Componentes del Control Interno COSO III

Despite the recent advances in understanding particular virulence mechanisms of D. nodosus (Kennan, et al., 2001; Han, et al., 2007, Parker, et al., 2006; Myers, et al., 2007; Han, et al., 2008; Kennan, et al., 2010) and in determining effective treatments (Wassink, et al., 2003; 2004; Green, et al., 2007; Kaler and Green, 2009;Wassink, et al., 2010b), the precise series of microbial succession events, between D. nodosus and F. necrophorum, leading to the initiation and development of ID and FR have not been determined, despite some recent advances (Calvo-Bado, et al., 2011b). In addition, the distribution of D. nodosus and F. necrophorum in the environment has yet to be determined.

A number of studies have detected D. nodosus and F. necrophorum in relation to disease presentation, however these studies were cross-sectional, non-quantitative and some used culture-dependent techniques, despite the fastidious nature of D. nodosus and F. necrophorum (Moore, et al., 2005a; Wani, et al., 2007; Bennett, et al., 2009). In addition, culture-dependent techniques have been shown to be less sensitive than PCR-

based methods for the detection of D. nodosus from ovine foot swabs (Moore, et al., 2005a). A number of reports have highlighted the difficulties associated with studying microorganisms in their natural environments, due to limited bacterial morphologies and the inability to grow certain microorganisms in a laboratory environment (Prosser, et al., 2007; Rogers, et al., 2009). In light of this, a series of culture-independent techniques were selected for the current study.

1.10.1. Culture-independent techniques; quantitative real-time PCR (qPCR).

Real-time quantitative PCR (qPCR) is a culture-independent, specific, sensitive and reproducible method for the detection and quantification of nucleic acids. It has revolutionised a number of fields, including molecular diagnostics and microbial ecology, because of its high-throughput and automated nature (Arya, et al., 2005). It has been used for a variety of clinical and environmental studies and for a wide range of sample types, including; soil, faeces, milk, water, tissue and swabs (Nogva, et al., 2000; Buttner, et al., 2001; Fujita, et al., 2002; Stelzel, et al., 2002; Kawaji, et al., 2011; Fredericks, et al., 2009;Pontiroli, et al., 2011;Calvo-Bado, et al., 2011b). It is a highly specific and sensitive approach and has been shown to be more sensitive than culture when detecting bacteria (Saukkoriipi, et al., 2003; Moore, et al., 2005a; Rampersad, et al., 2008). In addition, bacterial and viral loads have been shown to correlate temporally with disease development and presentation (Hill, et al., 2000; Hackett, et al., 2002; Dormans, et al., 2004; Sha, et al., 2005; Smith-Vaughan, et al., 2006). Despite this, bacterial DNA-based detection methods, such as qPCR, may overestimate bacterial load, by quantifying dead/membrane-compromised or dormant bacterial cells (Castillo, et al., 2006; Pathak, et al., 2012).

1.10.2. Culture-independent techniques; fluorescence in situ hybridisation (FISH). FISH is another culture-independent method for the in situ detection, identification, quantification and phylogenetic analysis of microbes. It was first introduced in the late 1980s and since then it has become one of the most widely used methods for examining microbial community composition in situ (Amann, et al., 1990; Amann and Fuchs 2008). A number of adaptations to FISH have been introduced, some using different gene targets; double labelling of oligonucleotide probes (DOPE-FISH) (Stoecker, et al., 2010), 16S rRNA gene clones (clone-FISH) (Schramm, et al., 2002), peptic nucleic acid probes (PNA-FISH) (Perry-O’Keefe, et al., 2001), combining microautoradiography with FISH (MAR-FISH) (Chua, et al., 2006) and using catalysed reporter deposition (CARD-FISH) (Eickhorst and Tippkötter, 2008), demonstrating the many applications of this method.

Despite this, the majority of FISH studies continue to target ribosomal RNA (rRNA) with fluorescently labelled oligonucleotide probes (Amann and Fuchs, 2008). The rRNA molecules are ideal targets because (i) each bacterial target cell contains many ribosomes and so are naturally amplified, (ii) they are more evolutionary conserved and (iii) by using rRNA as the target, one can link microbial ecology with microbial evolution. Using this method, bacterial classification is no longer based on morphology or physiology. In contrast to qPCR, FISH may provide information on the physiological state of microorganisms, because rRNA content has been shown to be directly correlated with growth rate (Kemp and LaRoche, 1993; Wallner, et al., 1993; Poulsen, et al., 1993), providing an advantage over qPCR.

Sample

Fixation

Hybridisation Fixed cells are

permeabilised

Fluorescently tagged target cell Fluorescently labelled oligonucleotide (probes) Visualisation and quantification 1. Epifluorescence microscopy 2. CLSM 3. Flow cytometry Washing Hybridised cells Target (ribosomal RNA)

Probe

The FISH procedure for bacterial targets consists of three basic steps (Figure 1.4.); cell

fixation, whereby cells are penetrated with a fixative (such as paraformaldehyde or

ethanol), which retains cells morphology and structure and also permeabilises the cell

membranes. Permeabilisation allows the labelled oligonucleotide probe to diffuse and

bind to its intracellular targets during hybridisation (usually 1-4 hours), the specific

hybrid formed is a heteroduplex (rRNA:DNA). Excess or unbound probe is then washed

away and labelled cells are visualised or quantified by a variety of methods (Amann and

Fuchs, 2008).

Figure 1.4. Outline of the core steps involved in fluorescence in situ hybridisation (FISH). The steps include fixation, permeabilisation, hybridisation, washing and visualisation. Modified from Figure 1 from Amann and Fuchs (2008).

FISH has been employed for a variety of clinical and environmental studies and has

been used on a number of samples types; faecal material, mammalian and plant tissue,

Ainsworth, et al., 2006; Kutter, et al., 2006; Boye, et al., 2006; Lefmann, et al., 2006 Klitgaard, et al., 2008; Whiley, et al., 2011), demonstrating again a broad range of applications. It is therefore suitable for the in situ detection, identification, localisation and quantification of bacterial species and complements the use of a DNA-based detection method, such as qPCR.