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Biochemical and molecular characterization of Campylobacter fetus isolates from bulls subjected to bovine genital campylobacteriosis diagnosis in Spain

BMC Veterinary Research volume 20, Article number: 131 (2024) Cite this article

Abstract

Background

Bovine genital campylobacteriosis (BGC) is caused by Campylobacter fetus subsp. venerealis (Cfv) including its biovar intermedius (Cfvi). This sexually transmitted disease induces early reproductive failure causing considerable economic losses in the cattle industry. Using a collection of well-characterized isolates (n = 13), C. fetus field isolates (n = 64) and saprophytic isolates resembling Campylobacter (n = 75) obtained from smegma samples of breeding bulls, this study evaluated the concordance of the most used phenotypic (H2S production in cysteine medium and 1% glycine tolerance) and molecular (PCR) methods for the diagnosis of BGC and assessed possible cross-reactions in the molecular diagnostic methods.

Results

Characterization at the subspecies level (fetus vs. venerealis) of C. fetus isolated from bull preputial samples using phenotypic and molecular (PCR targeting nahE and ISCfe1) methods showed moderate concordance (κ = 0.462; CI: 0.256–0.669). No cross-reactions were observed with other saprophytic microaerophilic species or with other Campylobacter species that can be present in preputial samples. Whole genome sequencing (WGS) of discrepant isolates showed 100% agreement with PCR identification. For the differentiation of Cfv biovars, comparison of the H2S test (at 72 h and 5 days of incubation) and a PCR targeting the L-cysteine transporter genes showed higher concordance when H2S production was assessed after 5 days (72 h; κ = 0.553, 0.329–0.778 CI vs. 5 days; κ = 0.881, 0.631–1 CI), evidencing the efficacy of a longer incubation time.

Conclusions

This study confirmed the limitations of biochemical tests to correctly identify C. fetus subspecies and biovars. However, in the case of biovars, when extended incubation times for the H2S test (5 days) were used, phenotypic identification results were significantly improved, although PCR-based methods produced more accurate results. Perfect agreement of WGS with the PCR results and absence of cross-reactions with non-C. fetus saprophytic bacteria from the smegma demonstrated the usefulness of these methods. Nevertheless, the identification of new C. fetus subspecies-specific genes would help to improve BGC diagnosis.

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Background

Campylobacter fetus subspecies venerealis (Cfv) and its biovar intermedius (Cfvi) [1] are the causative agents of Bovine Genital Campylobacteriosis (BGC), a sexually transmitted disease (STD) of cattle listed by the World Organisation for Animal Health (WOAH) [2,3,4,5] and included in the European legislation (Regulation (EU) 2016/429) that governs the trade of frozen bovine semen. The main route of Cfv transmission is during coitus, although transmission can also occur during artificial insemination via contaminated material or semen [4]. Bulls do not usually show clinical signs and can become chronic carriers. Heifers and cows often develop a self-limiting genital infection that causes endometritis and salpingitis, and can result in infertility, early reproductive failure and abortions [4, 6, 7]. Herds with BGC often have reduced reproductive efficiency due to lower pregnancy rates, higher number of services per conception and longer calving intervals, resulting in large economic losses in the beef cattle sector [3, 8, 9]. BGC is widely distributed in areas where cattle are extensively managed and natural breeding is practised [8, 9]. In some areas of Spain, BGC remains endemic with individual and herd prevalence of 7.7% and 12.2%, respectively [10]. However, the lack of monitoring programmes for this disease in many countries makes it difficult to estimate the prevalence rates of BGC world-wide.

The WOAH recommends microbiological culture followed by subsequent phenotypic characterization of C. fetus to ensure good health status before the movement of cattle [2]. However, Cfv is a fastidious growing microorganism with particular physiological characteristics and special nutritional requirements. These characteristics and the common presence of other competing saprophytic bacteria in preputial samples compromise the sensitivity and specificity of Cfv detection by bacterial culture [7, 11]. Moreover, another subspecies of C. fetus, C. fetus subsp. fetus (Cff) colonizes the intestinal tract of ruminants and occasionally the preputial cavity, causing sporadic abortion in cattle [7, 12]. The correct identification of Cfv and Cff is crucial for the implementation of effective BGC control and eradication programmes. C. fetus subspecies differentiation is possible by biochemical tests, with the 1% glycine tolerance test and hydrogen sulphide (H2S) production being the most relevant (Cfv is negative while Cff shows positive results to both tests) [1, 2]. However, since some Cfv strains can acquire glycine tolerance through phage-mediated transduction processes and mutations [13], these biochemical test may lead to misidentification. Furthermore, Cfvi strains exhibit biochemical characteristics intermediate between Cfv and Cff, which include low glycine tolerance and H2S production, that could lead to misidentification in diagnosis [1, 4].

Conventional culture-based methods are time-consuming and laborious, and several PCR methods have been developed for the BGC diagnosis. The most reliable PCR methods for C. fetus subspecies differentiation are those targeting the nahE gene specific of C. fetus and the Cfv-specific insertion element ISCfe1 [14, 15]. However, none of the methods so far described demonstrated complete accuracy in the identification of C. fetus strains at the subspecies level [7, 14] and cross-reactions were also found with Campylobacter hyointestinalis isolates that carry the ISCfe1 insertion element [14]. In addition, molecular differentiation between Cfv biovars is achieved by PCR targeting the L-cysteine (L-Cyst) ABC transporter genes, which are directly related to the H2S production typical of Cfvi isolates [16].

Comparative analyses of biochemical and molecular identification methodologies for C. fetus subspecies and Cfv biovars differentiation, using a wide range of field isolates, are scarce. Therefore, the aim of this study was to compare phenotypic methods and PCR assays based on the nahE and ISCfe1 [14, 15] and the L-Cyst ABC transporter gene [16] targets to differentiate Cff, Cfv and Cfvi, using a wide panel of C. fetus control and field isolates. In addition, we studied the impact of longer incubation time on H2S production on Cfvi identification. The isolates with discordant results were whole genome sequenced (WGS) for a more accurate identification. The performance of the PCR assays used for the identification of C. fetus subspecies was also evaluated using saprophytic bacteria with similar morphology and motility to Campylobacter isolated from the preputial cavity of breeding bulls to further examine possible occurrence of cross-amplification reactions.

Results

Phenotypic characterization of C. fetus isolates by biochemical tests

The expected results of biochemical tests for Cff, Cfv and related species as described by WOAH are detailed in Table S1. Based on the results of the 1% glycine tolerance and H2S production tests, 12 of the 13 C. fetus control strains tested (92.3%) were identified as Cff and 1 (7.7%) as Cfv. Notably, although the final identification of the control strains was the same regardless of the incubation time for the H2S test (72 h and 5 days), there were 3 Cff strains (Cff1, Cff2 and Cff-C0075) that at 72 h of incubation did not produce H2S, while at 5 days one of the strains was H2S-producer (Cff1). The only misidentified control isolate was Cfvi3, which showed tolerance to 1% glycine and H2S production after 5 days therefore being biochemically identified as Cff (Table 1). Finally, the C. hyointestinalis strain (C. hyo hyo) was classified as non-C. fetus (Table 1). Regarding the 64 C. fetus field strains, the analysis of 1% glycine tolerance and H2S production after 72 h of incubation, identified the 14.1% (9/64) of strains as Cff (one of them as non H2S-producer: Sal2), 29.7% (19/64) as Cfv and 56.2% (36/64) as Cfvi. However, 8 strains considered as Cfv at 72 h of incubation (H2S non-producers) were reclassified as Cfvi after 5 days of incubation (H2S producers), resulting in 17.2% (11/64) Cfv and 68.7% (44/64) Cfvi strains (Table 1).

Table 1 Phenotypic and molecular identification of C. fetus control and field isolates
Full size table

Regarding other phenotypic tests (growth at 42 °C, oxidase activity, catalase production and tolerance to 3.5% NaCl), the control strains Cfv ATCC 25922 and Cfvi3 showed no growth at 42 °C and in medium with 3.5% NaCl and were positive for oxidase and catalase activity. In contrast, all Cff control strains grew at 42 ºC and showed identical results to Cfv ATCC 25922 and Cfvi3 in all other tests (Table S2). Finally, the C. hyointestinalis strain tested positive to growth at 42 °C and oxidase activity and showed intolerance to 3.5% NaCl. However, it did not produce catalase, which is discordant with the expected result according to WOAH (Tables S1 and S2). Concerning the field isolates, all of them were oxidase and catalase producers and did not tolerate 3.5% NaCl. However, variable results were observed regarding their ability to grow at 42 °C. Thus, 45.5% (5/11) and 56.8% (25/44) of the field isolates biochemically characterized as Cfv and Cfvi, respectively, were able to grow at 42 °C, a typical characteristic of intestinal Campylobacter species. Finally, 100% of the Cff field isolates showed the same results as the control Cff isolates based on these tests (Table S2).

Identification of C. fetus subspecies and biovar by PCR assays

Identification of C. fetus control strains at the subspecies level produced the same results (2 Cfv and 11 Cff) by either using PCR-1 or PCR-2 (Table 1). The C. hyointestinalis control strain was considered as non-C. fetus by both PCRs. It should be noted that this strain showed cross-reaction in PCR-2, i.e., amplification of the ISC2 target (Table 1 and S2). For field isolates, 3 (4.7%) were identified as Cff and 61 (95.3%) as Cfv (Table 1). Isolates classified by PCR-1 and PCR-2 as Cfv were analysed by PCR-3 to identify them at biovar level. The control strain Cfv ATCC 25922 and Cfvi3 were identified properly by PCR-3. For the field isolates, 52 (81.3%) were identified as Cfvi and 9 (14%) as Cfv “sensu stricto” (Table 1).

Agreement between phenotypic and molecular identification tests

A moderate concordance (κ = 0.462; 0.256–0.669 CI) was observed between phenotypic and PCR identification tests for C. fetus subspecies identification (Table S3A). Discrepancies were found for 6 field isolates that were identified as Cfv by PCR but were classified phenotypically as Cff isolates (i.e., tolerance to 1% glycine and H2S production after 72 h and 5 days) (Table 1). Concerning the identification at biovar level, discordances were observed in the results obtained by the PCR-3 versus the H2S production at 72 h and 5 days. While at 72 h, 10 field isolates showed no H2S production, only 2 of these isolates (C1.43 and C3.5) remained as non-H2S producers at day 5 (Table 1). Therefore, the agreement between the PCR-3 and the H2S production test was moderate when H2S production was assessed after 72 h (κ = 0.553, 0.329–0.778 CI) and very good at day 5 (κ = 0.881, 0.631–1.000 CI) (Table S3B).

As described in the previous section, both PCR-1 and PCR-2 (Table 1) used for the identification of Cff and Cfv isolates showed 100% agreement (κ = 1.000; 0.755–1.000 CI) (Table S3C).

C. fetus subspecies identification by WGS

The complete genome of the eight field isolates that showed discordant results in their identification by the phenotypic and PCR tests were sequenced. Taxonomic identification by KmerFinder showed a query coverage (the percentage of input query k-mers that match the template) of over 94.5% with strain CfviADRI545 (RefSeq: GCF_011601375.2) for all of them (Table 2). These taxonomic results were 100% concordant with the results obtained in the identification of these isolates by PCR-1 and PCR-2. The genes that code for the L-Cyst transporter (ATP-Bp, yckJ and yckK) were present in all isolates (100% coverage) and shared 100% identity in all cases except for the yckK gene in isolate C1.43 that had a mutation at position 2827 (Table 2). This mutation involved a Cytosine (C) to Thymine (T) transition, resulting in an amino acid change from serine to proline. However, this mutation has been observed in other H2S producing isolates (personal communication), suggesting that it does not involve significant changes in the functionality of the L-Cyst transporter. These results obtained from taxonomy analysis are therefore in perfect agreement with the amplification results obtained by PCR-3. In addition, the cysK gene, involved in the pathway of cysteine synthesis and H2S production, showed no mutations in the coding region or in its putative promoter region.

Table 2 Comparison of subspecies identification results using the different techniques (biochemistry, PCR, WGS, and gene sequencing) for discrepant C. fetus isolates
Full size table

Identification of Campylobacter in smegma field samples by MALDI-TOF and evaluation of the specificity of C. fetus molecular diagnostic methods

Seventy-five field isolates with morphology and motility resembling Campylobacter were obtained from smegma samples from 750 breeding bulls and identified by MALDI-TOF analysis as Arcobacter butzleri (n = 2), Arcobacter cryaerophilus (n = 4), C. hyointestinalis (n = 2), C. sputorum (n = 62) and C. fetus (n = 5). No cross-amplification reactions were observed for the non-C. fetus isolates when analysed by PCR-1 and PCR-2 (Table S4).

Discussion

The accurate differentiation between the mammalian-associated C. fetus subspecies (Cff and Cfv/Cfvi) is crucial, since only Cfv and Cfvi cause BGC, a bovine sexually transmitted disease that produces infertility and great economic losses in the beef cattle sector [4, 5, 8, 17]. Several diagnostic methods are currently available to differentiate the C. fetus subspecies but none of them are 100% accurate due to the high homology between their genomes [2]. Biochemical tests (1% glycine tolerance and H2S production) [2, 18] and PCR amplification targeting the C. fetus-specific nahE gene and the Cfv-specific insertion element ISCfe1 [14, 15] are the methods most commonly used for subspecies identification. However, several studies have revealed inconsistencies between phenotypic and molecular typing methods for the identification of C. fetus subspecies [19,20,21,22].

In this study, 64 Spanish C. fetus field isolates from breeding bulls were identified by phenotypic and PCR methods and the consistency of the results was compared. Considering the variability among isolates within each subspecies, only the results of the 1% glycine tolerance test, crucial for C. fetus subspecies differentiation (Cff/Cfv), and the H2S production test, that differentiates the biovars (Cfv/Cfvi), were considered for the final identification, as recommended [1, 2]. Here, a moderate concordance was observed between phenotypic and PCR for subspecies identification. The field isolates showing incongruent results were those phenotypically identified as Cff due to their tolerance to glycine and H2S production, despite carrying the ISCfe1 insertion element characteristic of Cfv. This finding was also observed in the control strain Cfvi3, which is a glycine tolerant Cfvi strain. Previous studies have revealed the existence of Cfv isolates with the ability to grow on media supplemented with 1% glycine or higher, due to the acquisition of glycine tolerance related genes through phage mediated transduction or mutational processes [13, 19, 23]. In addition, glycine sensitive Cff isolates have also been described [23].

Cfvi is a phenotypic variant of Cfv with biochemical characteristics intermediate between Cfv and Cff [1]. H2S production allows differentiation between Cfv “sensu stricto” and Cfvi. C. fetus, and particularly Cfv, are slow-growing microorganisms that often need more than 48 h to develop well-formed colonies [1], therefore the WOAH recommends an incubation time of 72 h to assess H2S production [2]. In this study, we observed that the total number of H2S producing isolates increased by 13% when H2S production was measured after 5 days compared to 72 h, which could be related to its low metabolic activity. In fact, C. fetus has been described as a low or no H2S-producing species on standard media such as Triple Sugar Iron (TSI) and more sensitive and specific media are needed to detect its production [1, 24]. Therefore, the use of longer incubation periods under aseptic conditions seems to improve Cfv biovar identification. Actually, agreement between PCR-3 and H2S production was better when the latter was assessed at 5 days. However, after 5 days, two isolates still remained as H2S non-producers despite showing a Cfvi PCR pattern (i.e., 714 bp amplicon), suggesting the presence of a complete and functional L-Cyst transporter [16, 25].

In the present study, most Cfv isolates (81.3%) were identified as Cfvi, evidencing a predominance of this biotype in Spain, as described in South Africa and Argentina [26, 27]. A recent study, showed higher infectivity and long-term persistence of Cfvi in cattle compared to Cff and Cfv, suggesting better adaptation to the bovine genital tract or possible evasion of the immune response in cattle [28]. That study also reported differences in pregnancy rates in infected cows, being higher in Cfvi than in Cfv. Although genomic differences between Cfv and Cfvi have been identified [29], further studies are needed to determine which pathogenicity genes could be directly related to these differences in pregnancy rates.

Field isolates with inconsistent results were subjected to WGS. Several studies showed that WGS is an accurate method to correctly characterize C. fetus subspecies and to investigate their differences [5, 30, 31]. The taxonomic identification derived from genome analysis was in perfect agreement (100%) with the results obtained by PCR characterization of C. fetus subspecies. However, it is important to note that the ISCfe1 insertion element used in PCR for Cfv identification can be transferred to other bacteria through bacterial conjugation, which could result in specificity issues [14, 32]. In addition, genome sequencing of the two H2S non-producing Cfvi isolates showed that they carried the complete L-cysteine transporter with no mutations in neither the genes nor the promoter region that could explain the non-production of H2S. To investigate the lack of H2S-production of these isolates, we analysed the cysk gene, closely related to the cysteine biosynthesis pathway in C. jejuni or the degradation of cysteine excess and H2S production in Escherichia coli [33, 34] that is also present in C. fetus. However, no mutations were found in this gene or its promoter region. Alternatively, the non-production in these Cfvi isolates could be partly related to the low levels of biochemical activity of C. fetus [2]. Nonetheless, further investigations regarding this biosynthetic pathway are needed to explain these results.

Here, 75 isolates obtained from the preputial area of breeding bulls were identified by MALDI-TOF as A. butzleri, A. cryaerophilus, C. hyointestinalis, C. sputorum and C. fetus. In cattle, these species may be part of the commensal microbiota. Arcobacter and C. hyointestinalis are mainly isolated from faecal samples of healthy cattle, although Arcobacter isolates have also been detected in preputial and bovine abortions, suggesting their implication in reproductive disorders in cattle [35,36,37,38,39]. The presence of these species in preputial samples might be due to faecal contamination [35]. On the other hand, C. sputorum is a commensal bacterium often found in genital tissues of healthy cattle and usually shares an ecological niche with Cfv [40]. In this study the most frequently isolated species was C. sputorum (82.6%), while only 6.6% of the isolates were identified as C. fetus. This low proportion of C. fetus could be explained because most of the bulls were sampled for routine control and had no reproductive problems. In addition, the physiological conditions and special nutritional requirements of C. fetus make it a poor competitor compared to other species, hampering its isolation [7, 41]. MALDI-TOF has gained importance in recent years in the diagnosis of microorganisms, due to its speed, accuracy and low-cost [42]. In this study, the application of the MALDI-TOF could only identify C. fetus isolates to the species level. Emele et al. [42] successfully differentiated between C. fetus isolates from reptiles (C. fetus subsp. testudinum) and mammals (Cff/Cfv) using MALDI-TOF, but differentiation between Cff and Cfv is not currently possible, suggesting that they also share a high similarity in their ribosomal protein spectra.

The transference of genetic material between bacteria inhabiting the same environment can be challenging for PCR-based identification methods. In fact, specificity problems due to cross-reactivity have been reported in most PCR methods available for C. fetus subspecies differentiation [14, 32]. Even the most efficient methods, such as those targeting the nahE gene and the ISCfe1 insertion element, specificity problems have been reported associated to C. hyointestinalis isolates carrying the ISCfe1 insertion element [14]. Here, none of the 70 field isolates tested showed cross-amplification reactions by PCR. However, only 2 C. hyointestinalis isolates were analysed. Thus, although these molecular identification methods seem to be reliable, it is important to emphasise the importance of combining targets specific for C. fetus (nahE) and Cfv (ISCfe1) to avoid misidentification, which at farm level could result in increased costs due to unnecessary treatment and culling of animals.

Conclusions

This study confirmed the limitations of biochemical tests to correctly identify C. fetus subspecies and biovars. Yet, we showed that when prolonged incubation times (5 days) were used for H2S production tests, phenotypic identification was significantly improved. Instead, PCR-based methods (PCR-1 or PCR-2 for subspecies identification and PCR-3 for biovar identification) produced more accurate results. WGS and sequence analysis of the L-Cyst ABC Transporter and cysK genes, directly related to the H2S production, showed perfect agreement with PCR-based biovar identification results. WGS also confirmed the presence of the ISCfe1 insertion element in all Cf subspecies venerealis isolates with discordant biochemical-PCR results thus confirming the PCR results for subspecies identification. However, cross-reactions can occur when the ISCfe1 insertion element is acquired by other Campylobacter species, most commonly C. hyointestinalis. Nevertheless, other saprophytic microaerophilic bacteria that can be present in preputial samples did not cross-react. The identification of subspecies-specific genes would help to improve the diagnosis of BGC.

Methods

Campylobacter fetus isolates

A collection of 77 C. fetus strains were used to compare phenotypic and molecular methods for both C. fetus subspecies and biovar identification. The collection included a reference strain from the American Type Culture Collection (ATCC) and a panel of 12 well-characterized strains obtained from the Faculty of Veterinary Medicine (Utrecht University, The Netherlands, National Reference Laboratory for Animal Campylobacteriosis) and NEIKER (Basque Country, Spain), that were used as positive controls and comprised: 1 Cfv and 1 Cfvi strains from bovine genital mucosa, and 6 and 5 Cff strains from bovine and ovine faecal origin, respectively. In addition, a C. hyointestinalis strain isolated from pig intestine was used as a negative control in the biochemical and molecular identification tests of C. fetus subspecies (Table 1). Another 64 field isolates, primarily obtained from preputial samples from breeding bulls subjected to BGC diagnosis in Spain, were obtained from the isolate collection of SALUVET laboratory (which provide services for BGC and bovine trichomonosis diagnosis to private veterinary practitioners in Spain) and the Spanish Reference Laboratory for Animal Campylobacteriosis: Laboratorio Central de Veterinaria (LCV)-Algete (MAPA: Ministry of Agriculture, Fisheries and Food, Madrid, Spain). LCV provides the abovementioned diagnostic services to official artificial insemination centres to comply with the animal health and traceability requirements for the authorisation of intra-Community movements of bovine-derived germ products. Metadata and previous identification of all isolates used are summarized in Table S5.

Bacteriological culture and biochemical characterization of C. fetus isolates

All C. fetus isolates were subcultured on Columbia Agar plates supplemented with 5% sheep blood (Cos, Biomerieux, Marcy-l’Étoile, France) for 48 h at 37 ºC under microaerophilic conditions (5% O2, 10% CO2 y 85% N2, GENbox Microaer, Biomerieux, Marcy-l’Étoile, France) to obtain pure colonies. The phenotypical classification of the C. fetus isolates at the subspecies level was performed following standard protocols described by WOAH (Table S1) [2], as follows: growth at 42 °C, oxidase, catalase, H2S production in cysteine medium and tolerance to 3.5% sodium chloride (NaCl). The H2S production was measured both at 72 h and 5 days post inoculation. In order to avoid possible contamination that could alter the results, isolates were cultured under aseptic conditions in a hermetically closed anaerobiosis box, with an uninoculated vial as a negative growth control. Glycine tolerance was assessed on solid medium plates following the recommendations described elsewhere [43], with some modifications in medium composition: Nutrient Broth No. 2 (Oxoid, Hants, United Kingdom), 3% bacteriological agar (Bacto™ Agar, BD, Le Pont de Claix, France), 1% glycine (Glycine ≥ 99% molecular grade, Fisher Scientific, Waltham, MA USA) and 5% defibrinated sheep blood. For the growth test at 42 °C and tolerance to 1% glycine and 3.5% NaCl, all isolates showing a clear bacterial growth were considered positive. A colour changes from translucent to bluish was considered positive for the oxidase test. All isolates showing bubble formation on contact with H2O2 at 10 volumes were considered positive for catalase activity. Isolates that resulted in blackening of the lead acetate strips 5 days after inoculation were considered H2S producers.

Isolates were classified as Cfv when they showed negative results for both the 1% glycine tolerance and the H2S production tests; Cff when they showed 1% glycine tolerance independently of the H2S production; and Cfvi when they showed H2S production and glycine intolerance [1, 7, 19].

DNA extraction and PCR assays

DNA extraction was performed from single colony cultures following the protocol for gram-negative bacteria of the DNeasy Blood and tissue kit (QIAGEN, Hilden, Germany). Identification of the isolates at subspecies level was carried out by a PCR (PCR-1) targeting the C. fetus-specific nahE gene and the Cfv-specific ISCfe1 insertion element using the following conditions: 1 × PCR buffer, 2.5 mM MgCl2, 0.1 mM of each dNTP, 0.6 µM of each primer and 1 U of Platinum™ Taq DNA Polymerase, 5 μl of DNA template, adjusted to 25 μl with nuclease-free water [15]. PCR-1 was performed in separate reactions for each target on a BioRad T100 thermal cycler using the cycling parameters described in the original publication by Abril et al. [15]. In addition, a multiplex real-time PCR (PCR-2) was performed targeting both the nahE gene and a different region (ISC2) of the ISCfe1 insertion element to rule out any misidentification resulting from the presence of mutations in the primer binding site of the ISC1, as described previously by van der Graaf et al. [14]. Each PCR reaction contained 10 μl of 2 × TaKaRa Ex Taq® DNA, 0.2 μl of ROX, 600 nM of each primer, 200 nM of each probe and 2 μl of DNA template, adjusted to 20 μl with nuclease-free water. The PCR-2 was carried out in a QuantStudio™ 5 Real-Time PCR instrument (Applied Biosystems), using the following cycling parameters: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. Isolates were classified as Cff for each PCR when the nahE target was amplified in the absence of ISCfe1 amplification. They were identified as Cfv when positive for both targets, and as non-C. fetus when they were negative for both targets. Moreover, isolates negative for nahE and positive for ISCfe1 were considered as cross-reactions and therefore non-C. fetus, as previously described by Spence et al. [44].

Differentiation between Cfv and Cfvi isolates was performed by the PCR (PCR-3) described by Farace et al. [16] that targets the L-Cyst transporter containing ATP-Binding protein (ATP-Bp), permease protein (YckJ) and extracellular binding protein (YckK) coding genes. This transporter is directly related to H2S production and remains complete in Cfvi and Cff while Cfv strains show deletion of YckJ and partial deletion of YckK coding genes which makes them non-functional. The amplification mix consisted of 1 × PCR buffer, 1.5 mM MgCl2, 0.25 mM of each dNTP, 0.1 µM of each primer and 1.25 U of Platinum™ Taq DNA Polymerase, 5 μl of DNA template, adjusted to 25 μl with nuclease-free water. PCR-3 was performed on a BioRad T100 thermal cycler with the thermal cycler programme described in the original publication [16]. Isolates previously identified as Cfv by PCR-1 and PCR-2 were identified as Cfvi when they yielded a 714 bp amplicon in PCR-3 and as Cfv “sensu stricto” when the amplicon was 310 bp.

Whole-Genome Sequencing and C. fetus subspecies identification

The whole genome of all C. fetus field isolates showing discordant results (n = 8) between biochemical and molecular identification was sequenced in a commercial facility (Eurofins genomics, Ebersberg, Germany). Libraries based on the NEBNext Ultra™ II FS DNA Library Prep Kit (Illumina) were prepared and sequenced using Illumina NovaSeq 6000 System to generate 2 × 150 bp paired-end reads. The preprocessing of the raw reads (quality check and filtering) was carried out as previously described [45]. Genome de novo assembly was performed using SPAdes v.3.15.3 [46] and the quality of the assemblies was assessed with QUAST v.5.0.2 [47], discarding contigs < 500 bp with PRINSEQ v.0.20.4 [48]. C. fetus subspecies taxonomic classification of the genomes was carried out by exact alignment of k-mers with KmerFinder v.3.0.2 [49,50,51] (database version 2022–07-11) by submitting the draft genome to the CGE website (https://cge.cbs.dtu.dk/services/KmerFinder, DTU Bioinformatics, Technical University of Denmark). Only reference strains identified by KmerFinder as predicted species with sequence coverage values above 80% were considered.

In addition, BLASTn v.2.10.1 + [52] and ABRicate v.1.0.1 (T. Seemann, https://github.com/tseemann/abricate) were used to screen for the sequences that encode for the three molecular components of the L-Cyst transporter (ATP-Bp, YckJ and YckK) and the cysteine synthase (CysK). For this, a custom nucleotide database was previously generated by extracting target regions from the reference strain Cff 82–40 (specifically ATP-Bp locus tags CFF8240_RS03845, yckJ CFF8240_RS03850, yckK CFF8240_RS03855, and cysK CFF8240_0922). When the sequence identity of the genes encoding the L-Cyst transporter or cysK was less than 100%, the sequences were aligned to the Cff 82–40 reference strain using Geneious Prime® (2023.0.3 v11.0.15 + 10).

Isolation and identification of saprophytic microaerophilic bacteria in preputial samples from Spanish bulls

To further evaluate the specificity of the PCR-1 and PCR-2 assays for C. fetus subspecies identification, other saprophytic microaerophilic bacteria with morphology and motility resembling Campylobacter were isolated from smegma samples collected from 570 breeding bulls for routine STD diagnosis [10]. Briefly, the smegma samples were collected by preputial scraping of bulls in the field by veterinary practitioners, deposited in 5 ml tubes with Campylobacter transport medium, and sent to the SALUVET laboratory at room temperature within 24 h of collection. Campylobacter transport medium was formulated using thioglycolate broth (Sigma Aldrich) supplemented with Campylobacter growth supplement (Oxoid) and 7% foetal bovine serum (Thermo Fisher Scientific) without antibiotics. Upon arrival at the laboratory, samples were incubated at 36 °C for 24 h. After the incubation period, a sterile 0.65 µm filter was placed on Columbia Agar plates supplemented with 5% sheep blood (Cos, Biomerieux, Marcy-l'Etoile, France) and 200 µl of the upper portion of the Campylobacter transport medium were inoculated into the centre of the filter. The filter was then removed, and the filtered sample was spread evenly over the entire surface of the agar plate using a sterile inoculation loop. The plates were then incubated for 48 h at 37 ºC under microaerophilic conditions (5% O2, 10% CO2 y 85% N2, GENbox Microaer, Biomerieux, Marcy-l’Étoile, France). Grown colonies were microscopically examined at 40 × magnification. Those showing morphology and motility resembling Campylobacter spp. were subcultured on Columbia Agar plates supplemented with 5% sheep blood (Cos, Biomerieux, Marcy-l'Etoile, France) and incubated for 48 h at 37 ºC under microaerophilic conditions. Subcultured isolates were then analysed by matrix assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry at the Microbial Identification and Characterization Unit at the Complutense University of Madrid (Spain) following the procedures detailed in Perez-Sancho et al. [53].

Statistical analysis

Agreement between the results of the different assays for C. fetus subspecies and biovar differentiation was evaluated using the Cohen’s Kappa coefficient with a confidence level of 95%. To assess the strength of agreement between techniques the kappa values obtained were interpreted as follows: κ ≤ 0.20 = poor, 0.21–0.40 = fair, 0.41–0.60 = moderate, 0.61–0.80 = good and 0.81–1.00 = very good [54].

Availability of data and materials

The Whole Genome Sequencing project of Campylobacter fetus is deposited at GenBank under the BioProject accession number PRJNA1019261, which includes the 8 Campylobacter fetus genomes listed under the BioSample codes described in Table S6. Other datasets used and/or analysed during the current study are available from the corresponding author upon request.

Abbreviations

Cfv :

Campylobacter fetus subsp. venerealis

Cfvi :

Campylobacter fetus subsp. venerealis bv. intermedius

Cff :

Campylobacter fetus subsp. fetus

BGC:

Bovine Genital Campylobacteriosis

STD:

Sexual transmitted disease

WOAH:

World Organisation for Animal Health

H2S:

Hydrogen sulphide

L-Cyst:

L-cysteine

WGS:

Whole Genome Sequencing

NaCl:

Sodium chloride

ATCC:

American Type Culture Collection

LCV:

Laboratorio Central de Veterinaria

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Acknowledgements

The authors express their gratitude to Dr. Linda van der Graaf, from the WOAH reference laboratory for Campylobacteriosis at the University of Utrecht and to Montserrat Agüero from LCV-Algete (MAPA), for generously providing the bacterial strains. In addition, thanks to Zuriñe Pérez and Jose Carlos Ibabe (NEIKER diagnostic laboratory), for their invaluable technical support and assistance. We also express our more sincere gratitude to F.J. Javier Garcia-Peña (LCV-Algete) for the Campylobacter field strains isolation and to Professor Dr. L.M. Ortega-Mora (SALUVET) for the review of our manuscript. Thank you to both for being the pioneers in the study of BGC in Spain and their invaluable contribution to BGC knowledge. Finally, thanks to farmers and veterinarians for bull sampling, especially to Dr. J.M. San-Miguel (Zoetis, Spain S.L.).

Funding

This study was supported by several research projects (RTA2017-00076–00-00 funded by MCIN/AEI/10.13039/501100011033 and by FEDER “A way to do Europe” and PLATESA2-CM P2018/BAA-4370) and the grant for doctoral formation (Pre2018-086113 funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”) awarded to PhD student Nerea Pena Fernández.

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Author notes

  1. Esther Collantes-Fernández and Gorka Aduriz contributed equally to this work and share last authorship.

Authors and Affiliations

  1. SERIDA, Servicio Regional de Investigación y Desarrollo Agroalimentario, Carretera de Oviedo, S/N, Villaviciosa, 33300, Spain

    Nerea Pena-Fernández

  2. Department of Animal Health, NEIKER-Basque Institute for Agricultural Research and Development, Basque Research and Technology Alliance (BRTA), Parque Científico y Tecnológico de Bizkaia, P812, Derio, 48160, Spain

    Nerea Pena-Fernández, Nekane Kortabarria, Ana Hurtado, Medelin Ocejo & Gorka Aduriz

  3. SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria S/N, Madrid, 28040, Spain

    Marcelo Fort & Esther Collantes-Fernández

  4. Central Veterinary-Animal Health Laboratory (LCV), Ctra. Madrid-Algete Km. 8.00, Algete, 28110, Spain

    Iratxe Pérez-Cobo

  5. Faculty of Veterinary Sciences, SALUVET-Innova S.L, Complutense University of Madrid, Ciudad Universitaria S/N, Madrid, 28040, Spain

    Esther Collantes-Fernández

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Contributions

GA and ECF conceived the study and participated in its design. NPF performed the biochemical and PCR tests and interpreted the results with NK. NPF, MO and AH performed the whole genomes analysis and interpreted the results. MF isolated the Campylobacter spp. strains from bull preputial samples and identified them by MALDI-TOF. NPF and NK wrote the manuscript, with interpretation of results, material and methods and discussion inputs from GA, ECF, MO, AH, MF and IP. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Esther Collantes-Fernández or Gorka Aduriz.

Ethics declarations

Ethical approval and consent to participate

Informed oral consent was obtained from owners at the time of sample collection. The smegma samples were collected by preputial scraping of bulls in the field, following the WOAH recommended sampling method by certified veterinary practitioners as a part of normal veterinary care and diagnostic investigations. Therefore, ethical approval by an ethics committee was not necessary, as established in the Spanish RD 53/2013 of 1 February, article 2, Sect. 5, which establishes the basic rules applicable to the protection of animals used for experimental and other scientific purposes, including teaching, and the practices excluded from its scope of application.

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The authors declare no competing interests.

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Supplementary Information

Additional file 1: Table S1.

Differential characteristics of Campylobacter spp. published in The Manual of Diagnostic Tests and Vaccines for Terrestrial Animals: Bovine genital campylobacteriosis; Chapter 3.4.4 [2]. Table S2. Phenotypic and molecular results of C. fetus isolates included in this study. Table S3A. Assessment of the concordance between phenotypic and molecular (PCR) tests for C. fetus subspecies identification. Table S3B. Assessment of the concordance between phenotypic and molecular (PCR) tests for Cfv biovars identification. Table S3C. Assessment of the concordance between molecular (PCR) tests for C. fetus subspecies identification. Table S4. Identification of isolates recovered from bull preputial smegma with morphology and motility resembling Campylobacter used in this study. Table S5. Metadata of all C. fetus isolates used in this study. Table S6. NCBI accession data of the eight C. fetus isolates sequenced in this study.

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Pena-Fernández, N., Kortabarria, N., Hurtado, A. et al. Biochemical and molecular characterization of Campylobacter fetus isolates from bulls subjected to bovine genital campylobacteriosis diagnosis in Spain. BMC Vet Res 20, 131 (2024). https://doi.org/10.1186/s12917-024-03970-8

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