The strains of the MAI complex used for this study were all characterized for the presence or absence of IS900 and IS901 and are given in Table 1. All strains of M. paratuberculosis were characterized as sheep or cattle types on the basis of their RFLPs with IS900. Strains were cultured with standard mycobacterial media (3). Purified DNA was extracted as described previously (4). When DNA from strains of each type was subjected to PCR at an annealing temperature of 50°C with primers directed outward from each end of IS900, only DNA from sheep types gave a product between 300 and 400 bp. Subsequently, it was observed that the same 342-bp product was obtained if only one PCR primer (DMC136, Fig. ​Fig.1)1) was used. The PCR product was cloned into pBluescript KS II (Stratagene) and sequenced. Comparison of this sequence to the homologous region of a cattle type of M. paratuberculosis (National Center for Biotechnology Information database [http://www.ncbi.nlm.nih.gov/]) with BLAST revealed that the cattle type was not homologous to DMC136 at the 5′ end. Further investigation with a range of PCR primers (data not shown) indicated that the two types have similar sequences at the 3′ end but that only the sheep type has an IS900 site at the 5′ end. The 342-bp product was thus a result of DMC136 hybridizing perfectly to the terminal part of IS900 that comprises the 5′ end of the sheep sequence in Fig. ​Fig.11 and partially to the last 10 nucleotides at the 3′ end of this sequence. DNA from the cattle type has a similar sequence at the 3′ end, but it gives no product with DMC136 because it lacks a sequence matching DMC136 at the 5′ end. The site of insertion of IS900 is just downstream of the start codon for a putative gene on the complementary strand shown in lowercase letters in Fig. ​Fig.1.1. This gene has high homology to a Mycobacterium smegmatis gene whose product is involved in phage attachment (1). The other major difference in sequence between the two types is that the sheep type has a tandem repeat of a 12-bp sequence followed by a 4-bp linker, which together contain a 14-bp palindromic sequence (Fig. ​(Fig.1).1). Such sequences are commonly found in bacterial chromosomes, often as part of a promoter region (8), a terminator (15), or a phage attachment site (12). Analysis of the sequence around the tandem repeat site showed that it is inserted at the stop codon of a hypothetical gene in the cattle type that encodes a putative protein of 97 amino acids with no good homology to other proteins. This hypothetical gene is interrupted by insertion of IS900 in the sheep type, but a truncated version of it might be produced, as there are other putative start codons. The tandem repeat is just 19 bp before an open reading frame encoding a putative protein of 134 amino acids with good homology to two hypothetical proteins of Mycobacterium tuberculosis and a hypothetical protein encoded by a small Yersinia pestis virulence plasmid. The palindromic sequence might therefore be involved in controlling gene expression of this second gene, either by limiting read-through from the truncated first gene to the second gene or by acting as part of an independent promoter region for the second gene. These sequence differences between sheep and cattle types may therefore be important in determining the host preference of the two types.

A PCR assay was developed with a GeneAmp PCR System 9600 (Applied Biosystems) and the three primers DMC529, DMC531, and DMC533 (Fig. ​(Fig.1)1) under the following conditions: 1 cycle at 95°C for 3 min; 25 cycles at 60°C for 30 s, 72°C for 30 s, and 94°C for 30 s; and 1 cycle at 72°C for 7 min. DNA from all strains of the cattle type gave the expected product of 310 bp, while DNA from all strains of the sheep type gave the expected product of 162 bp (Fig. ​(Fig.2).2). A PCR product was not observed for any of the wide range of strains of the MAI complex (Table 1) that did not contain IS900 and were not M. paratuberculosis. The PCR was also negative for two isolates of Mycobacterium bovis and an isolate of M. tuberculosis.