3. RESULTS
3.3. Genotyping of MAP from South American countries
3.3.3. Combined analysis of MIRU–VNTR and MLSSR
Combined analysis of MIRU–VNTR and MLSSR revealed 9 different genotypes of MAP.
The genotype with the combination 1–A was the most common (n=14), followed by the combined genotype 1–B (n=5, Table 25).
Table 27. MLSSR genotypes of MAP from South American countries Genotype Number of repeats at SSR locus
1 (G–repeat) 2 (G–repeat) 8 (GGT–repeat)
A 7 10 4
B 7 11 4
C 7 >11 4
D 7 10 5
Ea 7 11 5
Fa >11 11 5
Ga 11 10 5
a Genotypes taken from the publication of Thibault et al. (2008).
The genotype 1–B was detected in two MAP isolates (cattle isolate 175 and hare isolate 79) from one property in Chile. From 6 Argentinean and from 1 Venezuelan isolates a combined genotype could not be determined due to the absence of MLSSR results in previous studies (Table 25).
3.4. Molecular characterization of MAP in Rhineland–Palatinate (RP), Germany
3.4.1. MIRU–VNTR
MIRU–VNTR analysis produced 11 different genotypes among 91 isolates analyzed (Table 28).
Table 28. Results of genotyping of MAP from dairy cattle in Germany and Luxembourg using MLSSR and MIRU–VNTR
Countiesa MIRU–
VNTRb Genotype
MLSSRc Genotype
Combined Genotype
Number of isolates
%
WIL, BIT, KUS, SIM, EMS, AC 1 A 1–A 8 8.8
AK, BIR, BIT, MYK, MZG, MZG, EMS, TR, TR
1 B 1–B 14 15.4
BIT 1 C 1–C 4 4,4
WIL, COC, BIT, GER, MZG 1 D 1–D 14 15.4
AK, KUS, NW, WW 2 A 2–A 4 4.4
KIB, BIT, SIM 2 B 2–B 6 6.6
BIT, LU, SIM, TR, DAU, WW 2 C 2–C 9 9.9
BIT 2 D 2–D 2 2.2
BIT, TR 2 E 2–E 3 3.3
TR 2 F 2–F 1 1.1
BIR, BIT 3 A 3–A 3 3.3
TR 3 B 3–B 1 1.1
BIT 3 C 3–C 3 3.3
AK, KUS, NW, PS 4 A 4–A 4 4.4
KIB, BIT 4 C 4–C 2 2.2
BIT, RÜD 5 A 5–A 3 3.3
COC 5 C 5–C 1 1.1
BIR, BIT 6 A 6–A 2 2.2
WIL 6 B 6–B 1 1.1
TR 6 C 6–C 1 1.1
PS 7 B 7–B 1 1.1
DAU 8 D 8–D 1 1.1
WW 9 D 9–D 1 1.1
Table 28. continued
NK 10 C 10–C 1 1.1
BIT 11 C 11–C 1 1.1
Total 11 6 25 91 100
Index of discrimination (D) d 0.74 0.78 0.93
C.I. 95% for De 0.66–0.81 0.76–0.80 0.91–0.95
a Counties: AC Städteregion Aachen (North Rhine–Westphalia), AK Altenkirchen-Westerwald, BIR Birkenfeld, BIT Eifelkreis Bitburg–Prüm, COC Cochem–Zell, DAU Vulkaneifel, EMS Rhein–Lahn–Kreis, GER Germersheim, KIB Donnersbergkreis, KUS Kusel, LU Luxembourg, MYK Mayen–Koblenz, MZG Merzig–
Wadern (Saarland), NK Neunkirchen (Saarland), NW Neuwied, PS Südwestpfalz, RÜD Rheingau–Taunus–
Kreis (Hesse), SIM Rhein–Hunsrück–Kreis, TR Trier–Saarburg, WIL Bernkastel–Wittlich, WW Westerwaldkreis.
b Performed according to Bull et al. (2003), Overduin et al. (2004), Thibault et al. (2007), Castellanos et al.
(2010).
c Performed according to Amonsin et al. (2004).
d Calculated for isolates without epidemiological relationship according to Hunter and Gaston (1988).
e Calculated according to Grundmann et al. (2001).
MIRU–VNTR genotypes were identified with numbers from 1 to 11 (Table 29). MIRU–
VNTR analysis revealed that the most common genotypes detected in the panel of MAP isolates were genotype 1 (INMV1) and genotype 2 (INMV 2), representing 44% (40/91) and 27.5% (25/91) of all isolates, respectively (Table 29). The MIRU–VNTR genotypes 5, 10, and 11 have not been assigned the INMV codification system or had not been detected before (Table 29).
Table 29. MIRU–VNTR genotypes obtained from MAP isolates in RP (Germany) MIRU–
VNTR genotype a
No. of isolates
% Number of copies of MIRU–VNTR INMV
Profileb 1 292 1658 25 47 3 7 10 32 259
1 40 44.0 3 4 2 3 3 2 2 2 8 2 1
2 25 27.5 3 3 2 3 3 2 2 2 8 2 2
3 7 7.7 3 3 2 5 2 2 2 2 8 2 33
4 6 6.6 3 4 2 3 3 2 1 2 8 2 19
5 4 4.4 3 4 3 3 3 2 1 2 8 2 Undet.
6 4 4.4 3 3 2 3 3 2 1 2 8 2 6
7 1 1.1 3 4 2 3 3 2 2 1 8 2 5
8 1 1.1 3 3 2 3 3 2 2 2 9 2 4
9 1 1.1 3 3 2 3 3 2 2 1 8 2 3
10 1 1.1 3 2 2 5 2 2 1 2 8 2 Undet.
11 1 1.1 3 2 2 5 2 2 2 2 6 2 Undet.
a Performed according to Bull et al. (2003); Overduin et al. (2004); Thibault et al. (2007); Castellanos et al., (2010).
b INMV profile based on loci MIRU–VNTR 292–1658 (alias X3)–25–47–3–7–10–32 (shaded area) according to Thibault et al. (2007)
Undet. Undetermined
The calculated numerical index of discrimination (D) for the MIRU–VNTR was 0.74 (95%
CI: 0.66–0.81, Table 28). Seven MIRU–VNTR loci (292, 1658, 25, 47, 7, 10, and 32) out of 10 loci analyzed presented allelic variation. No isolate produced allelic variation by MIRU–1, MIRU–VNTR 3 and MIRU–VNTR 259. The calculation of the allelic diversity (h) for individual MIRU–VNTR loci revealed differences ranging from 0.03 (MIRU–VNTR 32) to 0.50 (MIRU–VNTR 292, Table 31).
3.4.2. MLSSR
MLSSR were analyzed in the same panel of isolates genotyped by MIRU–VNTR. MLSSR analysis detected six different genotypes among the 91 isolates analyzed (Table 27). MLSSR genotypes were identified with letters from A to F (Table 30). The genotype A (>11g–4ggt) was the most common MLSSR genotype found, representing 26.4% (24/91) of all MAP isolates (Table 30).
Table 30. MLSSR genotypes obtained from MAP isolates in RP (Germany)
MLSSR Genotypea No. of isolates % Number of copies of SSR Loci 2 (g) 8 (ggt)
A 24 26.4 >11 4
B 23 25.3 11 4
C 22 24.2 10 4
D 18 19.8 9 4
E 3 3.3 10 5
F 1 1.1 9 5
a Performed according to Amonsin et al. (2004).
The calculated numerical index of discrimination (D) for MLSSR was 0.78 (95% CI: 0.76–
0.80, Table 28). The two SSR–loci analyzed produced allelic variation. Locus 2 presented the highest allelic variation with four alleles, while locus 8 presented two alleles. The allelic diversity (h) was 0.03 for locus 8 and 0.75 for locus 2 (Table 31).
Table 31. MIRU–VNTR and MLSSR allelic distribution of MAP in RP (Germany)
Locus No. of isolates with the allele Allelic
diversity (h)a 0 1 2 3 4 5 6 7 8 9 10 11 >11
MIRU–VNTR 292 2 38 51 0.50
7 15 76 0.29
47 9 82 0.11
10 2 89 0.11
25 82 9 0.10
1658 87 4 0.08
32 1 89 1 0.03
MLSSR 2 19 25 23 24 0.75
8 87 4 0.03
a Calculated only for isolates without epidemiological relationship according to Selander et al. (1986).
3.4.3. Subdivision of MIRU–VNTR and MLSSR genotypes
Six MIRU–VNTR genotypes (genotypes 1 to 6) were subdivided by MLSSR. Conversely four MLSSR genotypes (A, B, C, and D) were subdivided by MIRU–VNTR (Table 32). The most common MIRU–VNTR genotypes 1 (INMV 1) and 2 (INMV 2) were subdivided by
MLSSR into four and six different MLSSR–genotypes, respectively. The most common MLSSR genotype A was divided by MIRU–VNTR into six different genotypes, while MLSSR genotype B, C, and D were subdivided into five, eight, and four MIRU–VNTR genotypes, respectively (Table 32). Adjusted Rand index was 0.054. The Wallace coefficient (W) for MIRU–VNTR → MLSSR, and for MLSSR →MIRU–VNTR was 0.261 (95% C.I.
0.20–0.312) and 0.321 (95% C.I. 0.239–0.403), respectively. The expected W value if the classifications are independent (Wi) was 0.275 and 0.224 for MIRU–VNTR and MLSSR, respectively (Table 32).
Table 32. Subdivision of genotypes obtained by MIRU–VNTR and MLSSR in RP (Germany)
Genotype No of isolates Subdivided by
MIRU–VNTR MLSSR
1 40 A, B, C, D
2 25 A, B, C, D, E, F
3 5 A, B, C
4 6 A, C
5 4 A, C
6 4 A, B, C
Wia 0.275
Wa 0.261
C.I. 95% of Wa 0.20–0.312
A 24 1, 2, 3, 4, 5, 6
B 23 1, 2, 3, 6, 7
C 22 1, 2, 3, 4, 5, 6, 10, 11
D 18 1, 2, 8, 9
Wi 0.224
W 0.321
C.I. 95% of W 0.239–0.403
a The Wallace coefficient (W), the 95% C.I., and the expected W value if the classifications are independent (Wi) were calculated according to Pinto et al. (2008)
3.4.4. Combined analysis of MIRU–VNTR and MLSSR
Combined analysis of MIRU–VNTR and MLSSR produced 25 genotypes among the 91 isolates analyzed (Table 28). The combined genotypes of every isolate were expressed as the combination of the numbers and letters of MIRU–VNTR and MLSSR results, respectively.
The calculated numerical index of discrimination (D) for the combined methods was 0.93
(95% CI: 0.91–0.95, Table 28). Combined genotypes 1–B and 1–D were detected in 14 out of 91 isolates (15.4%) each, being the most detected genotypes among the isolates analyzed.
Genotypes 2–C (9.9%) and 1–A (8.8%) were detected in nine and eight isolates, respectively Remaining isolates were found in six or fewer isolates (Table 28).
3.4.5. Molecular epidemiology of MAP in Rhineland–Palatinate
Results of combined MIRU–VNTR and MLSSR were used to analyze the molecular epidemiology of MAP in Rhineland–Palatinate, as only both methods combined achieved a minimum discriminatory ability needed for epidemiological studies (D ≥0.90) but not reached by application of each method independently. Diverse combined genotypes were widely distributed on the counties of the federal state of Rhineland–Palatinate, Luxembourg, and in isolates of the neighboring German federal states of Saarland, Nordrhein–Westphalia and Hesse (Table 28). Although in some counties of Rhineland–Palatinate fewer isolates were obtained, probably related to a lower concentration of cattle reporting clinical cases of paratuberculosis along time, diverse combined genotypes were also detected (Table 28). In RP, all genotypes excepting genotype 10-C were found in isolates of RP (Table 28).
The distribution of MAP genotypes in RP correlates with the cattle population density in the counties (Anonymous, 2011b). The three counties with the highest cattle population (BIT, DAU, and WILL) concentrate 64% of all genotypes detected in the whole study. However, some genotypes detected in low–populated counties were not detected in the most populated.
For example, genotypes 2–A and 4–A were detected in the counties Altenkirchen (Westerwald, AK), Neuwied (NW), Kusel (KUS), Westerwaldkreis (WW) and Südwestpfalz (PS), but were not detected in Eifelkreis Bitburg–Prüm (BIT, Table 33).
Table 33. Distribution of MAP genotypes in the counties of RP, Germany Countya Cattle
population
Communes Herds Isolates Genotypes
BIT 99.162 27 32 42 1-A, 1-B, 1-C, 1-D,
2-B, 2-C, 2-D, 2-E, 3-A, 3-C, 4-C, 5-A, 5-A, 6-A, 8-D, 11-C
DAU 31.366 1 1 1 2-C
WIL 24.474 3 4 4 1-A, 6-B, 1-D
WW 24.154 3 3 3 9-D, 2-C, 2-A
TR 21.080 9 9 9 E, 1-B, 6-C, F,
2-C, 3-B
AK 19.115 3 3 3 4-A, 1-B, 2-A
SIM 16.777 3 3 3 2-C, 2-B, 1-A
PS 15.135 2 2 2 7-B, 4-A
NW 13.497 2 2 2 2-A, 4-A
BIR 13.383 3 3 4 3-A, 1-B, 6-A
KUS 12.769 3 3 3 2-A, 1-A, 4-A
EMS 11.950 2 2 2 1-A, 1-B
MYK 9.439 1 1 1 1-B
COC 8.202 1 1 1 1-D
KIB 7.035 2 2 2 4-C, 2-B
Total 329.182 65 71 82 25
a Counties: AK Altenkirchen-Westerwald, BIR Birkenfeld, BIT Eifelkreis Bitburg–Prüm, COC Cochem–Zell, DAU Vulkaneifel, EMS Rhein–Lahn–Kreis, GER Germersheim, KIB Donnersbergkreis, KUS Kusel, MYK Mayen–Koblenz, NW Neuwied, PS Südwestpfalz, SIM Rhein–
Hunsrück–Kreis, TR Trier–Saarburg, WIL Bernkastel–Wittlich, WW Westerwaldkreis.
Eight isolates from Saarland, North Rhine–Westphalia, and Luxembourg produced the common genotypes 1–A, 1–B, 1–D and 2–C, while two isolates from Hesse (county Rheingau–Taunus–Kreis, RÜD) and Saarland (county Neunkirchen, NK) produced the two uncommon genotypes 5–A and 10–C, respectively. Genotypes 5–A was also found in other herds of Rhineland–Palatinate, while genotype 10–C was only found in the isolate from Saarland (Table 28).