MAREK SELWET¹, TOMASZ CŁAPA¹, MARIOLA GALBAS²*, RYSZARD SŁOMSKI² and FILIP PORZUCEK²

¹Department of General and Environmental Microbiology, Poznań University of Life Sciences, Poznań, Poland
²Department of Biochemistry and Biotechnology, Poznań University of Life Sciences, Poznań, Poland

*Corresponding author: M. Galbas, Department of Biochemistry and Biotechnology, Poznań University of Life Sciences, Poznań, Poland; e-mail: mariolagalbas@gmail.com

Submitted 16 July 2014, revised 18 November 2014, accepted 15 December 2014

Abstract

This study was conducted to determine the prevalence of Campylobacter spp. isolated from dogs’ faecal samples. From June 2012 to June 2013, a total of 210 faecal samples from pet dogs living in different kennels (n = 210) were collected by the owners in Greater Poland Voivodeship, Poznań District, Poland. The study revealed that 105 out of 210 faecal samples (50%) contained Campylobacter. The highest prevalence of Campylobacter spp. occurred in spring (81%), followed by winter (64%). The cadF gene was found in 100% of the isolates tested. The occurrence of the other genes was variable. The isolates from young dogs were characterised by higher occurrence of virulence genes.

Keywords: Campylobacter spp., dogs, virulence genes

Salmonella spp., Escherichia coli and Campylobacter spp. are the main causes of diarrhoea both in animals and humans all over the world (Silva et al., 2011). According to the EFSA (EFSA, 2014), campylobacteriosis is the most frequently reported zoonotic disease. The most common sources of infection are products of animal origin: meat (primarily poultry), milk and water from natural reservoirs (Acke et al., 2011). The most frequently isolated species from dogs in Denmark are: Campylobacter upsaliensis 75%, Campylobacter jejuni 19.4% and Campylobacter coli 0.7% (Hald et al., 2004). These Campylobacter species are more often isolated from dogs aged between 3 and 12 months than from older dogs (Hald et al., 2004). Moreover, some researchers also reported seasonal differences in prevalence (Rahimi and Saljooghian Esfahani, 2010; Andrzejewska et al., 2013). The factors which are associated with the pathogenicity of Campylobacter include: motility, chemotaxis as well as adhesion, and invasiveness (Bang et al., 2001; Krutkiewicz, 2008) and toxicity. These traits are associated with certain virulence genes identified from these bacteria, for example: flaA, cadF, iam and cdtB (Biswas, 2011; Selwet and Galbas, 2012a). The investigations which have been carried out so far have revealed varying distribution of these virulence genes among strains. The aim of this study was to determine the frequency of occurrence of C. upsaliensis, C. jejuni and C. coli isolated from two different age groups of healthy dogs and from dogs with the symptoms of diarrhoea in Greater Poland Voivodeship, Poznań District. Apart from that, the frequency of occurrence of selected virulence genes was determined in the collected isolates. From June 2012 to June 2013, a total of 210 samples of faeces from pet dogs living in 5 kennels were collected by their owners in Greater Poland Voivodeship Poznań District, Poland. All the samples were obtained by veterinarian by means of swab kits with a transport substrate (Euro Tubo Collection Swab Rubi, Spain). The animals were aged as follows: 105 dogs were adult (> 12 months), 105 dogs were younger than 1 year. Diarrhoea was reported in 25 dogs. The owners of the other dogs did not report clinical symptoms of diarrhoea. The faecal samples were cultured at 42±1°C in Campy Selective Agar Base (Preston) (Neogen Europe, Scotland UK) for 48 h under microaerophilic conditions. DNA was extracted by means of CHELEX-100 chelating resin (Bio-Rad, CA, USA). Bacterial colonies were suspended in 100 µl Tris buffer and 45 µl 20% CHELEX and boiled for 10 min. The samples were immediately placed on ice for 1 min. and centrifuged at 13.000 g for 10 min. at room temperature. A PCR was used for the detection of C. upsaliensis, C. jejuni and C. coli. (Andrzejewska et al., 2011). The following positive controls were included in the PCR: C. upsaliensis ATCC 43954, C. jejuni ATCC 33560 and C. coli ATCC 33559.

Table I
The prevalence of Campylobacter spp. isolated from diarrhoeic and healthy dogs

Sources		No. of samples	Campylobacter spp. positive	Campylobacter upsaliensis	Campylobacter jejuni	Campylobacter coli
Adult dogs	Healthy	95	37 (38.9%)	18 (48.6%)	15 (40.5%)	4 (10.8%)
	Diarrhoeic	10	5 (50%)	1 (20%)	2 (40%)	2 (40%)
Young dogs	Healthy	90	54 (60%)	29 (53.7%)	18 (33.3%)	7 (13.0%)
	Diarrhoeic	15	9 (60%)	2 (22.2%)	5 (55.5%)	2 (22.2%)

The presence of the flaA, cadF, cdtB and iam genes was determined with the primers according to the authors (Nachamkin et al., 1993; Konkel et al., 1999; Bang et al., 2001; Carvalho et al., 2001). The statistical analysis was performed according to the GLM procedure of the SAS program (SAS, 1999) and the significance of differences was verified with Tukey’s test. The prevalence of Campylobacter spp. was determined in the 210 faecal samples. PCR revealed the presence of Campylobacter spp. in 105 samples (50%). Campylobacter spp. was isolated from 42 samples (40%) from adult individuals and from 63 samples (60%) from young dogs. The numbers of isolates differed significantly between the age groups (P < 0.05). The most frequent species in the group of adult dogs were: 45.2% C. upsaliensis, 40.5% C. jejuni and 14.3% C. coli. In the group of young dogs the isolation percentage was similar, i.e. 49.2% C. upsaliensis, 36.5% C. jejuni and 14.3% C. coli. There were significant differences observed in the prevalence of C. upsaliensis between the age groups of the dogs under study (P < 0.05). Out of the 210 samples examined 25 samples derived from individuals with diarrhoea: 10 samples from adult dogs and 15 samples from young individuals (Table I). As far as the adult dogs are concerned, the predominant species were: 45.2% C. upsaliensis, 40.5% C. jejuni and 14.3% C. coli whereas in the young dogs these were: 49.2% C. upsaliensis 36.5% C. jejuni and 14.3% C. coli. In summer and autumn the frequency was similar with: 37.6% and 39.6%, respectively (Table II).

Table II
The seasonal prevalence of Campylobacter spp. isolated from dogs

Seasons	Sources		Total
	Adult dogs	Young dogs
Spring	14/22 (63.6%)	20/20 (100%)	34/42 (80.9%)
Summer	12/45 (26.7%)	20/40 (50%)	32/85 (37.6%)
Autumn	10/28 (35.7%)	13/30 (43.3%)	23/58 (39.6%)
Winter	6/10 (60%)	10/15 (66.7%)	16/25 (64%)
Total	42/105	63/105	105/210

The analyses of the percentages of the selected virulence genes (Table III) revealed that the cadF gene was found in 100% of all the species. Also, the flaA gene was determined in 100% of C. jejuni and C. coli. As far as the cdtB and iam genes are concerned, they were detected more frequently in the younger individuals. Although poultry is considered to be the main source of infection with Campylobacter spp. in humans, according to Biswas et al. (2011), the role of other animals as vectors spreading this bacterium should also be recognised. Parsons et al. (2010) in UK and Andrzejewska et al. (2013) in Poland reported that the frequency of Campylobacter spp. isolation in dogs ranges from 17% in Brazil and Argentina to 76.2% in Denmark. According to the study by Workman et al. (2005), the age of animals may also exert significant influence on the degree of occurrence of Campylobacter spp. Andrzejewska et al. (2013) reported the highest prevalence of Campylobacter spp. in dogs less than one year of age. Westgarth et al. (2008) observed that younger dogs were more likely to harbour C. upsaliensis and C. jejuni than adult animals. It may be related with younger dogs’ lower immunity. So far the relationships between the occurrence of C. upsaliensis and gastritis as well as diarrhoea in dogs and humans have not been sufficiently explained. However, younger individuals are believed to be the main source of occurrence of gastritis and diarrhoea, as Rahimi et al. (2012) noted. Salihu et al. (2010), claim that age is not an indicator of the risk of contamination with C. jejuni. In our study C. upsaliensis was found significantly more often in younger dogs than adult animals whereas there was no such difference observed with C. jejuni and C. coli. Infections caused by Campylobacter spp. are usually correlated with the seasons of the year, although there are reports indicating that the isolations of Campylobacter spp. from dogs were more frequent in spring (Sandberg et al., 2002). Rahimi et al. (2012) recorded an increase of Campylobacter spp. isolations from young dogs aged under 1 year as well as from individuals which were over 1 year of age in warm months: in spring – 38.5% and in summer – 38.9%. In our study, the frequency of isolation of Campylobacter spp. was higher in spring and in winter.

Table III
The number and percentages of virulence genes in Campylobacter spp. isolated from dogs

Isolates/Animals	Genes
	cadF	flaA	cdtB	iam
C. upsaliensis
Adult dogs (n=19)	19 (100%)	18 (94.7%)	17 (89.5%)	18 (94.7%)
Young dogs (n=31)	31 (100%)	30 (96.8%)	30 (96.8%)	30 (96.8%)
C. jejuni
Adult dogs (n=17)	17 (100%)	17 (100%)	16 (94.1%)	15 (88.2%)
Young dogs (n=23)	23 (100%)	23 (100%)	23 (100%)	21 (91.3%)
C. coli
Adult dogs (n=6)	6 (100%)	6 (100%)	5 (83.3%)	5 (83.3%)
Young dogs (n=9)	9 (100%)	9 (100%)	8 (88.9%)	8 (88.9%)

The study by Rizal et al. (2010) showed that the following factors are involved in pathogenicity of Campylobacter spp.: motility and chemotaxis as well as adhesion and invasiveness. Nowadays it is believed that the following genes are responsible for the potential pathogenicity of Campylobacter spp.: the flaA gene influencing motility, cadF – affecting adhesion, cdtB – responsible for toxin production (cytolethal distending toxin) and iam – determining invasiveness (Krutkiewicz, 2008). In our investigations the cadF gene was determined in 100% in C. upsaliensis, C. jejuni and C. coli. Biswas et al. (2011) confirmed the occurrence of this gene in 100% of the examined C. jejuni strains derived from human clinical studies and cow faeces. Selwet and Galbas (2012a; 2012b) observed that the cadF and flaA genes were found in 100% of C. coli and C. jejuni isolated from broilers, porkers, calves and piglets. All the strains of C. jejuni, C coli C. upsaliensis under analysis carried the cdtB gene encoding the protein exhibiting toxic properties, i.e. cytolethal distending toxin. This exotoxin causes the inhibition of the cell cycle and DNA degradation in the host (Lara-Tejero, 2001) and it may cause the death of sensitive eukaryotic cells (Heywood et al., 2005). CDT is composed of three subunits: CdtA, CdtB and CdtC, which are encoded by three genes: cdtA, cdtB and cdtC. All the three subunits are required for full activity (Rozynek et al., 2005). In our study the iam gene, which is responsible for invasiveness, was found in all the strains of Campylobacter spp. Carvalho et al. (2001) observed in their studies that the iam gene was detected most frequently in the strains of C. jejuni rather than in C. coli. The PCR analysis of stool isolates collected from dogs on farms in Wielkopolska (Greater Poland) region revealed the presence of Campylobacter spp. with the predominance of C. upsaliensis, which occur more frequently in young animals. The presence of C. jejuni and C. coli was frequently identified in adult dogs with the symptoms of diarrhoea, whereas C. jejuni was identified in young dogs. The correlation between diarrhoea and the presence of these bacteria should be taken into consideration in veterinary practice.

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