR run included a positive control and a non-template control of nuclease free water. For each reaction the melt curve was analysed and a positive MAC was confirmed with a melting temperature (Tm) of 88 ± 1 °C, 82·5 ± 1 °C, 85 ± 1 °C respectively. MAC qPCR limit of detection was 50 copies/reaction (Whiley et al. Reference Whiley, Keegan, Fallowfield and Bentham2014).
To determine the presence of environmental inhibitors in the extracted DNA, qPCR reactions were conducted in triplicate for both undiluted DNA extract and 1 : 10 dilution UltraPureTM DNase/RNase-Free Distilled Water (Invitrogen) of the same sample. If the cycle threshold (CT) value for the 1 : 10 dilution of DNA extract was less than approximately 3·3 (representing approximately 1-log10 concentration value) than the pure DNA extract then it was assumed that environmental inhibitors were present (Whiley et al. Reference Whiley, Keegan, Fallowfield and Bentham2014).
Results and discussion
20 different brands of commercially available pasteurised milk from around Australia were tested in triplicate for the presence of MAC using qPCR. This process was repeated three times throughout the year resulting in a total of 180 milk samples. All qPCR runs were performed in triplicate. All the milk samples were negative for MAC DNA. The limit of detection for the qPCR method described by Whiley et al. (Reference Whiley, Keegan, Fallowfield and Bentham2014) was 50 copies of DNA per reaction. There was no presence of environmental inhibitors in the milk and the spiked milk sample returned a positive result for MAC DNA demonstrating that the DNA extraction method was appropriate for detecting MAC from Milk.
To theoretically increase the concentration of any potentially viable MAC present in the milk samples they were incubated for 3 weeks. These were then tested using qPCR in triplicate and all were negative.
These results demonstrate that MAC was absent from the commercial milk tested or at concentrations lower than the limit of detection. A possible reason for this is the diluted factor that occurs in bulk tank milk. It was demonstrated by Slana et al. (Reference Slana, Liapi, Moravkova, Kralova and Pavlik2009) that milk collected from individual cows may test positive for MAP but due to the dilution factor and the limit of detection, the bulk tank milk will test negative. Alternatively, simply a larger sample size may have yielded a positive result. However, another explanation could be due to the strict quarantine regulations and Johne's disease surveillance programs in ruminant animals in Australia (Citer & Kennedy, Reference Citer and Kennedy2009).
Van Brandt et al. (Reference Van Brandt, Van der Plancken, De Block, Vlaemynck, Van Coillie, Herman and Hendrickx2011) and Grant et al. (Reference Grant, Ball and Rowe1998) have also experimentally demonstrated MAP ability to survive the milk pasteurisation process. However there has been limited research into the survival of other MAC organisms during the pasteurisation process. As all milk samples tested in this study were negative for MAC, it warrants further investigation to determine if this is representative of all Australian commercial milk and to determine if this can be attributed to Johne's disease surveillance programs reducing the incidence of MAP in raw milk.
Author Contributions
SA and TC performed laboratory work and wrote the manuscript. HW conceived the study and wrote the manuscript. All authors commented on manuscript drafts.
Conflicts of Interest
The authors declare no conflict of interest.
