# A novel real-time PCR assay for quantitative detection of Campylobacter fetus based on ribosomal sequences

Gregorio Iraola, Ruben Pérez, Laura Betancor, Ana Marandino, Claudia Morsella, Alejandra Méndez, Fernando Paolicchi, Alessandra Piccirillo, Gonzalo Tomás, Alejandra Velilla, et al.

► To cite this version:

Gregorio Iraola, Ruben Pérez, Laura Betancor, Ana Marandino, Claudia Morsella, et al. A novel real-time PCR assay for quantitative detection of Campylobacter fetus based on ribosomal sequences. BMC Veterinary Research, 2016, 12 (1), pp.286. 10.1186/s12917-016-0913-3. pasteur-01880470

HAL Id: pasteur-01880470

https://riip.hal.science/pasteur-01880470

Submitted on 24 Sep 2018

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
<figure><img src="image_2.png" /></figure>

# A novel real-time PCR assay for quantitative detection of *Campylobacter fetus* based on ribosomal sequences

Gregorio Iraola<fnref n="1" />,², Ruben Pérez<fnref n="1" />, Laura Betancor⁴, Ana Marandino<fnref n="1" />, Claudia Morsella³, Alejandra Méndez³, Fernando Paolicchi³, Alessandra Piccirillo⁵, Gonzalo Tomás<fnref n="1" />, Alejandra Velilla³ and Lucía Calleros<fnref n="1" />*

**Abstract**

**Background:** *Campylobacter fetus* is a pathogen of major concern for animal and human health. The species shows a great intraspecific variation, with three subspecies: *C. fetus* subsp. *fetus*, *C. fetus* subsp. *venerealis*, and *C. fetus* subsp. *testudinum*. *Campylobacter fetus* *fetus* affects a broad range of hosts and induces abortion in sheep and cows. *Campylobacter fetus venerealis* is restricted to cattle and causes the endemic disease bovine genital campylobacteriosis, which triggers reproductive problems and is responsible for major economic losses. *Campylobacter fetus testudinum* has been proposed recently based on genetically divergent strains isolated from reptiles and humans. Both *C. fetus* *fetus* and *C. fetus testudinum* are opportunistic pathogens for immune-compromised humans. Biochemical tests remain as the gold standard for identifying *C. fetus* but the fastidious growing requirements and the lack of reliability and reproducibility of some biochemical tests motivated the development of molecular diagnostic tools. These methods have been successfully tested on bovine isolates but fail to detect some genetically divergent strains isolated from other hosts. The aim of the present study was to develop a highly specific molecular assay to identify and quantify *C. fetus* strains.

**Results:** We developed a highly sensitive real-time PCR assay that targets a unique region of the 16S rRNA gene. This assay successfully detected all *C. fetus* strains, including those that were negative for the *cstA* gene-based assay used as a standard for molecular *C. fetus* identification. The assay showed high specificity and absence of cross-reactivity with other bacterial species. The analytical testing of the assay was determined using a standard curve. The assay demonstrated a wide dynamic range between 10² and 107 genome copies per reaction, and a good reproducibility with small intra- and inter-assay variability.

**Conclusions:** The possibility to characterize samples in a rapid, sensitive and reproducible way makes this assay a good option to establish a new standard in molecular identification and quantification of *C. fetus* species.

**Keywords:** *Campylobacter fetus*, Molecular detection, Real-time PCR

**Background**

Members of the genus *Campylobacter* are gram-negative epsilon-proteobacteria highly adapted to vertebrate hosts. Most species are pathogens of a wide range of livestock species and have extensive reservoirs in wildlife [1–3].

The species *Campylobacter fetus* shows a remarkable level of intraspecific variation, with three subspecies: *C.*

*fetus* subsp. *fetus*, *C. fetus* subsp. *venerealis*, and *C. fetus* subsp. *testudinum*. *Campylobacter fetus* *fetus* and *C. fetus venerealis* are classified on the basis of their mechanisms of transmission, clinical presentations and two key biochemical tests (tolerance to glycine and H₂S production) [4, 5]. *Campylobacter fetus* *fetus* infects the intestinal tract of several mammalian species and induces abortion in cattle and sheep [2, 5, 6]. In humans, it is an opportunistic pathogen that mainly infects immune-compromised patients [7, 8]. *Campylobacter fetus* *venerealis* is a cattle-restricted pathogen with tropism for

<footnote>* Correspondence: calleros@fcien.edu.uy</footnote>

<footnote n="1">¹Sección Genética Evolutiva, Facultad de Ciencias, Iguá 4225, Montevideo
11400, Uruguay</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
genital tissues and is the etiological agent of bovine genital campylobacteriosis (BGC), a serious reproductive disease that causes infertility and abortion [9]. *C. fetus venerealis* includes a variant, namely *C. fetus venerealis* biovar intermedius that reacts differently to the H₂S test and also causes BGC [5]. *Campylobacter fetus testudinum* has been proposed recently to cluster some reptilian and human strains of putative reptilian origin on the basis of notorious genetic divergence from *C. fetus fetus* and *C. fetus venerealis* [10].

Biochemical tests remain as the gold standard for identifying *C. fetus* and differentiating between *C. fetus fetus* and *C. fetus venerealis*, but the fastidious growth requirements and the lack of reliability and reproducibility of some assays [11], due in part to the genetic heterogeneity of some strains, motivated the development of alternative diagnostic methods.

Several studies have endeavored in determining the suitability of different genetic methods for identifying the species *C. fetus* using end-point PCRs. In particular, the multiplex-PCR assay designed by Hum et al. [12] has been vastly used for species identification. Detection of *C. fetus* in this assay is achieved using PCR primers that target signature regions of the *cstA* gene, and *C. fetus venerealis* identification is based on the *parA* gene. However, genetic divergence in the *cstA* gene could prevent their detection by this assay, as occur in reptilian strains, and thus fails as a general diagnostic tool to identify the species [10].

Other assays for *C. fetus* identification were later designed to target additional genes, like *cpn60*, which encodes the universal 60-kDa chaperonin, and *nahE*, which encodes a sodium/hydrogen exchanger protein [13, 14]. The *cpn60* and *nahE* gene-based methods have been updated to real-time PCR assays using different technologies [14–17]. Both real-time assays have been designed to detect *C. fetus* on bovine isolates, and successfully tested on this kind of samples, but may fail to detect some genetically divergent strains, particularly of reptilian origin, which have distinctive nucleotide variants in many genes. Therefore, detection of *C. fetus* can be improved by developing new real-time PCR assays able to detect strains from all subspecies and hosts. These assays should be designed to target highly stable genomic regions that are characteristic for the species. Ribosomal genes are one of the most common DNA regions used to design PCR assays for the identification and detection of microorganisms. The 16S rRNA gene-targeted molecular tools are widely used as its variability has been thoroughly described in all *Campylobacter* species [18–23]. The sequence of the 16S rRNA gene is species-specific within the genus and *C. fetus* has several unique nucleotide markers [24, 25]. Moreover, ribosomal genes are homogeneous for *C. fetus* subspecies and have three identical copies per genome allowing a better

detection. Despite the obvious advantages of these genes, so far, there is not a real-time PCR assay targeting ribosomal sequences for the specific detection of *C. fetus*.

The aim of the present study was to develop a highly sensitive real-time PCR assay, to detect and quantify *C. fetus* strains.

## Results

Strains were assigned to *C. fetus* and its subspecies using standard bacteriological methods (Table 1). Additionally, we performed the molecular characterization in the same collection of strains (Table 1). The results of bacteriological and molecular classification do not always match, particularly at the subspecies level One bovine (INTA 89/222) and the reptilian isolate (RA8/Italy/2011) were phenotypically identified as *C. fetus* but were negative for the *cstA* gene amplicon that is currently used as a marker for *C. fetus*. The bovine isolate was positive for the subspecies (*C. fetus venerealis*) markers of both tests and the reptilian isolate was negative. The assignment of these isolates to the species *C. fetus* was confirmed by sequencing a fragment of the 16S rRNA gene, which unequivocally discriminates between *Campylobacter* species and from other bacterial species [21, 24].

The 16SPb probe is species specific and has a minimum of one mismatch with a single sequence from *C. hyointestinalis*, and a maximum of nine differences with other *Campylobacter* species (e.g. *C. rectus* and *C. showae*). The forward primer's sequence is species specific and has a minimum of one and a maximum of four mismatches with other *Campylobacter* species (Figs. 1 and 2, Additional file 1). The reverse primer's sequence is identical in some *Campylobacter* species but has one or two differences with others. The combination of primers and probe only matches perfectly with the 16S rRNA gene of *C. fetus*.

All PCR reactions using template DNA from *C. fetus*, *C. fetus venerealis*, *C. fetus venerealis* bv. intermedius, and *C. fetus testudinum* yielded a VIC signal corresponding to the *C. fetus*-specific probe. This result indicates a 100% clinical sensitivity and 95% confidence interval of 94–100% (Clopper-Pearson interval).

The analytical performance of the assay was determined using a standard curve (Fig. 3). The linear dynamic range of the assay was established between 10² and 10⁷ genome copies per reaction. The amplification efficiency and the coefficient of determination (R²) were 93% and 0.9973, respectively. Intra- and inter-assay reproducibility was calculated using the coefficient of variation (CV), which showed considerable low values, being the highest 2.19% (Table 2).

No fluorescent signal was observed using template DNA from non-*C. fetus* bacterial species used as negative controls (i.e. *C. geochelonis*, *C. hyointestinalis*, *C. jejuni*, *C. coli* and *C. sputorum*). This result corresponds to a clinical
<table><caption>**Table 1** Isolates analyzed, discriminated by host, source, country and year of isolation</caption><thead><tr><th>Isolate</th><th>Host</th><th>Source</th><th>Country</th><th>Year</th><th>Phenotypic typing<sup>a</sup></th><th>Multiplex PCR A<sup>b</sup></th><th>Multiplex PCR B<sup>c</sup></th><th>Real-time PCR</th></tr></thead><tbody><tr><td>A28</td><td>Bovine</td><td>U</td><td>Australia</td><td>1978</td><td>Cff</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>063</td><td>Bovine</td><td>Prepuce</td><td>Uruguay</td><td>1980</td><td>Cff</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>0835</td><td>Bovine</td><td>U</td><td>Uruguay</td><td>U</td><td>Cff</td><td>Cfv</td><td>Cff</td><td>+</td></tr><tr><td>F106</td><td>Bovine</td><td>U</td><td>Uruguay</td><td>U</td><td>Cff</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>71098</td><td>Bovine</td><td>Fetal abomasal content</td><td>Uruguay</td><td>1998</td><td>Cff</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>INTA 97/C1N3<sup>d</sup></td><td>Bovine</td><td>Vaginal mucus</td><td>Argentina</td><td>1997</td><td>Cff</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>INTA 04/554</td><td>Bovine</td><td>Fetal abomasal content</td><td>Argentina</td><td>2004</td><td>Cff</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>INTA 90/189</td><td>Bovine</td><td>Fetal lung</td><td>Argentina</td><td>1990</td><td>Cff</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 89/222</td><td>Bovine</td><td>Prepuce</td><td>Argentina</td><td>1989</td><td>Cff</td><td>No Cf/Cfv</td><td>No Cf/Cfv</td><td>+</td></tr><tr><td>INTA 01/165</td><td>Bovine</td><td>Vaginal mucus</td><td>Argentina</td><td>2001</td><td>Cff</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>INTA 12/218</td><td>Bovine</td><td>Fetal abomasal content</td><td>Argentina</td><td>2012</td><td>Cff</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 99/801</td><td>Bovine</td><td>Prepuce</td><td>Argentina</td><td>1999</td><td>Cff</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>INTA 01/064</td><td>Bovine</td><td>Vaginal mucus</td><td>Argentina</td><td>2001</td><td>Cff</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>INTA 04/875</td><td>Bovine</td><td>Vaginal mucus</td><td>Argentina</td><td>2004</td><td>Cff</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>INTA 08/328</td><td>Bovine</td><td>Fetal lung</td><td>Argentina</td><td>2008</td><td>Cff</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>INTA 05/622</td><td>Bovine</td><td>Fetal abomasal content</td><td>Argentina</td><td>2005</td><td>Cff</td><td>Cff</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 11/262</td><td>Bovine</td><td>Fetal abomasal content</td><td>Argentina</td><td>2011</td><td>Cff</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 11/295</td><td>Bovine</td><td>Fetal abomasal content</td><td>Argentina</td><td>2011</td><td>Cff</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 11/685A</td><td>Bovine</td><td>Vaginal mucus</td><td>Argentina</td><td>2011</td><td>Cff</td><td>Cfv</td><td>Cff</td><td>+</td></tr><tr><td>INTA 11/685B</td><td>Bovine</td><td>Fetal abomasal content</td><td>Argentina</td><td>2011</td><td>Cff</td><td>Cfv</td><td>Cff</td><td>+</td></tr><tr><td>INTA 11/677</td><td>Bovine</td><td>Fetal abomasal content</td><td>Argentina</td><td>2011</td><td>Cff</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>INTA 11/501</td><td>Bovine</td><td>Vaginal mucus</td><td>Argentina</td><td>2011</td><td>Cff</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>INTA 11/408</td><td>Bovine</td><td>Fetal abomasal content</td><td>Argentina</td><td>2011</td><td>Cff</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>INTA 11/356</td><td>Bovine</td><td>Fetal abomasal content</td><td>Argentina</td><td>2011</td><td>Cff</td><td>Cff</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 11/360</td><td>Bovine</td><td>Fetal lung</td><td>Argentina</td><td>2011</td><td>Cff</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>NCTC10354<sup>T</sup></td><td>Bovine</td><td>U</td><td>England</td><td>1962</td><td>Cfv</td><td>Cff</td><td>Cfv</td><td>+</td></tr><tr><td>D78</td><td>Bovine</td><td>U</td><td>Australia</td><td>1978</td><td>Cfv</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>660</td><td>Bovine</td><td>Fetal abomasal content</td><td>Uruguay</td><td>2010</td><td>Cfv</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>3726</td><td>Bovine</td><td>Fetal abomasal content</td><td>Uruguay</td><td>2010</td><td>Cfv</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>2733</td><td>Bovine</td><td>Fetal abomasal content</td><td>Uruguay</td><td>2006</td><td>Cfv</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>2740</td><td>Bovine</td><td>Fetal abomasal content</td><td>Uruguay</td><td>2006</td><td>Cfv</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>MCR03</td><td>Bovine</td><td>Prepuce</td><td>Uruguay</td><td>2009</td><td>Cfv</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>3837</td><td>Bovine</td><td>Fetal abomasal content</td><td>Uruguay</td><td>2010</td><td>Cfv</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>1198</td><td>Bovine</td><td>U</td><td>Uruguay</td><td>U</td><td>Cfv</td><td>Cff</td><td>Cfv</td><td>+</td></tr><tr><td>3598</td><td>Bovine</td><td>U</td><td>Uruguay</td><td>U</td><td>Cfv</td><td>Cff</td><td>Cfv</td><td>+</td></tr><tr><td>2432</td><td>Bovine</td><td>U</td><td>Uruguay</td><td>2010</td><td>Cfv</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>2370P</td><td>Bovine</td><td>Fetal abomasal content</td><td>Uruguay</td><td>2011</td><td>Cfv</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>2374C</td><td>Bovine</td><td>Fetal abomasal content</td><td>Uruguay</td><td>2011</td><td>Cfv</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>27460P</td><td>Bovine</td><td>Fetal abomasal content</td><td>Uruguay</td><td>2011</td><td>Cfv</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 97/608<sup>d</sup></td><td>Bovine</td><td>Placenta</td><td>Argentina</td><td>1997</td><td>Cfv</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 83/371</td><td>Bovine</td><td>Vaginal mucus</td><td>Argentina</td><td>1983</td><td>Cfv</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 90/264</td><td>Bovine</td><td>Fetal abomasal content</td><td>Argentina</td><td>1990</td><td>Cfv</td><td>Cff</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 05/355</td><td>Bovine</td><td>Fetal abomasal content</td><td>Argentina</td><td>2005</td><td>Cfv</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 95/258</td><td>Bovine</td><td>Vaginal mucus</td><td>Argentina</td><td>1995</td><td>Cfv</td><td>Cff</td><td>Cfv</td><td>+</td></tr></tbody></table>
<table><caption>**Table 1** Isolates analyzed, discriminated by host, source, country and year of isolation (Continued)</caption><tr><td>INTA 08/382</td><td>Bovine</td><td>Fetal abomasal content</td><td>Argentina</td><td>2008</td><td>Cfv</td><td>Cff</td><td>Cfv</td><td>+</td></tr><tr><td>021</td><td>Bovine</td><td>U</td><td>Australia</td><td>1978</td><td>Cfvi</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 98/BL472</td><td>Bovine</td><td>Fetal abomasal content</td><td>Argentina</td><td>1998</td><td>Cfvi</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 99/541</td><td>Bovine</td><td>Prepuce</td><td>Argentina</td><td>1999</td><td>Cfvi</td><td>Cff</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 97/384</td><td>Bovine</td><td>Fetal abomasal content</td><td>Argentina</td><td>1997</td><td>Cfvi</td><td>Cff</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 98/472</td><td>Bovine</td><td>Fetal abomasal content</td><td>Argentina</td><td>1998</td><td>Cfvi</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 00/305</td><td>Bovine</td><td>Fetal abomasal content</td><td>Argentina</td><td>2000</td><td>Cfvi</td><td>Cff</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 02/146</td><td>Bovine</td><td>Vaginal mucus</td><td>Argentina</td><td>2002</td><td>Cfvi</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 03/596</td><td>Bovine</td><td>Fetal abomasal content</td><td>Argentina</td><td>2003</td><td>Cfvi</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>INTA 07/379</td><td>Bovine</td><td>Fetal abomasal content</td><td>Argentina</td><td>2007</td><td>Cfvi</td><td>Cff</td><td>Cfv</td><td>+</td></tr><tr><td>INTA 06/341</td><td>Bovine</td><td>Fetal lung</td><td>Argentina</td><td>2006</td><td>Cfvi</td><td>Cfv</td><td>Cfv</td><td>+</td></tr><tr><td>H1-UY</td><td>Human</td><td>Blood</td><td>Uruguay</td><td>2013</td><td>Cf</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>HC</td><td>Human</td><td>Blood</td><td>Uruguay</td><td>2014</td><td>Cf</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>70 L</td><td>Human</td><td>Cerebrospinal fluid</td><td>Uruguay</td><td>2014</td><td>Cf</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>70H</td><td>Human</td><td>Blood</td><td>Uruguay</td><td>2014</td><td>Cf</td><td>Cff</td><td>Cff</td><td>+</td></tr><tr><td>RA8/Italy/2011</td><td>Turtle</td><td>Cloaca</td><td>Italy</td><td>2011</td><td>Cft</td><td>No Cf</td><td>No Cf</td><td>+</td></tr><tr><td>RC7</td><td>Turtle</td><td>Cloaca</td><td>Italy</td><td>2011</td><td>C. geochelonis</td><td>No Cf</td><td>No Cf</td><td>-</td></tr><tr><td>RC11</td><td>Turtle</td><td>Cloaca</td><td>Italy</td><td>2011</td><td>C. geochelonis</td><td>No Cf</td><td>No Cf</td><td>-</td></tr><tr><td>RC20</td><td>Turtle</td><td>Cloaca</td><td>Italy</td><td>2011</td><td>C. geochelonis</td><td>No Cf</td><td>No Cf</td><td>-</td></tr><tr><td>INTA 08/209</td><td>Bovine</td><td>Prepuce</td><td>Argentina</td><td>2008</td><td>C. sputorum</td><td>No Cf</td><td>No Cf</td><td>-</td></tr><tr><td>CcHB41</td><td>Human</td><td>Feces</td><td>Uruguay</td><td>2010</td><td>C. coli</td><td>No Cf</td><td>No Cf</td><td>-</td></tr><tr><td>CjHB32</td><td>Human</td><td>Feces</td><td>Uruguay</td><td>2010</td><td>C. jejuni</td><td>No Cf</td><td>No Cf</td><td>-</td></tr><tr><td>CjCP3</td><td>Chicken</td><td>Cecal content</td><td>Uruguay</td><td>2010</td><td>C. jejuni</td><td>No Cf</td><td>No Cf</td><td>-</td></tr><tr><td>CcCP60</td><td>Chicken</td><td>Cecal content</td><td>Uruguay</td><td>2009</td><td>C. coli</td><td>No Cf</td><td>No Cf</td><td>-</td></tr><tr><td>INTA 99/243</td><td>U</td><td>Vaginal mucus</td><td>Argentina</td><td>1999</td><td>C. hyointestinalis</td><td>No Cf</td><td>No Cf</td><td>-</td></tr><tr><td>NCTC 11562</td><td>Pork</td><td>U</td><td>England</td><td>1983</td><td>C. hyointestinalis</td><td>No Cf</td><td>No Cf</td><td>-</td></tr></table>

Cft Campylobacter fetus subsp. testudinum, Cff Campylobacter fetus subsp. fetus, Cfv Campylobacter fetus subsp. venerealis, Cfvi Campylobacter fetus subsp. venerealis biovar intermedius, Cf Campylobacter fetus, U unknown, ND not determined

specificity of 100 and a 95% confidence interval of 59–100% (Clopper-Pearson interval).

## Discussion

Campylobacter fetus is a pathogen of great relevance for the cattle industry and public health. It is mandatory to report the presence *C. fetus venerealis* to the World Organization for Animal Health (OIE). In humans it is necessary to detect this opportunistic pathogen to achieve a better treatment and for epidemiological surveys. Detection of *C. fetus* in humans is difficult because both *C. fetus* *fetus* and *C. fetus testudinum* are potential pathogens and well-established methods would fail to detect strains of reptilian origin [10]. Therefore, cost-effective, automated and straightforward tools for the unambiguous identification of *C. fetus* are of paramount importance.

Bacteriological analysis, like culture isolation and biochemical tests, are well standardized and extensively used but challenging by the slow growing and few differential phenotypic properties of *C. fetus* [26]. These methods are also laborious and time-consuming, a disadvantage when processing samples at large-scale or delivering a fast diagnosis. To improve the quality and complement the gold-standard bacteriological methods for *C. fetus* detection, some end-point PCR methods have been designed based on the presence of species-specific amplicons [12, 27–29]; these assays fulfill various criteria such as accuracy, high detection probability and well-standardized protocols for its application and interpretation. Real-time PCR methods have been also designed with the same purpose [14–17] and have provided additional technical improvements to *C. fetus*

<footnote>a in C. fetus, glycine tolerance and H2S production, see text for details</footnote>

<footnote>b As described in Hum et al. [12]</footnote>

<footnote>c As described in Iraola et al. [41]</footnote>

<footnote>d These strains were assayed both starting from a resuspended culture and directly from bovine samples of placenta or vaginal mucus, without a previous isolation step</footnote>
<figure><img src="image_3.png" /><figcaption>**Fig. 1** Multiple alignment of partial sequences of 16S gene obtained from databases. Sequences of all species of the genus from which information is available are shown. The sequences of the primers and probe are shaded</figcaption></figure>

detection protocols, like the prevention of cross contamination and the minimization of manipulation and running times. However, both end-point and real-time PCR methods described to date are designed to identify *C. fetus* in bovine samples and do not deal with the intra-specific genetic variability of the bacteria that is found in diverse hosts. In comparison to conventional PCR methods, real-time PCR assays provide increased sensitivity and an accurate quantification of target DNA to study the dynamics of the bacteria in different hosts and tissues. To the best of our knowledge, there is not a real-time PCR method that uses ribosomal sequences for the identification and quantification of *C. fetus*.

Here, we have improved the current molecular methods for *C. fetus* detection by designing a new real-time PCR assay that targets the multi-copy 16S rRNA gene. The variability of these sequences within *Campylobacter* species supports its suitability as a target for amplification-based methods using fluorescent probes. The inclusion in the assay of a TaqMan-MGB probe provides higher specificity, sensitivity and accuracy than traditional TaqMan probes and discriminates between sequences that differ in just one nucleotide [30–32].

Our assay was compared to the *cstA* gene end-point PCR proposed by Hum et al. [12] and currently used as standard for molecular diagnosis of *C. fetus*. The bovine
<figure><img src="image_4.png" /><figcaption>**Fig. 2** Mean number of differences in probe sequence of non-*C. fetus* species 16S gene</figcaption></figure>

sample INTA 89/222 and the reptilian RA8/Italy/2011 could not be detected by Hum's PCR (Table 1), revealing that the sensitivity of this method for bovine isolates is not complete as previously reported [12, 17, 33–36]. These isolates were confirmed as belonging to *C. fetus*

<figure><img src="image_5.png" /><figcaption>**Fig. 3** Standard curve of developed TaqMan-MGB real-time PCR for *C. fetus* detection. Each point represents the mean Ct of nine different measures (three independent reactions, three replicates each). The curve equation (y), coefficient of determination (R²) and amplification efficiency (E) are indicated</figcaption></figure>

by sequencing a fragment of the 16S rRNA gene; therefore the lack of amplification of the *cstA* gene could be due to the absence of the target *cstA* gene in these strains, or the presence of sequence variations that prevent the correct annealing of primers. Our attempt to amplify a larger region including Hum's PCR target region also failed, indicating the absence of this gene in these strains or an even greater sequence divergence within the *cstA* gene (data not shown). To test this hypothesis, it would be necessary to conduct the whole genome analysis of these strains. This notion is supported by the presence of several differences in Hum's primers binding sites in the complete genome of the reptilian strain *C. fetus* subsp. *testudinum* 03-427 (GenBank Acc. number NC_022759). This explains why the 13 isolates used for the description of this subspecies, and the RA8/Italy/2011 strain analyzed here, were negative for Hum's method based on the *cstA* gene [10]. Given the importance of this gene in the metabolism of nitrogen, and in the interaction with the host in *C. jejuni* [37], it is necessary to continue investigating its variations and possible roles in *C. fetus*.

Our novel real-time PCR assay detected all *C. fetus* tested in this study, but was negative for other *Campylobacter* species. The complete identity of primer and probe targets in all *C. fetus* strains deposited in the GenBank database (including reptilian isolates) supports that our
<table><caption>**Table 2** Intra- and inter-assay reproducibility for the detection of *C. fetus*</caption><thead><tr><th rowspan="2">Genome copies/reaction</th><th colspan="2">Intra-assay variations</th><th colspan="2">Inter-assay variations</th></tr><tr><th>Mean Ct (from - to)</th><th>CV (from - to)</th><th>Mean Ct</th><th>CV</th></tr></thead><tbody><tr><td>1 × 10<sup>1</sup></td><td>-<sup>a</sup></td><td>-</td><td>-</td><td>-</td></tr><tr><td>1 × 10<sup>2</sup></td><td>36.57-37.69</td><td>0.97-2.1</td><td>37.13</td><td>2.19</td></tr><tr><td>1 × 10<sup>3</sup></td><td>33.68-34.11</td><td>0.48-1.15</td><td>33.89</td><td>1.05</td></tr><tr><td>1 × 10<sup>4</sup></td><td>30-30.07</td><td>0.25-0.16</td><td>30.03</td><td>0.23</td></tr><tr><td>1 × 10<sup>5</sup></td><td>26.37-26.46</td><td>0.14-0.27</td><td>26.41</td><td>0.26</td></tr><tr><td>1 × 10<sup>6</sup></td><td>22.62-22.86</td><td>0.18-0.7</td><td>22.74</td><td>0.73</td></tr><tr><td>1 × 10<sup>7</sup></td><td>19.02-19.23</td><td>0.5-0.83</td><td>19.12</td><td>0.86</td></tr></tbody></table>

CV coefficient of variation of Ct values [%]

<sup>a</sup>Ct value out of dynamic range

assay is expected to detect the currently described subspecies from diverse hosts (Fig. 1). These results indicate the excellent sensitivity and specificity of the assay. In addition, the primers and probe sequences are conserved in the 16S rRNA gene of the three subspecies (Fig. 1), in contrast with what happens with primers that amplify the *cstA* gene.

The assay here described has some advantages over other real-time PCR methods described in the literature. The *nahE* assay reported by Van der Graaf-van Bloois et al. [17] uses a TaqMan probe that provides high sensitivity and detection capability, but its quantification capability has not been ascertained using a standard curve. It is also uncertain whether this assay would detect reptilian *C. fetus testudinum* isolates, for which it was not designed, as the probe and the forward PCR primers have two mismatches each with respect to the *C. fetus testudinum* reference strain 03-427. The hybridization of primers and probes to the *nahE* gene could be also affected because it is embedded in a region that shows genomic rearrangements in most of the complete genome sequences available in the databases (not shown). The methodology to detect the *cpn60* gene described by Chaban et al. [14] uses specific primers and SYBR green chemistry to identify *C. fetus* species, but its performance is sub-optimal in samples with low bacterial concentrations [15], such as the uncultured samples that were successfully tested in the present assay (Table 1).

## Conclusions

The 16S rRNA gene-targeted assay here developed is excellent for the accurate detection and quantification of *C. fetus* in clinical samples and pure cultures. The possibility to characterize a large number of samples in a rapid, sensitive and reproducible way makes this assay a suitable tool for routine testing and research. For these reasons, this method has the potential to become a new standard in molecular identification of *C. fetus* species.

## Methods

### Real-time PCR design

The assay is based on a set of primers that amplifies a 78-bp sequence of the 16S rRNA gene (16SFw: 5'-GC ACCTGTCTCAACTTTC-3' and 16SRv: 5'-CCTTACCT GGGCTTGAT-3') and a TaqMan-MGB probe (16SPb: 5'-VIC-ATCTCTAAGAGATTAGTTG-MGB/NFQ-3'), which targets a 19-bp polymorphic region that discriminates strains of *C. fetus* from the remaining *Campylobacter* species and other bacteria. This polymorphic region (Fig. 1) was detected by visual inspection of over 3859 partial and complete 16S rRNA gene sequences aligned with T-Coffee [38]. The constructed alignment comprised sequences from all recognized *Campylobacter* species and from unassigned strains belonging to the genus, which were obtained from the SILVA database [39]. An alignment of 1907 representative sequences (removing identical sequences) is shown in Additional file 1. BLAST algorithm [40] was used to check *in silico* the specificity of primers and probe sequences, and to evaluate the occurrence of non-specific matches within the genomes of *C. fetus* and other bacterial species.

### Bacterial strains: species and subspecies identification

The real-time PCR assay was tested with a collection of *C. fetus* strains isolated from cattle, humans and reptiles. Two of the strains (INTA 97/C1N3 and INTA 97/608) were assayed also directly from bovine samples of placenta or vaginal mucus, without a previous isolation step. Ten additional strains from four non-*fetus* *Campylobacter* species that occasionally occur in bovine samples were used to verify the specificity of the assays (Table 1).

Strains were previously typed using bacteriological methods to test the assay specificity. Samples were grown in Brucella semi-solid Broth and *Campylobacter* selective medium under microaerophilic conditions (85% H₂, 5% O₂, 10% CO₂) for 48 h at 37 °C. The presumptive *Campylobacter* colonies were tested by catalase and oxidase tests, and grown in Brucella broth (Sigma-Aldrich, St.
Louis, USA) with 1, 1.3, 1.5 and 1.9% glycine (Sigma-Aldrich), without glycine and in Brucella broth with NaCl and cysteine (Sigma-Aldrich) to detect H₂S production with a lead acetate paper (Sigma-Aldrich). Sodium selenite reduction test was also performed. Colonies that grew in 1% glycine were classified as *C. fetus fetus* or *C. fetus testudinum* by their positive or negative H₂S production, respectively. Glycine-sensitive colonies were assigned to the subspecies *C. fetus venerealis* (H₂S negative) or *C. fetus venerealis* by intermedius (H₂S positive) (Table 1). Out of a total of 60 strains, 25 were *C. fetus fetus*, 20 *C. fetus venerealis*, 10 *C. fetus venerealis* by intermedius, one was *C. fetus testudinum*, and four were not analyzed.

Strains were further characterized using the multiplex-PCR assays designed by Hum et al. [12] and Iraola et al. [41]. Both assays use the same species-specific primers to detect the *cstA* gene and different genes to identify the subspecies. The first method includes a fragment of the *parA* gene as a *C. fetus venerealis* marker, and the second uses a fragment of the *virB11* gene (Table 1) [42].

In cases where multiplex-PCR based methods failed to identify the isolates, molecular identification of species was confirmed by sequencing a fragment of the 16S rRNA gene, which was amplified using the C412F and C1288R primers described by Linton et al [21].

## Real-time PCR assays

DNA was extracted from 500 µL of a suspension of live bacteria in a phosphate-buffered saline pH 7.4 solution (1 × 10⁸ CFU/mL), or from 1 mL of preputial washing or vaginal mucus. The QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) was used for all DNA extractions and the DNA purity was measured as the ratio of absorbance at 260 and 280 nm (A260/280) using a Nanodrop 2000 (Thermo Scientific, Waltham, USA).

Real-time PCR was carried out in a 25-µL reaction containing 1 × TaqMan Genotyping Master Mix (Applied Biosystems, Foster City, USA), 1 × Custom TaqMan SNP Genotyping Assay (0.9 µM each primer and 0.2 µM probe), and 1 µL genomic DNA. Thermocycling was performed on an ABI Prism 7500 (Applied Biosystems) and consisted of a 5 min incubation step at 50 °C, denaturation for 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C, and a final step of 5 min at 70 °C. Fluorescence measurements from VIC fluorophore was collected at the 5 min initial incubation stage, at the 60 °C step of each cycle, and at the end of the run.

## Standard curve generation for analytical testing

To construct the standard curve for the ribosomal probe we generated 10-fold serial dilutions containing 10⁰–10⁷ genome copies/µL. Number of genome copies was determined by the following formula: Y (genome copies/µL) = [X (g/µL) DNA/ (nt genome length × 660)] × (6.022 × 10²³)

using the DNA concentration of the dilution (X) and the genome size of the strain Cff 82-40 (1.77 Mb; GenBank accession number NC008599). The log dilution series of *C. fetus* genomes and negative controls containing nuclease-free water were tested with real-time PCR in triplicate and in three independent runs.

Standard curve was generated by plotting threshold cycle (Ct) values per three replicates per standard dilution versus the logarithm of the bacterial genome copies to determine analytical sensitivity and efficiency of the assay. The amplification efficiency was calculated with the equation E = (10^(-1/k)) - 1, where (k) is the slope of the linear regression line [43, 44]. A value of 1 corresponds to 100% amplification efficiency. The coefficient of determination (R²) was also assessed and was considered to be suitable when it was higher than 0.980 in a single run [45, 46]. The coefficients of variation (CVs) of Ct values were assessed separately for each standard bacterial dilution by analyzing the replicates of the same analytical run (intra-assay) and the repeated analyses from different analytical runs (inter-assay).

## Additional files

**Additional file 1:** Alignment of 16S rRNA gene sequences from *Campylobacter* species obtained from the SILVA database, in FASTA format. (TXT 4882 kb)

### Acknowledgments

LC and GI acknowledge support from the "Comisión Sectorial de Investigación Científica" (CSIC), and "Agencia Nacional de Investigación e Innovación" (ANII) fellowship programs from Uruguay. This work was partially financed by ANII-FSSA-2014-1-105252 Project Grant from the ANII agency.

### Funding

This work was partially funded by ANII-FSSA-2014-1-105252 Project Grant from the ANII agency.

### Availability of data and materials

The datasets supporting the conclusions of this article are included within the article (and its Additional file 1).

### Authors' contributions

GI, LC and RP conceived and designed the experiments; GI, LC, LB, AMa, AMé, CM, GT and AV performed the experiments; GI, LC, AMa, RP and GT analyzed the data; GI, LB, LC, FP, AP and RP contributed reagents/materials/ analysis tools; GI, LC and RP wrote the paper. All authors revised and approved the final version of the manuscript.

### Competing interests

The authors declare that they have no competing interests.

### Consent for publication

Not applicable.

### Ethics approval and consent to participate

This study was carried out in compliance with the veterinary best practice and the informed owner consent in the case of animal samples sent to investigation diagnostics in a Laboratory of Bacteriology. Human bacterial isolates were already part of a strain collection from a diagnostics center, therefore no ethics approval was considered necessary.
Author details

¹Sección Genética Evolutiva, Facultad de Ciencias, Iguá 4225, Montevideo 11400, Uruguay. ²Unidad de Bioinformática, Instituto Pasteur Montevideo, Montevideo, Uruguay. ³Laboratorio de Bacteriología, Unidad Integrada INTA-Universidad Nacional de Mar del Plata, Balcarce, Argentina.

⁴Departamento de Bacteriología y Virología, Instituto de Higiene, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. ⁵Dipartimento di Biomedicina Comparata e Alimentazione, Università degli Studi di Padova, Padova, Italy.

Received: 20 February 2016 Accepted: 6 December 2016

Published online: 15 December 2016

References

1. Man SM. The clinical importance of emerging *Campylobacter* species. Nat Rev Gastroenterol Hepatol. 2011;8:669-85.

2. On SLW. Isolation, identification and subtyping of *Campylobacter*: where to from here? *J Microbiol Methods*. 2013;95:3-7.

3. On SLW. Taxonomy of *Campylobacter*, *Arcobacter*, *Helicobacter* and related bacteria: current status, future prospects and immediate concerns. *J Appl Microbiol.* 2001;90:15-155.

4. OIE. Código sanitario para los animales terrestres. CA PÍTULO 11. 3. Campilobacteriosis genital bovina. 2014. p. 10-1. Spanish version.

5. Veron M, Chatelain R. Taxonomic Study of the Genus *Campylobacter* Sebald and Veron and Designation of the Neotype Strain for the Type Species, *Campylobacter fetus* (Smith and Taylor) Sebald and Veron. *Int J Syst Bacteriol.* 1973;23:122-34.

6. Garcia MM, Ruckerbauer GM, Eaglesom MD, Boisclair WE. Detection of *Campylobacter* fetus in artificial insemination bulls with a transport enrichment medium. *Can J Comp Med*. 1983;47:336-40.

7. Wagenaar JA, van Bergen MAP, Blaser MJ, Tauxe RV, Newell DG, van Putten JPM, van Bergen MAP, Robert V, Van Putten JPM, Diseases M, Consultancy FZ, Birches S. *Campylobacter* fetus infections in humans: exposure and disease. *Clin Infect Dis*. 2014;58:1-26.

8. Iraola G, Betancor L, Calleros L, Gadea P, Algorta G, Galeano S, Muxi P, Greif G, Pérez R. A rural worker infected with a bovine-prevalent genotype of *Campylobacter* fetus subsp. *fetus* supports zoonotic transmission and inconsistency of MLST and whole-genome typing. *Eur J Clin Microbiol Infect Dis*. 2015;34:1593-6.

9. Mshelia GD, Amin JD, Woldehiwet Z, Murray RD, Egwu GO. Epidemiology of bovine venereal campylobacteriosis: geographic distribution and recent advances in molecular diagnostic techniques. *Reprod Domest Anim.* 2010;45:e221-30.

10. Fitzgerald C, Tu ZC, Patrick M, Stiles T, Lawson AJ, Santovenia M, Gilbert MJ, van Bergen M, Joyce K, Pruckler J, Stroika S, Duim B, Miller WG, Loparev V, Sinnige JC, Fields PI, Tauxe RV, Blaser MJ, Wagenaar JA. *Campylobacter* fetus subsp. *testudinum* subsp. nov., isolated from humans and reptiles. *Int J Syst Evol Microbiol*. 2014;64(Pt 9):2944-8.

11. Alves TM, Stynen APR, Miranda KL. Campilobacteriose genital bovina e tricomonose genital bovina : epidemiologia, diagnóstico e controle. Pesqui Veterinária Bras. 2011;31:336-44.

12. Hum S, Quinn K, Brunner J, On SL. Evaluation of a PCR assay for identification and differentiation of *Campylobacter* fetus subspecies. Aust Vet J. 1997;75:827-31.

13. Abril C, Vilei EM, Brodard I, Burnens A, Frey J, Miserez R. Discovery of insertion element ISCF1: a new tool for *Campylobacter* fetus subspecies differentiation. *Clin Microbiol Infect.* 2007;13:993-1000.

14. Chaban B, Musil KM, Hinsworth CG, Hill JE. Development of cpn60-based real-time quantitative PCR assays for the detection of 14 *Campylobacter* species and application to screening of canine fecal samples. *Appl Environ Microbiol.* 2009;75:3055-61.

15. Chaban B, Chu S, Hendrick S, Waldner C, Hill JE. Evaluation of a *Campylobacter fetus* subspecies venerealis real-time quantitative polymerase chain reaction for direct analysis of bovine preputial samples. *Can J Vet Res.* 2012;76:166-73.

16. Selim AM, Elhaig MM, Gaede W. Development of multiplex real-time PCR assay for the detection of *Brucella* spp., *Leptospira* spp. and *Campylobacter* *foetus*. Vet Ital. 2014;50:269-75.

17. van der Graaf-van Bloois L, van Bergen MAP, van der Wal FJ, de Boer AG, Duim B, Schmidt T, Wagenaar JA, van Bloois LVDG. Evaluation of molecular assays for identification *Campylobacter* fetus species and subspecies and

development of a *C. fetus* specific real-time PCR assay. *J Microbiol Methods*. 2013;95:93-7.

18. Blom K, Patton CM, Nicholson MA, Swaminathan B. Identification of *Campylobacter* fetus by PCR-DNA probe method. *J Clin Microbiol.* 1995;33:1360-2.

19. Cardarelli-Leite P, Blom K, Patton CM, Nicholson MA, Steigerwalt AG, Hunter SB, Brenner DJ, Barrett TJ, Swaminathan B. Rapid identification of *Campylobacter* species by restriction fragment length polymorphism analysis of a PCR-amplified fragment of the gene coding for 16S rRNA. *J Clin Microbiol.* 1996;34:62-7.

20. Kolbert CP, Persing DH. Ribosomal DNA sequencing as a tool for identification of bacterial pathogens. Curr Opin Microbiol. 1999;2:299-305.

21. Linton D, Owen RJ, Stanley J. Rapid identification by PCR of the genus *Campylobacter* and of five *Campylobacter* species enteropathogenic for man and animals. *Res Microbiol*. 1996;147:707-18.

22. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. *J Bacteriol.* 1991;173:697-703.

23. Wesley IV, Wesley RD, Cardella M, Dewhirst FE, Paster BJ. Oligodeoxynucleotide probes for *Campylobacter* fetus and *Campylobacter jejunalis* based on 16S rRNA sequences. *J Clin Microbiol.* 1991;29:1812-7.

24. Gorkiewicz G, Feierl G, Schober C, Dieber F, Köfer J, Zechner R, Zechner EL. Species-specific identification of campylobacters by partial 16S rRNA gene sequencing. *J Clin Microbiol.* 2003;41:2537-46.

25. Oyarzabal OA, Wesley TV, Harmon KM, Barbaree JM, Lauerman LH, Backert S, Conner DE. *Campylobacter* fetus by PCR targeting variable regions of the 16S rDNA, vol. 58. 1997. p. 61-71.

26. On SLW. Identification methods for campylobacters, helicobacters, and related organisms. *Clin Microbiol Rev.* 1996;9:405-22.

27. Tramuta C, Lacerenza D, Zoppi S, Goria M, Dondo A, Ferroglio E, Nebbia P, Rosati S. Development of a set of multiplex standard polymerase chain reaction assays for the identification of infectious agents from aborted bovine clinical samples. *J Vet Diagn Invest.* 2011;23:657-64.

28. van Bergen MA, Simons G, van der Graaf-van Bloois L, van Putten JP, Rombout J, Wesley I, Wagenaar JA. Amplified fragment length polymorphism based identification of genetic markers and novel PCR assay for differentiation of *Campylobacter* fetus subspecies. *J Med Microbiol.* 2005;54(Pt 12):1217-24.

29. Wang G, Clark CG, Taylor TM, Pucknell C, Barton C, Price L, Woodward DL, Rodgers FG. Colony multiplex PCR assay for identification and differentiation of *Campylobacter jejuni*, *C. coli*, *C. lari*, *C. upsaliensis*, and *C. fetus* subsp. *fetus*. *J Clin Microbiol.* 2002;40:4744-7.

30. Alonso R, Mateo E, Cisterna R. Detection of parC mutations in *Streptococcus pneumoniae* by Real-time PCR and Taqman-MGB probes. *J Microbiol Methods*. 2007;69:214-7.

31. Kutyavin IV, Afonina IA, Mills A, Gorn VV, Lukhtanov EA, Belousov ES, Singer MJ, Walburger DK, Lokhov SG, Gall AA, Dempcy R, Reed MW, Meyer RB, Hedgpeth J. 3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res. 2000;28:655-61.

32. Rousselon N, Delgenès JP, Godon JJ. A new real time PCR (TaqMan® PCR) system for detection of the 16S rDNA gene associated with fecal bacteria. *J Microbiol Methods*. 2004;59:15-22.

33. Schmidt T, Venter EH, Picard JA. Evaluation of PCR assays for the detection of *Campylobacter* fetus in bovine preputial scrapings and the identification of subspecies in South African field isolates. *J S Afr Vet Assoc.* 2010;81:87-92.

34. Schulze F, Bagon A, Müller W, Hotzel H. Identification of *Campylobacter* fetus subspecies by phenotypic differentiation and PCR. *J Clin Microbiol.* 2006;44:2019-24.

35. van der Graaf-van Bloois L, Miller WG, Yee E, Rijnsburger M, Wagenaar JA, Duim B. Inconsistency of phenotypic and genomic characteristics of *Campylobacter* fetus subspecies requires reevaluation of current diagnostics. *J Clin Microbiol.* 2014;52:4183-8.

36. Willoughby K, Nettleton PF, Quirie M, Maley MA, Foster G, Toszeghy M, Newell DG. A multiplex polymerase chain reaction to detect and differentiate *Campylobacter* fetus subspecies *fetus* and *Campylobacter* fetus subspecies *venerealis*: use on UK isolates of *C. fetus* and other *Campylobacter* spp. *J Appl Microbiol.* 2005;99:758-66.

37. Rasmussen JJ, Vegge CS, Frøkiær H, Howlett RM, Krogfelt KA, Kelly DJ, Ingmer H. *Campylobacter jejuni* carbon starvation protein A (CstA) is involved in peptide utilization, motility and agglutination, and has a role in stimulation of dendritic cells. *J Med Microbiol.* 2013;62:1135-43.

38. Notredame C, Higgins DG, Heringa J. T-coffee: a novel method for fast and accurate multiple sequence alignment. *J Mol Biol.* 2000;302:205-17.
39. Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glockner FO. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2013;41:D590–6.

40. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–10.

41. Iraola G, Hernández M, Calleros L, Paolicchi F, Silveyra S, Velilla A, Carretto L, Rodríguez E, Pérez R. Application of a multiplex PCR assay for *Campylobacter* *fetus* detection and subspecies differentiation in uncultured samples of aborted bovine fetuses. J Vet Sci. 2012;13:371–6.

42. Moolhuijzen PM, Lew-Tabor AE, Wlodek BM, Agüero FG, Comerci DJ, Ugalde RA, Sanchez DO, Appels R, Bellgard M. Genomic analysis of *Campylobacter fetus* subspecies: identification of candidate virulence determinants and diagnostic assay targets. BMC Microbiol. 2009;9:86–97.

43. Ginzinger DG. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol. 2002;30:503–12.

44. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29:e45.

45. Buh Gasparic M, Cankar K, Zel J, Gruden K. Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms. BMC Biotechnol. 2008;8:26.

46. Bustin SA, Nolan T. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech. 2004;15:155–66.

Submit your next manuscript to BioMed Central
and we will help you at every step:

* We accept pre-submission inquiries

* Our selector tool helps you to find the most relevant journal

* We provide round the clock customer support

* Convenient online submission

* Thorough peer review

* Inclusion in PubMed and all major indexing services

* Maximum visibility for your research

Submit your manuscript at
www.biomedcentral.com/submit

BioMed Central