3.3. Genotyping

The availability of standardized techniques that allow the discrimination of different P. larvae strains is essential for studying the epidemiology of AFB. This will allow scientists to identify outbreaks of the disease, determine the source of infection, determine the relationship between outbreaks, recognize more virulent strains, and monitor prevention and treatment strategies.

To date different techniques have been used in order to evaluate the diversity of P. larvae isolates. Some of them are based on the analysis of phenotypic characteristics, such as study of cell and colony morphology, analysis of whole bacterial proteins by SDS-PAGE or biochemical profile, among others (Hornitzky and Djordevic, 1992; Neuendorf et al., 2004; de Graaf et al., 2006a; Genersch et al., 2006; Antúnez et al., 2007). During the last decade methods based on genetic analysis have gained more attention. Different strategies have been used to evaluate the genetic diversity of P. larvae, including restriction endonuclease fragment patterns (Djordjevic et al., 1994; Alippi et al., 2002), pulsed-field gel electrophoresis (Wu et al., 2005; Genersch et al., 2006), amplified fragment length polymorphism (de Graaf et al., 2006b), ribotyping and denaturing gradient gel electrophoresis (Antúnez et al., 2007). Nevertheless, some of these techniques differentiating P. larvae genotypes, have not been adopted by the scientific community.

An appropriate genotyping method should be highly discriminatory, but also demonstrate interlaboratory and intralaboratory reproducibility. It should be easy to use and interpret (Genersch and Otten, 2003). For these reasons, the most utilized method is rep-PCR, or PCR amplification of repetitive elements (Versalovic et al., 1994), although presently its reproducibility in other labs has not been proven. There are three sets of repetitive elements randomly dispersed in the genome of bacteria, enterobacterial repetitive intergenic consensus (ERIC) sequences, repetitive extragenic palindromic (REP) elements, and BOX elements (which includes boxA, boxB, and boxC). Primers to amplify those elements have been reported and proved to be useful for subtyping of Gram positive and Gram negative bacteria (Versalovic et al., 1994; Olive and Bean, 1999).

rep-PCR has been widely used for the study of P. larvae (Alippi and Aguilar, 1998a, 1998b; Genersch and Otten, 2003; Alippi et al., 2004; Antúnez et al., 2007; Peters et al., 2006; Loncaric et al., 2009). The most useful pair of primers are ERIC1R-ERIC2, which allowed the differentiation of four different genotypes (ERIC I, II, III and IV) (Genersch et al., 2006). Genotypes ERIC I and II corresponds to the former subspecies P. l. larvae while genotypes ERIC III and IV corresponds to the former subspecies P. l. pulvifaciens (Genersch, 2010).

P. larvae genotype ERIC I is the most frequent genotype and is present in Europe and in America, genotype ERIC II seems to be restricted to Europe and genotypes ERIC III and IV have not been identified in field for decades, but exist as few isolates in culture collections (Genersch, 2010). In order to enhance the discrimination of strains, the analysis using ERIC primers can be complemented with the use of other primers. The use of BOXA1R primer allowed the discrimination of four banding patterns in America, all of them belonging to genotype ERIC I (Alippi et al., 2004; Antúnez et al., 2007) and three in Europe (Genersch and Otten, 2003; Peters et al., 2006; Loncaric et al., 2009). Primers BOX B1 and BOX C1 did not amplify P. larvae DNA (Genersch and Otten, 2003). When REP primers were used, four banding patterns were found in America and Europe although results could not be compared since different pairs of primers (REP1R-I and REP2-I and MBO REP1 primers) were used (Alippi et al., 2004; Kilwinski et al., 2004; Loncaric et al., 2009). Protocols for subtyping of P. larvae are provided below.

Restriction fragment length polymorphic (RFLP) analysis of bacterial genomes, as visualized via pulsed-field gel electrophoresis (PFGE), is also a very effective procedure for bacterial genotyping (PFGE-typing). PFGE-typing of 44 P. larvae isolates, obtained from honey bee larval smears and honey samples collected in Australia and from Argentinean honey, has demonstrated resolution of this bacterium into 12 distinct genotypes when using restriction endonuclease XbaI (Wu et al., 2005). Outlined below is a PFGE-typing procedure for P. larvae.  This procedure is presented as a three-part operation of genomic DNA preparation, restriction digestion of DNA, and then electrophoresis of the digested DNA.  Performance of PFGE-typing is labour intensive. Also, many factors can contribute to an unsuccessful electrophoresis run. Therefore, troubleshooting PFGE and helpful hints for performing this technique can be found in the protocols section of the Bio-Rad website (http://www.bio-rad.com/evportal/en/US/LSR/Solutions/LUSORPDFX/Pulsed-Field-Gel-Electrophoresis).

3.3.2. Pulsed-field gel electrophoresis