PCR for the detection of Streptobacillus moniliformis

Abstract

SummaryStre pto b a c illus m o nil ifo rm is is a Gram-negat ive bac terium found in various laboratory anim al species and is the cause of rat bit e fever and Haverhill fever in man. In order to evaluate a polymerase chain reaction (PCR ) for the detection of this zoonotic bact erium in anim al tissues a set of primers was designed based on the DNA base sequence of part of the 16S rRNA gene from 11 S. m o nili fo rm is strains. T he PCR detected as few as 2±6 copies of S. m o nilifo rm is DNA. A 296 bp DNA fragm ent was ampli®ed from S. m o nilifo rm is strains from rodents, humans and turkeys. Amplicons of about the same size were obtained from T he PCR detected S. m o ni lifo rm isinfection in all four orally-and four intravenously-infected C57BL/6 mice and the bac terium was cultured from all but one mouse. T he PCR detected S. m o nilifo rm is infection in all 12 orally-infected WU rats, and in ®ve of eight rats exposed to natural infection. Enzyme linked immunosorbent assay (ELISA) and PCR were equally successful in detecting infection in rats but S. m o ni lifo rm is was not detected by using culture. Keywords S. m o nilifo rm is; PCR; mouse; rat; health monitoring Stre pto b a c illus m o nil ifo rm isis a Gramnegative bac terium that can occur in various laboratory animal species including gerbils, guineapigs, mice and rats. Humans may become infected through ingestion of contam inated water or through a bit e or a scratch of an animal, leading to Haverhill fever (HF) and rat bite fever (RBF), respectively (Wullenweber 1995). It is commonly believed that with the use of modern maintenance system s streptobac illosis has been eradicated from laboratory animal colonies. T here are however recent reports on the isolation of S. m o nilifo rm is from laboratory rodents (Wullenweber e t a l. 1990, Koopman e t a l. 1991, Kirchner e t a l. 1992, Wullenweber e t a l. 1992, Glastonbury e t a l. 1996 ), and serological observations using an ELISA (Boot e t a l. 1993 ) suggest that latent infection may be common in rats (unpublished observations). As the culture of S. m o ni lifo rm is from healthy animals is dif®cult, we developed and evaluated a PCR for the detection of the bacterium in tissues of mice and rats. Materials and methods Mic ro o rga nism sSe ve nte e n S. m o nilifo rm is strains were obtai ned from mice (3 ), rats (3 ), a spinifex hopping mouse (1 ), turkeys (4 ) and from cases of HF (1 ) and RBF (5 ) in humans (Table 1).

Rat bite fever caused byStreptobacillus moniliformisin a child: human infection and rat carriage diagnosed by PCR

“…3-5 8 9 Recently, molecular methods have been used to identify S moniliformis directly in human samples, 6 8 in addition to cultures 5 and organ biopsies of experimentally infected mice and rats. 2 Although S moniliformis could not be isolated here in well individualised colonies by culture from pet specimens, its presence was strongly suggested both by the Gram colouration of cultures and molecular biology data (PCR restriction fragment length polymorphism) on DNA extracts from the cultures and rat biopsy. To our knowledge, the detection of S moniliformis by molecular methods in the pet of a human suffering from RBF has not been reported previously.…”

“…The 296 bp DNA fragment was amplified and restricted using BfaI, as described previously. 2 This yielded supernumerary fragments but a S moniliformis signature (128, 92, and 76 bp fragments) was detected in the culture and biopsy DNA when compared with the isolated strain (fig 2).…”

Rat Bite Fever andStreptobacillus moniliformis

“…Although these assays are sufficiently sensitive, the increasing demand for rapid, more-sensitive tests likely has detracted from their utility. Molecular methods such as PCR show promise and have been used successfully with humans and laboratory animals (4,7,11,43,85). A PCR assay specific for S. moniliformis has been described by Boot et al (11); it uses primers designed on the basis of 16S rRNA gene base sequence data of human and rodent strains of S. moniliformis (forward primer, 5Ј GCT TAA CAC ATG CAA ATC TAT 3Ј; reverse primer, 5Ј AGT AAG GGC CGT ATC TCA 3Ј).…”

“…The PCR assay generated a 296-bp product which, when discriminated by BfaI restriction enzyme treatment, generated three fragments (128, 92, and 76 bp) specific to S. moniliformis. This assay has been used by others to examine both human-and animal-derived specimens and has been found to distinguish S. moniliformis from other organisms with great accuracy (4,11,85). However, until such an assay becomes more readily available, a diagnosis of S. moniliformis-associated rat bite fever requires a high clinical index of suspicion coupled with the appropriate use of culture and attention to ruling out alternate diagnoses.…”

Approved and novel strategies in diagnostics of rat bite fever and otherStreptobacillusinfections in humans and animals

“…10,12,97,[102][103][104] The method by Boot et al 102 amplified a 296 bp fragment and showed considerable sensitivity but also some flaws in specificity due to amplicon sequence similarities with Leptotrichia sp., Fusobacterium necrogenes and Sebaldella termitidis. 102,103 Although these non-specificities could be solved by macro restriction with the endonuclease BfaI, Kimura et al 10 advanced this PCR with respect to specificity by improving oligonucleotide primers according to Table 3.…”