MLVA 16 fingerprinting
The genetic diversity among B. melitensis isolates (n=41) using HGDI values, was estimated for each MLVA-16 locus subsets (Table1). Based on the HGDI classification by Sola et al. (2013), the markers of Panel 1 were monomorphic displayed single alleles in all B. melitensis isolates (n=41) under the field of this study with no discrimination and HGDI (0). Similarly, Bruce19 and Bruce 21 of panel 2 A displayed the same as Panel1 except for Bruce18 that showed different copy numbers of the tandem repeats with poor discrimination (HGDI=0.109). On the contrary, Bruce04 and Bruce16 of panel 2 B were highly discriminatory in B. melitensis (HGDI > 0.7) while, Bruce07 and Bruce09 of panel 2 B were poorly discriminatory. The remaining locus (Bruce30) exhibited only a single allele with no discrimination.
MLVA-16 analysis of the 41 B. melitensis isolates (Figure 1) from Kafrelsheikh governorate showed 19 different genotypes with 9 singletons (unique) genotypes. MLVA-16 data analysis illustrates highly consistent results among the local B. melitensis strains. All these genotypes along with the B. melitensis bv3 reference strain (Ether) were clustered together into one cluster with a genetic similarity of approximately 97% cut-off value. The nine singleton genotypes were isolated from animals belonging to groups E11(M1_K.E), E5 (M4_K.E), two genotypes (M6_K.E and M7_K.E) of G(6), C1 (M8_K.E), G7 (M12_K.E), E7 (M19_K.E), E8 (M15_K.E, and 88 (M11_K.E). M17_K.E is the most common genotype (eight identical strains), followed by M16_K.E (five identical strains), M10_K.E (four identical strains) and, M9_K.E (three identical strains). Complete data of the performed MLVA-16 analysis is shown in (Table 2) and is also available on the MLVAdatabase, “http://microbesgenotyping.i2bc.paris-saclay.fr”.
Interestingly, there were different genotypes isolated from samples of the same animal; three genotypes were isolated from each animal E(3), E(7), and E(8), and two genotypes were isolated from each animal G(6), E(2), E(4), E(5), E(6), and E(11). Among the shared B. melitensisgenotypes (n=10), three genotypes were common between two different animal species. Two of these shared genotypes belonged to different herds (M5_K.E and M13_K.E) while the remaining one belonged to the same herd (M2_K.E). Moreover, these shared genotypes were M2_K.E (cow; NED17/14919 and ewe; NED17/14920), M5_K.E (ewe; NED17/14930 and buffalo; NED17/14940), and M13_K.E (cow; NED17/14914 and ewe; NED17/14947).