Virulence and antibiotic resistance genes in Campylobacter spp. in the Czech Republic

Summary

Objective:
Thermotolerant species of the genus Campy-lobacter are the important agents causing human foodborne infections throughout the world. The aims of this study were to evaluate the presence of nine putative virulence genes in Campylobacter spp. isolated from patients and from foods (poultry meat, pork liver), to determine the resistance of Campylobacter isolates to eight antibiotic agents and to detect four resistance genes.

Matherial and methods:
The presence of the virulence genes cdtA, cdtB, cdtC, virB11, ciaB, wlaN, iam, dnaJ and racR was detected by polymerase chain reaction (PCR) in 94 Campylobacter spp. isolates from humans and 123 campylobacters from foods. The phenotypic resistance to selected antimicrobial agents was tested with microdilution method in 82 human isolates and 91 food isolates. The isolates with antibiograms were tested for the presence of bla_OXA-61, tet(O), aph-3-1 and cmeB genes by PCR with specific primers.

Results:
In both human and food C. jejuni isolates the preva-lence of the studied virulence genes, especially dnaJ, racR, ciaB genes and the toxigenic genes cdtA, cdtB, cdtC, was considerably higher than in C. coli isolates. The only exception was the iam gene identified in only C. coli. The tested isolates of both C. jejuni and C. coli were highly resistant to quinolone antibiotics. Additionally, C. coli was also more resistant to erythromycin, streptomycin and, in case of isolates from pork liver, to tetracycline. High prevalence rates of genes encoding antibiotic resistance was noted for the bla_OXA-61 and tet(O) genes in both Campylobacter species.

Conclusions:
The presented study is the first to assess the presence of genes for virulence and resistance to antibiotics in thermotolerant Campylobacter spp. isolated from humans and foods in the Czech Republic. The resistance of Campylobacter isolates to eight antibiotic agents was also assessed. The prevalence of genes responsible for virulence and resistance is rather varied in thermotolerant Campylobacter spp.

KEYWORDS:
Campylobacter – foodborne infections – virulence genes – resistance genes

INTRODUCTION

Members of the genus Campylobacter are the main pathogens responsible for acute bacterial gastroenteritis in humans throughout the world. Most of the infections are caused by Campylobacter jejuni (approximately 90%), followed by C. coli (approximately 10%). Foodborne infections due to C. lari and C. upsaliensis are sporadic [1]. Worldwide, approximately 400 million people a year contract the infection. In developing countries, up to 60% of children younger than 5 years become ill with Campylobacteriosis [2].

The infectious dose of Campylobacter infections is relatively low and has been estimated at as few as 500 cells [3, 4]. The typical incubation period ranges between 3 to 5 days. The pathogenesis of this human foodborne infection has not been fully elucidated. The survival of Campylobacters in the acid stomach environment upon ingestion can be affected by the buffer capacity of the consumed food. The colonization starts in the jejunum and upper ileum and then spreads to the rest of the ileum and colon. Campylobacters have to overcome the intestinal mucosa, adhere to the epithelial cells and enter them. It is assumed that this is essential for inducing diarrhea [1].

The abilities of Campylobacter spp., mainly to adhere to, colonize and invade the intestinal wall and to produce toxins, are encoded by numerous genes responsible for the virulence of their strains. For instance, the genes flaA, cadF, racR, and dnaJ are responsible for intestinal adherence and colonization. The genes virB11, ciaB, iam and pldA are responsible for invasiveness [5, 6]. In vitro studies have shown that virB11 gene of C. jejuni strains is associated with both adherence and invasion [7]. The gene wlaN is responsible for mimicry leading to post-infectious complications in the form of Guillain-Barré syndrome [5]. Another virulence factor that has been proposed to play a role in the pathogenesis is the cytolethal distending toxin (CDT). This cytotoxin consists of three subunits which are encoded by cdtA, cdtB a cdtC genes, and all three subunits are necessary for full activity [8, 6].

The treatment of human Campylobacter infections is usually symptomatic; in case of antibiotic therapy, the recommended agents are macrolides or possibly ciprofloxacin [1]. However, many studies in Europe reported high percentages of Campylobacter spp. strains isolated from both humans and animals that were completely resistant to quinolones [9]. Tetracyclines have been mentioned as alternative antibiotics for therapy, but they are not used in practice [10].

The targets of quinolone antibiotics are enzymes (namely gyrases/topoisomerases) playing an important role in bacterial synthesis of DNA. The resistance to quinolones in Campylobacter spp. is usually caused by mutations in specific regions of target enzymes, but there are some other mechanisms of resistance such as efflux pumps. These systems also play a role in resistance to other antimicrobials, for example macrolides [11]. In Campylobacter spp., the CmeABC pump has been described as the main efflux mechanism causing resistance to several classes of antibiotics (beta-lactams, erythromycin, tetracycline) [12]. The resistance of Campylobacter spp. to antimicrobial agents is not associated with mutations of target structures or efflux systems only as several resistance genes have been described as well. These are, for example, genes encoding modifying enzymes (aph), beta-lactamase (bla_OXA-61) or ribosomal protection proteins (tet(O)) which are linked with resistance to aminoglycosides, beta-lactams and tetracyclines, respectively [10].

The aims of this study were to evaluate the presence of nine putative virulence genes in Campylobacter spp. isolated from patients and from foods (poultry meat, pork liver), to determine the resistance of Campylobacter isolates to eight antibiotic agents and to detect four resistance genes.

MATERIALS AND METHODS

Sampling

Between May 2013 and December 2014, samples of fresh chicken, frozen chicken, fresh pork liver and raw cow’s milk were regularly collected at 2-month intervals for Campylobacter spp. testing. Both poultry (n = 209) and pork liver (n = 103) samples were collected in randomly selected large supermarkets and raw cow’s milk samples (n = 110) were obtained from milk vending machines. Over the above period, samples were taken on 10 occasions, with each set comprising approximately 20 poultry, 10 liver and 11 milk samples. All the meat and milk samples to be tested were normally purchased from supermarkets and vending machines in Moravia, the eastern part of the Czech Republic. Thus, commodities directly entering the consumers’ food chain were included in the study. Between May 2013 and December 2014, human isolates of Campylobacter spp. (n = 235) were obtained from rectal swabs taken from patients with diarrhea. The isolates originated from hospital laboratories (University Hospital Olomouc, University Hospital Brno, St. Anne´s University Hospital Brno, Hospital Prostějov) and laboratories performing tests to detect diarrheal diseases in the above community (Mikrochem Laboratories Olomouc, Laboratories IFCOR-99 Brno). The territory of operation of these laboratories are The Olomouc Region and The South Moravian region, Czech Republic. From each patient, only one isolate was included. Although data on gender, age and primary diagnosis were available for all patients, these were not evaluated in the study.

Detection, isolation and identification of thermophilic Campylobacter

Food samples were always delivered to the laboratory on the day of collection. The method for Campylobacter spp. detection was based on ISO 10272-1 (qualitative testing) [13]. The samples were in the form of 25 g of skin collected from chicken necks, 25 g of pork liver or 25 ml of raw milk. Culture media as recommended by the above norm were manufactured by Trios and Oxoid. For identification purposes, suspected isolates were inoculated onto blood agar (Trios), and following 48-hour microaerophilic incubation at 42.5 °C, they were identified using the MALDI-TOF MS method (Biotyper Microflex, Bruker). In case of inconclusive results (identification scores < 2), PCR methods with a commercial kit for real-time PCR (Taq Man Campylobacter spp. Kit, AB Applied Biosystems) were used [14, 15]. Human isolates of Campylobacter spp. had already been identified by the external laboratories that provided them. Prior to further testing, however, their identification was confirmed by MALDI-TOF MS. Quality control was performed using the C. jejuni reference strain ATCC 33560.

Detection of virulence genes

To test the presence of the particular virulence genes, a total of 94 human isolates (C. jejuni – 73, C. coli – 21) and 123 food isolates (C. jejuni – 70, C. coli – 53) were selected. The food isolates were only from poultry and from pork liver as no Campylobacter spp. were detected in milk samples. Genetic detection of 9 selected virulence genes which play a part in Campylobacter virulence was performed using PCR with a set of specific primers for determining the presence of the cdtA, cdtB, cdtC, virB11, wlaN, ciaB, iam, dnaJ and racR genes. The PCR conditions for all the above genes have been previously described [5].

Antibiotic susceptibility

In confirmed Campylobacter spp. isolates, resistance to selected antimicrobial agents was tested with the microdilution method [16]. The tests were performed on microtitration plates in solutions of the particular antibiotics and Mueller-Hinton broth with 2.5% lysed horse blood (Trios). The inoculated plates were incubated in a microaerophilic atmosphere (GENbox microaer, BioMérieux) at 37 °C for 48 hours. Resistance to the following selected antibiotics was tested: erythromycin, ciprofloxacin, tetracycline, streptomycin, gentamicin, chloramphenicol, ampicillin and nalidixic acid. The parameters for individual antibiotics, including interpretation criteria, were based on recommendations issued by the EU Reference Laboratory – Antimicrobial Resistance and Antibiogram Committee of the French Microbiology Society [17, 18]. Quality control was performed at regular intervals using the C. jejuni reference strain ATCC 33560. The above approach was used to test 82 human isolates (C. jejuni – 59, C. coli – 23) and 91 food isolates (C. jejuni – 48, C. coli – 43). The detailed parameters for testing are shown in Tables 1 and 2.

Detection of antibiotic resistance genes

The isolates with antibiograms were tested for the presence of blaOXA-61, tet(O), aph-3-1 and cmeB genes. These genes were detected using PCR with specific primers [19]. These tests were carried out in 59 human, 48 poultry and 2 pork liver isolates of C. jejuni and 23 human, 30 poultry and 13 pork liver isolates of C. coli. Given the small number of C. jejuni isolates obtained from pork liver, the relationship between phenotypic and genotypic resistance was not further assessed in this subgroup.

Statistical analysis

Fisher’s exact test was used to compare the frequencies of virulence genes in C. jejuni and C. coli in human and poultry isolates. The same test was used to compare the frequencies of the bla_OXA-61 gene in ampicillin-susceptible and -resistant human and poultry isolates as well as of the tet(O) gene in tetracycline-susceptible and -resistant isolates from patients, poultry and pork liver. For multiple comparisons, Fisher’s exact test with Bonferroni correction was used. IBM SPSS Statistics version 22 was used to analyze the data. A significance level less than 0.05 was considered statistically significant (p < 0.05).

RESULTS

Prevalence of virulence genes

A total of 56% of poultry samples (68% of fresh chicken, 44% of frozen chicken) were found to contain Campylobacter spp. Pork liver samples were contaminated in 24% of cases. The raw cow’s milk samples were negative. Campylobacters identified in individual foods showed species specificity. While C. jejuni was more prevalent in poultry meat (67.2% of isolates), C. coli prevailed in pork liver (80.0% of isolates). Among 235 human isolates, most belonged to C. jejuni (88.9%). That is why the tested human isolates of C. coli are low in numbers. The percentages of the virulence genes in selected human and food C. jejuni and C. coli isolates are shown in Table 3.

The results suggest that the studied virulence genes are more prevalent in C. jejuni than in C. coli. This is particularly apparent in genes encoding CDT, which were detected in the vast majority of the tested isolates of C. jejuni obtained from both humans and foods. Most C. jejuni isolates carried 5 or 6 virulence genes simultaneously (83.6% of human isolates, 90.0% of food isolates), as opposed to C. coli isolates in which, with a few exceptions, only the iam gene was detected (85.7% of human isolates, 88.7% of food isolates). There were no considerable differences in the frequency of most virulence genes between isolates of the same species of the tested Campylobacters obtained from humans and foods. The only statistically significant difference in the prevalence of virulence genes was noted in case of racR in C. jejuni. The prevalence was higher in food isolates (84.9% of human isolates vs. 95.6% of food isolates; p = 0.046).

Antimicrobial resistance

Phenotypic resistance to the selected antibiotics is shown in Tables 2 and 3, clearly showing high resistance to quinolone antibiotics and ampicillin in both tested Campylobacter species. Moreover, the tested C. coli isolates showed high resistance to streptomycin and increased resistance to erythromycin (as much as 23% in case of pork liver isolates). Only 14 (8%) out of the 175 tested Campylobacter spp. samples were susceptible to all antibiotics. The data of multiple antimicrobial resistance are shown in Table 4.

Prevalence of resistance genes and the genotype-phenotype relationship

The testing of phenotypic resistance to antibiotics was followed by detecting the selected resistance genes (bla_OXA-61, tet(O), cmeB and aph3-1). The most frequent gene was bla_OXA-61, detected in 74.6% and 74.0% of the tested C. jejuni isolates obtained from humans and foods, respectively. In case of C. coli isolates, the same gene was detected in 78.3% of human isolates and 67.4% of food isolates. The gene was more prevalent in isolates resistant to ampicillin (C. jejuni 87.5% of human isolates and 84.6% of food isolates; C. coli 90.9% and 97.7%, respectively). Comparison of the frequencies of the blaOXA-61 gene between ampicillin-susceptible and -resistant human isolates of C. jejuni revealed that the gene was significantly more common in ampicillin-resistant human isolates (p = 0.003). The difference was not significant in poultry isolates (p = 0.302). The opposite was true for ampicillin-resistant isolates of C. coli. The difference was not significant in human isolates (p = 0.317). The bla_OXA-61 gene was significantly more frequent in ampicillin-resistant isolates of C. coli obtained from pork liver and poultry (p = 0.029 and p = 0.009, respectively). The tet(O) gene, responsible for resistance to tetracycline, was detected in 37.3% of human and 20.0% of food isolates of C. jejuni and 34.8% of human and 39.5% food isolates of C. coli. The rates were higher in isolates with phenotypic resistance (C. jejuni 58.1% and 76.9%, respectively; C. coli 87.5% and 57.1%, respectively). The tet(O) gene was significantly more frequent in tetracycline-resistant isolates of C. jejuni obtained from both patients and poultry (p = 0.001 and p < 0.0001, respectively). Similarly, there was a significant difference in tetracycline-resistant human and poultry isolates of C. coli (p < 0.0001 and p = 0.042, respectively). The difference was not significant in pork liver isolates of C. coli (p = 0.128). The cmeB gene (efflux pump) was detected in only one human isolate of C. jejuni but in 91.3% of human and 76.7% of food isolates of C. coli. The aph3-1 gene was detected in one human C. jejuni isolate and in one C. coli isolate obtained from pork liver.

Relationships between the presence of selected genes responsible for mechanisms of resistance linked to particular antibiotics and phenotypic resistance to the antibiotics are shown in Tables 5 and 6. It is apparent from the tables that the assessed resistance genes are more frequent in isolates with phenotypically determined resistance. This is particularly true for Campylobacter spp. resistant to tetracycline which carried the tet(O) gene significantly more frequently that isolates susceptible to the antibiotic.

DISCUSSION

This study provides the first insights into the prevalence of nine virulence genes important in the pathogenesis of C. jejuni and C. coli in the Czech Republic. In the tested C. jejuni isolates, the most prevalent virulence genes were those responsible for the production of CDT (90–100%). But their prevalence in C. coli isolates was low (up to 10%). High prevalence rates of these genes in C. jejuni isolated from both humans and poultry (100%) were also reported by Datta et al. [5] or Ripabelli et al. [20]. Slightly lower rates of prevalence of cdtA (64%), cdtB (82%) and cdtC (84%) in poultry isolates of C. jejuni were found by Polish authors who, in contrast to the present study, also reported high rates of these genes in C. coli isolates (cdtA – 100%, cdtB – 91% and cdtC – 100%) [21]. However, another Polish study found CDT genes in only 5.6% of human isolates of C. coli [22]. Genes responsible for the invasiveness of Campylobacter include virB11. In the present study, the gene was detected in 4% of poultry isolates of C. jejuni and 6% of C. coli isolates obtained from pork liver. Krutkiewicz and Klimuszko [21] identified the gene in 14% of C. jejuni and 9% of C. coli isolates from poultry and 42% and 0% of pig isolates, respectively. Another gene, ciaB, was only detected in C. jejuni isolates (humans – 62%, poultry – 60% and pork liver – 100%) but in none of C. coli isolates from either patients or foods. Datta et al. [5] found the gene in 98% of human and 100% of poultry isolates of C. jejuni. The gene wlaN contributing to post-infection complications in the form of Guillain-Barré syndrome was only detected in C. jejuni, namely in 4% of human isolates and 2% of poultry isolates. Cha et al. [23] found the gene in 35% of C. jejuni isolates obtained from Korean patients with Campylobacteriosis and 23% of isolates from Southeast Asia. Feodoroff et al. [24] identified the gene in 23% of 166 human isolates of C. jejuni. Talukder et al. [6] found the wlaN gene in 9% of samples in a set of 40 human isolates of C. jejuni. Datta et al. [5] performed a study of virulence factors in clinical human isolates, poultry meat, broiler feces and bovine feces. The detection rates for the wlaN gene were 25.0%, 23.8%, 4.7% and 7.7%, respectively. In the present study, the iam gene was only confirmed in C. coli isolates (humans – 92%, poultry – 97% and pork liver – 82%). Wieczorek and Osek [25] stated that the gene was present in 31% of C. jejuni isolates and 27% of poultry isolates of C. coli. In another study, Wieczorek [26] reported 100% prevalence of the gene in poultry isolates of C. jejuni and 15% prevalence in C. coli isolates. The considerable difference in the genetic makeup of C. jejuni and C. coli strains tested in the present study was also apparent in case of the genes dnaJ and racR detected in the majority of C. jejuni isolates (85–100%). In C. coli, the dnaJ gene was found in 6% of isolates from pork liver; the racR gene was detected in 9% of C. coli isolates from patients, 3% of poultry isolates and 6% of isolates from pork liver. In a study by Thakur et al. [27], for instance, the dnaJ gene was present in approximately 16% of human and 11% of poultry isolates of C. jejuni. The authors found dnaJ in 72% of poultry C. coli isolates but failed to detect the gene in human isolates of C. coli. It is therefore clear that the prevalence of genes responsible for virulence and production of toxins is rather varied in thermotolerant Campylobacter spp. This fact was also shown in a study by Wieczorek et al. [28] comparing the presence of 8 selected virulence genes in Campylobacter isolates from Poland, Australia and Malaysia. The genes differed in prevalence, depending on the country of origin. Genetic diversity is definitely influenced by geographical location and sources of isolates, including climate, approach to agriculture and use of antibiotics. For example, five of the genes (ciaB, racR, ceuE, cdtB and cdt gene cluster) were least prevalent in isolates from Malaysia. However, the authors also pointed to genetic diversity in the subgroup of isolates from Poland which could not be clearly explained.

Another parameter analyzed in the present study was phenotypic resistance of thermotolerant Campylobacter spp. isolated from foods and humans to eight selected antibiotics. Europe is typically characterized by high resistance of Campylobacter spp. to quinolones. For poultry isolates of C. jejuni, the 2012 rates of resistance to ciprofloxacin were 89% in Poland, 82% in Hungary, 81% in Romania and 63% in Austria. The mean resistance to ciprofloxacin in the EU was 60% in the same year. The mean rate of resistance to nalidixic acid calculated from data provided by individual countries was 58% [9]. In the present study, the resistance of poultry isolates of C. jejuni to ciprofloxacin and nalidixic acid was 69% and 58%, respectively; for C. coli isolates, the rates were 67% and 58%, respectively. For isolates of C. coli obtained from pork liver, the resistance to both quinolones was roughly identical at approximately 61%. Comparison of resistance of C. coli isolates obtained from poultry and pork liver to tetracycline (57% vs. 85%) and streptomycin (57% vs. 92%) showed higher rates in pig isolates. This may be explained by the fact that tetracycline antibiotics are more frequently used in pig farming. In 2012, high resistance of pig C. coli isolates to tetracycline was reported, for example, in Spain (100%), France (92%) or Hungary (89%) [9]. Since macrolides are the drug of choice when antibiotic therapy of human Campylobacteriosis is needed, attention was paid to testing resistance to erythromycin. In the present study, 2% of poultry isolates of C. jejuni, 3% of poultry isolates of C. coli and 23% of C. coli isolates from pork liver were resistant to erythromycin. Human isolates of C. jejuni were more resistant to all antibiotics than poultry isolates. By contrast, human isolates of C. coli were less resistant to most antibiotics than food isolates; the only exception was ciprofloxacin, with there being a slightly higher resistance of human isolates to this antibiotic (70% vs. 61% and 68%, respectively). Resistance of human isolates of C. coli to erythromycin (9%) was higher than that of poultry isolates (3%) but lower than resistance of isolates obtained from pork liver (23%).

Antibiotic resistance is encoded by numerous genes that may, but do not have to, be expressed in the form of phenotypic resistance tested In vitro by, for example, the microdilution method and manifested in vivo by failed antibiotic therapy. The present study focused on detecting of four genes, three of which (blaOXA-61, tet(O) and aph3-1) are thought to be associated with resistance to a particular group of antibiotics and one is linked to efflux pump activity (cmeB). The aph3-1 gene was only detected in two isolates. In another two genes encoding the mechanism of resistance linked to a particular antibiotic, the study tried to assess relationships between the presence of a particular gene and phenotypic resistance to that antibiotic. Those were the blaOXA-61 gene linked to resistance to ampicillin and the tet(O) gene linked to resistance to tetracycline (see Tables 5 and 6). In all cases, the genes were more frequent in resistant isolates as compared with susceptible isolates. This was particularly true for the tet(O) gene participating in protection of bacterial ribosomes against the effects of tetracyclines. For instance, the gene was highly prevalent in resistant isolates of C. jejuni from poultry and C. coli from pork liver (77% and 73%, respectively) but it was not detected in similar isolates susceptible to tetracycline. Abdi-Hachesoo et al. [29] detected the tet(O) gene in as many as 93% of C. coli isolates and 74% of C. jejuni isolates from poultry. In the present study, less striking differences in the prevalence of the tet(O) gene were found between resistant and susceptible human isolates of C. jejuni. Isolates resistant and susceptible to tetracycline carried the tet(O) gene in 58% and 14%, respectively.

In accordance with detection of high phenotypic resistance to ampicillin, high prevalence rates of the blaOXA-61 gene contributing to resistance to this antibiotic were found in both Campylobacter species. The prevalence rates of the gene in both C. jejuni and C. coli isolated from both foods and humans were relatively similar, ranging from 67% to 78%. Although the blaOXA-61 gene was more frequently detected in ampicillin-resistant isolates (88% vs. 56%), its presence is not necessarily linked with resistance [30, 31]. This is confirmed by the fact that in the present study, the gene was detected in 48% of susceptible C. jejuni isolates and 67% of susceptible C. coli isolates obtained from humans. The blaOXA-61 gene was also carried by 68% of poultry isolates of C. jejuni.

The remaining gene, cmeB, is responsible for mechanisms potentially causing resistance of bacteria to a broader range of antibiotics. Therefore, it is more difficult to determine its significance with respect to phenotypic resistance to a particular antibiotic group. This gene encodes the inner membrane transporter which is a part of efflux pump and can be associated with resistance to several antibiotic classes such as quinolones [12]. Although the microdilution method showed high resistance of both Campylobacter species to quinolones, the prevalence of the cmeB gene in C. jejuni isolates was rather low (2% in human isolates; undetected in food isolates). This observation is in accordance with previously published data suggesting that resistance to quinolones is primarily associated with other resistance mechanisms, especially mutation in genes encoding DNA gyrase and DNA topoisomerase [10, 32]. Higher prevalence of cmeB in C. coli than C. jejuni isolates was also observed by Obeng et al. [19]. But this fact may be related to higher sequence variability of the cmeB gene in the tested isolates.

Conclusion

In conclusion, high percentages of both human and food isolates of C. jejuni carried the toxigenic genes cdtA, cdtB and cdtC. Conversely, the prevalence of these genes in C. coli isolated from all types of samples included in the study was low. The genes ciaB, dnaJ and racR were found to be highly prevalent in both human and food isolates of C. jejuni. The studied virulence genes were considerably more prevalent in C. jejuni isolates than in C. coli isolates. This fact was particularly apparent in the dnaJ and racR genes. The only exception was the iam gene identified in only C. coli.

The tested isolates of both C. jejuni and C. coli, irrespective of their origin, showed high levels of phenotypic resistance to mainly quinolone antibiotics as well as higher levels of resistance to ampicillin, tetracycline and streptomycin. C. coli isolates from pork liver had 23% resistance to erythromycin. Both Campylobacter species showed high prevalence rates of blaOXA-61 and tet(O), genes encoding resistance to antibiotics. There were considerable differences in the prevalence of the cmeB gene between C. jejuni and C. coli. There was a significant difference between a high prevalence of the resistance-encoding gene tet(O) in tetracycline-resistant isolates and its low prevalence in isolates susceptible to the drug. The present study is the first in the Czech Republic to assess the prevalence of virulence and antibiotic resistance genes in Campylobacter isolated from humans and foods.

Acknowledgements

This work was supported by the grant project of ÚMTZ LO1304. We thank Dr. Jana Zapletalová for statistical analysis.

Do redakce došlo dne 29. 5. 2016.

Adresa pro korespondenci:

doc. MVDr. Jan Bardoň, Ph.D., MBA

Státní veterinární ústav Olomouc

Jakoubka ze Stříbra 1

779 00 Olomouc

e-mail: jbardon@svuol.cz