Paratuberculosis is a chronic infectious disease caused by Mycobacterium avium subsp. paratuberculosis (Map), an intracellular, slow-growing bacterial pathogen. Paratuberculosis mainly affects domestic and wild ruminants, is a generally sub-clinical condition, and characterized by a long incubation period. Clinically affected animals develop granulomatous enteritis, diarrhea, and loss of body weight, eventually leading to death or culling. The disease is present worldwide and considered a production-limiting factor in domestic ruminants [7]. Paratuberculosis decreases milk production as well as the growth and productive lifetime of cattle [14]. Additionally, an association between Map and Crohn's disease in humans has been suggested [13], raising the concern in the livestock industry of potential trade barriers or decreased consumption of animal products [7].

A diagnosis of paratuberculosis is based either on detection of the causative agent or immune response of the host. Tissue or fecal culturing is considered the most accurate diagnostic test available. However, the use of this diagnostic method has been hampered by costs that greatly exceed those of other diagnostic tests such an Enzyme-Linked ImmunoSorbent Assay (ELISA). One way to overcome the costs associated with fecal culturing for individual animals and minimizing the probability of false-positive results (at the herd level) is to pool fecal samples. The lack of a highly reliable diagnostic test for measuring Map infection is one of the most significant shortcomings that hinder paratuberculosis control [16].

Previously acquired evidence indicates that culture methods using liquid media have greater analytical and diagnostic sensitivity than counterpart techniques which utilize solid media [1,6]. In addition, bacterial growth can be detected sooner using liquid culture modalities [5,25]. However, confirmation of the organism is more difficult with liquid culture because the appearance of colonies and mycobactin-dependence are not observable, and the growth of other non-pathogenic mycobacteria needs to be identified. Nevertheless, once bacterial growth is detected in the broth tube, acid-fast staining, sub-culturing on solid media, or PCR are options for confirming the presence of Map in a sample. PCR represents a rapid and specific means of confirming Map in broth culture [3,12,15,23] and eliminates the need to visualize Map colonies.

Isolation and DNA purification are key steps for the majority of protocols in molecular biology [10]. For most mycobacteria species, the simplest way to obtain DNA from a mycobacterial suspension for PCR assays is boiling for 10 to 15 min in distilled water [19,21]. Herthnek et al. [9] reported that incubating a bacterial suspension at 99℃ or room temperature results in an insignificant DNA yield, suggesting the presence of free DNA. Moreover, free DNA is probably present in liquid culture suspensions as indicated by Sweeney et al. [20] who were able to detect Map organisms in liquid cultures by direct transfer of culture medium to PCR tubes. Extraction of genomic DNA from Map is challenging since this microorganism has one of the slowest growth rates among members of the genus Mycobacterium, and possesses a robust and waxy cell wall. These characteristics render Map cells difficult to lyse.

Published protocols for mycobacterial DNA preparation and commercially available extraction kits are available for PCR applications [9,22]. For these procedures, DNA for Map PCR testing is harvested by proteinase K digestion, phenol-chloroform extraction, and column purification using commercial kits. These procedures are deemed necessary for the release of DNA from mycobacterial cells and separating DNA from PCR inhibitors that are potentially contained in the culture media. Major disadvantages of methods for harvesting DNA from broth culture for subsequent real-time PCR confirmation of Map are high cost as well as substantial time and labor demands. Due to the lack of a simple protocol for extracting DNA from Map liquid cultures, the goal of the present study was to develop a simple and efficient Map DNA harvesting method based on mechanical Map cell disruption and ethanol DNA precipitation. This novel technique was compared to two established methods.

Out of 517 sampled animals (from 15 herds) and 104 fecal pools tested, 12 (12%) pools from eight (53%) herds were positive. The number of sampled animals and pools tested per herd are presented in Table 1. The three DNA extraction-purification protocols produced similar results. Only one significant difference between the three purification methods was observed based on the final PCR results. According to the QIAamp kit, sample 11 (H6P5) was negative while the other two protocols indicated that this sample was positive (Table 2). The cost per processed sample was lowest for the UACH method (Table 2).

In our study, we created a new DNA extraction method (UACH) for confirming the presence of Map with PCR. The goal of our investigation was to develop a simple technique with uncompromised analytical sensitivity and low cost. PCR and statistical analysis results suggested that the UACH method is a useful alternative for harvesting Map DNA from broth culture. This technique is cheaper and less complex than either of QIAamp and CTAB. In the literature, several DNA extraction methods for harvesting DNA from mycobacteria, including phenol extraction protocols, have been described but these procedures commonly involve multiple, time-consuming steps, including the handling of hazardous chemicals [10]. Genomic DNA extraction from Map cells is especially demanding since this microorganism is among the most extreme slow growers belonging to the genus Mycobacterium. Additionally, a robust and waxy cell wall renders Map cells difficult to lyse. Previous studies have evaluated and proposed improvements to methods for Map DNA extraction and purification from liquid cultures for PCR confirmation [12,15,20]. In these investigations, Map DNA was harvested using commercial kits and involved proteinase K digestion along with phenol-chloroform or column purification. These procedures were assumed to be necessary for the release of DNA from mycobacterial cells and separating DNA from PCR inhibitors that may be present in the culture media. In the present study, the DNA extraction protocols we evaluated produced similar results with the exception of sample H6P5 for which the QIAamp method rendered a negative result while those for CTAB and UACH were positive. This false-negative result from the QIAamp kit could most likely be attributed to the elusion column system. This kit was designed to obtain a highly purified DNA template. However, some DNA is lost during the purification process and may potentially affect PCR sensitivity.

It was our intention to assess the effect of the purification protocols on the accuracy of detecting Map infection in the field (southern Chile) rather than for a highly positive or clearly negative laboratory panel. We concede that the number of pools that were positive was relatively low and more robust results would require a larger sample size. However, similar results were produced by one of the standard methods (CTAB) and the one we created (UACH). Additionally, CP values of CTAB and UACH protocols were significantly different from those of the commercial QIAamp kit. This finding could be explained by a lower yield but recovery of more pure DNA when the QIAamp protocol was used. Furthermore, the QIAamp kit is associated with minimal PCR inhibition. Egg yolk is one of the components of the culture media that inhibits PCR [20]. It was removed by centrifuging the samples as proposed by Whittington et al. [24].

Evidence gathered in this study suggests that the UACH protocol represents a better technique than the two reference methods based on similar results and lower cost. This finding coincides with a study in which a protocol for extracting Mycobacterium ulcerans DNA was optimized [11]. For this procedure, a combination of mechanical disruption along with chemical solubilization of the waxy lipid and mycolic acid-containing cell wall was performed to effectively release the DNA. Once the balance between mechanical disruption and chemical solubilization was altered, the DNA quantity and/or quality were compromised.

The key step in the protocol established in the current study is bead-beating that allows a large amount of DNA to be harvested from the bacteria. Purification steps (column-based or chloroform extraction) performed for the QIAamp and CTAB methods are associated with more pure DNA suitable for Map strain-typing methods [4,18]. Bead-beating has been previously performed to lyse Map cells using small zirconia beads [17]. Importantly, pre-treatment with a proteolytic enzyme and lysis buffer helps break tough cell walls with forceful shaking in a cell disrupter (bead-beater). We believe that bead-beating as a means of mechanical disruption along with simple purification and precipitation with ethanol was enough to obtain a higher DNA yield via the UACH method indicated by a lower CP value. The proposed method involved mechanical disruption of the bacterial wall as well as the use of ethanol for purifying and washing the DNA with no need for special or expensive equipment. The technique we have developed will allow diagnostic laboratories to easily extract DNA and improve the ability to accurately detect Map infection.