ARS | Publication request: DIFFERENTIATION OF SERPULINA SPECIES BY NADH OXIDASE GENE (NOX) SEQUENCE COMPARISONS AND NOX-BASED POLYMERASE CHAIN REACTION TESTS

Submitted to: Veterinary Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 22, 1999
Publication Date: N/A

Interpretive Summary: Helical-shaped bacteria, called spirochetes, cause intestinal diseases in pigs, chickens, and possibly, in humans. The worldwide disease swine dysentery caused by an intestinal spirochete is estimated to cost the U.S. pork industry $100 million per year. The names of two of these bacteria are Serpulina hyodysenteriae and Serpulina pilosicoli. These bacteria are difficult to culture in the laboratory and it is difficult to rapidly and accurately identify these organisms in a veterinary diagnostic laboratory. Consequently, we searched for some difference between the various Serpulina species that could be used to develop a rapid identification test. We found that the different Serpulina species have a different DNA sequence for one of their genes. That gene is called NADH oxidase and can be abbreviated as "nox". We found that a molecular test (polymerase chain reaction) could be used to identify the nox gene of each species. The identification of the particular nox gene tells us the identity of the Serpulina species that contains it. These findings are important because they provide an easy, rapid method for identifying intestinal spirochetes in the veterinary diagnostic laboratory.

Technical Abstract: The NADH oxidase genes (nox) of 18 strains of intestinal spirochetes were partially sequenced (1246 bases). Strains examined included 17 representatives from six species of the genus Serpulina, and the type strain 513AT of the human intestinal spirochaete Brachyspira aalborgi. A phenogram produced based on sequence dissimilarities was in good agreement with the current classification of species in the genus Serpulina, although an atypical strongly beta-hemolytic porcine strain (P280/1), previously thought to be S. innocens, appeared distinct from other members of this species. Primer pairs were developed from the nox sequence alignments for use in polymerase chain reaction (PCR) identification of the pathogenic species S. hyodysenteriae (NOX1), S. intermedia (NOX2), and S. pilosicoli (NOX3), and for the combined non- pathogenic species S. innocens and S. murdochii (NOX4). The PCRs were optimized using 80 strains representing all currently described species in the genus Serpulina, as well as the type strain of B. aalborgi. Tests NOX1 and NOX4 specifically amplified DNA from all members of their respective target species, whilst tests NOX2 and NOX3 were less sensitive. NOX2 amplified DNA from all 10 strains of S.intermedia from pigs but from only 4 of 10 strains from chickens, whilst NOX3 amplified DNA from only 18 of 21S. pilosicoli strains, even at low stringency. Tests NOX1 and NOX4 should prove useful in veterinary diagnostic laboratories whilst NOX2 and NOX3 require refinement.