Identification of Campylobacter fetus Subspecies by Phenotypic Differentiation and PCR

Abstract

The species Campylobacter fetus is divided into the subspecies C. fetus subsp. venerealis and C. fetus subsp. fetus, which differ in their epidemiologies and clinical importance. The differences between these subspecies make accurate distinction between the two essential. First, the value of seven key tests for the traditional differentiation of C. fetus was investigated. Afterwards, the results of the phenotypic differentiation and PCR were compared to address the question of the reliability of this PCR assay. Altogether, 103 C. fetus isolates were investigated, including the type strains of C. fetus subsp. fetus and C. fetus subsp. venerealis. Depending on the result of the glycine tolerance test, the isolates could be separated into 81 C. fetus subsp. venerealis isolates (glycine intolerant) and 22 C. fetus subsp. fetus isolates (glycine tolerant). For all C. fetus subsp. venerealis strains tested, the results of the selenite reduction assay and sensitivity to metronidazole and cefoperazone completely agreed with the results of the glycine tolerance test (correspondence, 100%). Seventy-three C. fetus subsp. venerealis isolates did not grow at 42°C (correspondence, 90.1%), but eight isolates showed a faintly discernible, flat, dark gray growth. For 22 C. fetus subsp. fetus isolates, the results of additional phenotypic tests only partly agreed with the results of the glycine tolerance test. For C. fetus subsp. fetus the results of the glycine tolerance test showed a relatively good correspondence with those of the selenite reduction assay (correspondence, 81.8%), assays for cefoperazone resistance (correspondence, 86.4%), and assays for growth at 42°C (correspondence, 81.8%). The results of the glycine tolerance test and PCR completely agreed for the 103 C. fetus isolates tested. We conclude that at present the traditional phenotypic characterization of C. fetus subspecies under strongly defined conditions remains indispensable, but this PCR assay constitutes a valuable adjunctive technique for the confirmation of phenotypic results.

Fatal Relapse of a Purulent Pleurisy Caused by Campylobacter fetus subsp. fetus

“…Nevertheless, because of inappropriate standardization of biochemical tests, accurate identification of Campylobacter strains at the species level should include molecular methods. This is particularly true for C. fetus, which furthermore is divided into two different subspecies that have dramatic differences in their epidemiological and clinical implications (20). To the best of our knowledge, this is the first reported case of recurrent pleurisy caused by accurately identified C. fetus subsp.…”

Molecular diagnostic protocols for bovine genital campylobacteriosis using comparative genomics and virulence studies

“…Existence of atypical and mutant strains highlights that biochemical testing is not a reliable method for the differentiation of C. fetus subspecies and emphasises the need to perform more than one biochemical test to limit misidentification (Chang and Ogg, 1971;Hum et al, 1997;On, 1996;On and Harrington, 2001;On and Holmes, 1991a;Salama et al, 1992;Schulze et al, 2006;Vargas et al, 2003;Wagenaar et al, 2001 (Hazeleger et al, 1992;Hodinka and Gilligan, 1988;Nachamkin and Barbagallo, 1990). Of the three techniques, Microscreen appeared to be superior because of its ability to recognise a larger number of species, the absence of cross-reactions with non-Campylobacter spp.…”

“…A recent study obtained 100% correlation between PCR and biochemical phenotyping of 103 C. fetus isolates by using optimal inoculum size and two subsequent PCR assays instead of a multiplex PCR . Some studies reported that C. fetusspecific primers (MG3F and MG4R, Table 2) produced a fragment of 750 bp although others obtained a 960 bp amplicon (Hum et al, 1997;On and Harrington, 2001;Schulze, et al 2006;Vargas et al, 2003;Wagenaar, et al 2001, Willoughby, et al 2005. Sequencing determined the accurate C. fetus product size as 764 bp, targeting a carbon starvation protein (Hum et al, 2010).…”

“…The multiplex PCR designed by Hum et al (1997) has been widely adopted; and appeared to correlate well with biochemical phenotyping of C. fetus isolates (Hum et al, 1997;On and Harrington, 2001;Schulze et al, 2006;Vargas et al, 2003). Subsequently, a sensitive real-time PCR assay designed from the subspecies-specific parA gene targeted by Hum's PCR revealed a much higher prevalence of C. fetus subsp.…”

“…fetus (n=2) phenotypes based solely on glycine tolerance and H 2 S detection tests. Due to the lack of differential properties between C. fetus subspecies and the existence of atypical and mutant strains, the need to characterise C. fetus subspecies with more than one biochemical test to limit misidentification has previously been emphasised (Chang and Ogg, 1971;Hum et al, 1997;Jones et al, 1985;On, 1996;On and Harrington, On and Holmes, 1991b;Salama et al, 1992;Schulze et al, 2006;Vargas et al, 2003;Véron and Châtelain, 1973). This large and diverse collection of Australian field cultures will allow further investigation of C. fetus subsp.…”