Article Figures & Data
Figures
- FIG. 1.
Distribution of MPIL and AFLP fingerprints of M. avium subsp. paratuberculosis isolates by U.S. states and Ohio counties. The numbers in parentheses indicate the numbers of isolates with each specific fingerprint. Isolates analyzed by both fingerprinting techniques are hyphenated. On the Ohio county map, the first number indicates the M. avium subsp. paratuberculosis isolates available from those regions, a subset of which was fingerprinted. Five of the states—Iowa, Illinois, Indiana, Pennsylvania, and Florida—had more than one fingerprint. The superscript letters a to e indicate fingerprint combinations as follows: a, A1, A2, A5, A10, A15-Z2 (3), A18-Z1 (3), A18-Z2 (8), A18-Z21, A18 (17), and B1; b, A18-Z2 and A18 (2); c, A18-Z1 (2) and A18-Z2; d, A1, A12, A18-Z2, and A18; (4); and e, A3, A13-Z15, A14, A18-Z10, and A18 (3). Similarly, Ohio counties with more than one fingerprint are indicated by superscript letters as follows: f (Auglaize), A1 (2), A2 (2), A6, and A18-Z1; g (Hardin), A3, A18-Z2, and B3; h (Holmes), A1 (4), A2, A3, A6-Z2, A18-Z1, A18-Z2, A18 (2), and B4; i (Medina), A12, A18 (8), B1 (2), B2-Z2, and B4; and j (Columbiana), A2 (2), A6, A8, A18-Z1, A18-Z2 (3), A18-Z21, and A18 (2). Isolates not included in the map are strains from Nova Scotia, Argentina, and areas for which the source was not known, including ATCC 19851.
- FIG. 2.
Multiplex PCR of IS900 loci of M. avium subsp. paratuberculosis isolates selected from bovine (lanes 1, 4, 7, 10, and 13), ovine (lanes 2, 5, 8, 11, and 14), and human (lanes 3, 6, 9, 12, and 15) hosts. Lanes 1, 2, and 3 depict the IS900 integrations amplified from the right end of the insertion. The left-end integration sites were analyzed as separate sets termed as 4L1 (lanes 4 to 6), 4L2 (lanes 7 to 9), 3L3 (lanes 10 to 12), and 3L4 (lanes 13 to 15).
- FIG. 3.
MPIL cluster analysis by UPGMA. Cluster analysis of 247 isolates identified 90 ETs. Clusters at a proportion distance of <0.1 U were grouped together and assigned a fingerprint. Most of the bovine isolates clustered into a major group (A18) containing 154 isolates that included the caprine, murine, and deer isolates, indicating limited diversity. On the other hand, the sheep isolates were more polymorphic in that they were distributed in several nodes of the tree. Similarly, human isolates were distributed without clustering into any specific nodes. Unless specified, all isolates are M. avium subsp. paratuberculosis.
- FIG. 4.
AFLP cluster analysis. A total of 104 isolates including type strains M. avium subsp. paratuberculosis strain K-10 and M. avium subsp. avium strain 104 were fingerprinted by using AFLP and analyzed by UPGMA. Analysis separated the isolates into 25 different fingerprints, which were arbitrarily designated Z1, Z2, and Z4 to Z26. Isolates with identical fingerprints (100% match) were assigned the same fingerprint. Unless otherwise specified, all isolates are M. avium subsp. paratuberculosis. Majority of the bovine isolates, including the caprine and murine isolates, clustered into Z1 and Z2. One of the four ovine isolates analyzed clustered with the bovine isolates in Z2, while the other two were in close proximity to a New York bovine isolate, and the fourth ovine isolate fell on a distinct branch. One of the human isolates appeared in closer proximity to the node containing most of the animal M. avium subsp. paratuberculosis isolates (Z1 to Z9), whereas the other one had a diverse fingerprint. This illustrates the limited variation in bovine isolates from a variety of hosts and the high degree of diversity among the ovine and human isolates. The MAC isolates from CD patients and unknown hosts fell into distinct branches.
- FIG. 5.
Typical IS1311 PCR-REA patterns with undigested and HinfI-digested amplicons in adjacent lanes. Shown are bovine M. avium subsp. paratuberculosis (lanes 1 and 2), ovine M. avium subsp. paratuberculosis (lanes 3 and 4), and PCR-negative control (lanes 5 and 6) isolates and a bovine isolate with several mutations resulting in multiple restriction sites (lanes 7 and 8).
Tables
- TABLE 1.
Bacterial isolates used for analysis
Strain and source No. of isolates used in each analysis L1-L9 PCRa Locus 251 PCR hsp65 PCR-REA IS1311 PCR-REA MPIL AFLP Mycobacterium avium subsp. paratuberculosisa ATCC (700535, 19698, and 19851) 3 3 3 2 3 Cattle 254 71 105 78 165 72 Sheep 18 11 18 10 16 4 Goat 19 19 8 10 17 7 CD patients 7 7 7 7 7 2 Deer 1 1 1 1 1 Mouse 1 1 1 1 1 1 K-10 1 MAC strains TIGR 104 1 1 1 1 CD patients 31 15 30 24 20 10 Unknown host 53 45 53 30 17 6 Mycobacterium tuberculosis (human) 25 21 5 6 Mycobacterium bovis (cattle and deer) 9 3 2 2 Mycobacterium bovis BCG 4 3 3 3 Atypical mycobacteriab (ATCC) 6 6 6 6 Staphylococci 10 10 4 2 Salmonella enterica subsp. (cattle) 3 1 1 Escherichia coli (cattle) 20 20 4 4 Mannheimia hemolytica (cattle) 2 2 2 2 Uncharacterized nonmycobacteria (human and animal) 133 89 19 Total 600 328c 272c 190c 247c 104c ↵ a PCR amplification and hybridization for IS900 integration loci L1 (left-side integration site of IS900 into an unknown ORF) and L9 (left-side integration of IS900 into alkA).
↵ b Atypical mycobacteria include M. chelonae, M. xenopi, M. celatum, M. marinum, M. scrofulaceum, and M. smegmatis.
↵ c The numbers represent a convenient subset of isolates analyzed for L1-L9 integration loci.
