Prevalence of thermotolerant Campylobacter in pheasants (Phasianus colchicus)

Abstract

The present study was undertaken with the aim to evaluate the prevalence of thermotolerant Campylobacter spp. in living pheasants in Italy. To achieve this goal, a total of 240 living pheasants, equally shared between female and male birds, were examined. Thermotolerant Campylobacter spp. was isolated in 104 out of 204 (43.3%) living pheasants analysed. Campylobacter coli (100%) and Campylobacter jejuni (13.5%) were identified by polymerase chain reaction. Adult pheasants showed a significantly higher prevalence value (P < 0.05) than younger pheasants.

Introduction

Thermotolerant Campylobacter spp., primarily Campylobacter jejuni and Campylobacter coli, are among the most common bacteria causing acute human gastroenteritis in industrialized countries (Friedman et al., 2000). Both species colonize the intestinal mucosa of most warm-blooded animals, including food-producing species and humans (Newell, 2001). However, the favoured environment appears to be the intestinal tract of several avian species where Campylobacter spp. colonizes as a commensal organism (Newell & Fearnley, 2003).

Current scientific knowledge on the presence of Campylobacter spp. in pheasants (Phasianus colchicus) is scarce and fragmentary, although this microorganism has been reported in pheasant in Germany (Atanassova & Ring, 1999), Russia (Stern et al., 2004), and Czech Republic (Nebola et al., 2007).

To address this lack of information, the present study was undertaken with the aim to evaluate the prevalence of these microorganisms in commercially reared pheasants in southern Italy.

Materials and Methods

A total of 240 living pheasants were examined from a game-birds farm located in the province of Napoli (Southern Italy). One hundred and twenty birds were examined with an age ranged between 0 and 1 month (young group). Subsequently, the same flock (a further 120 pheasants) was examined when the birds exceeded 1 month of age (adult group). These sample sizes (n = 120 pheasants) were calculated using the formula proposed by Thrusfield (1995) for a large (theoretically “infinite”) population using the following values: expected prevalence (8%), confidence interval (95%) and desired absolute precision (5%).

With respect to sex, there were 120 male birds (60 young and 60 adult) and 120 female birds (60 young and 60 adult). The pheasants were apparently in healthy body conditions.

Cloacal swabs were collected from all 240 pheasants, stored in Amies Transport Medium (Oxoid) at 4°C, transported to the laboratory and analysed within 1 h of collection. Samples were inoculated into Campylobacter selective enrichment broth (Oxoid) and incubated at 42°C for 48 h under microaerobic conditions provided by CampyGen (Oxoid). Subsequently, each sample was streaked onto Campylobacter blood-free selective agar (CCDA; Oxoid). After incubation at 42°C for 48 h under microaerobic conditions, the plates were examined for typical Campylobacter colonies. Suspected colonies were subcultured on sheep blood agar (Oxoid) and were finally incubated for 24 h at 42°C.

Under phase contrast microscopy, colonies comprising curved or spiral motile rods were presumptively identified as Campylobacter spp. and submitted to a multiplex polymerase chain reaction (PCR).

DNA was extracted from isolated colonies on sheep blood agar using the PrepMan sample reagent (Applied Biosystems, Foster City, California, USA), following the manufacturer's protocol. The specific detection of the Campylobacter genus was based on PCR amplification of the cadF gene using oligonucleotide primers cadF2B (5′-TTG AAG GTA ATT TAG ATA TG-3′) and cadR1B (5′-CTA ATA CCT AAA GTT GAA AC-3′) as described by Konkel et al. (1999). All DNA extracts were also examined for the presence of C. jejuni and C. coli species using oligonucleotide primers C-1 (5′-CAA ATA AAG TTA GAG GTA GAA TGT-3′) and C-4 (5′-GGA TAA GCA TAG CTA GCT GAT-3′), and COL1 (5′-ATG AAA AAA TAT TTA GTT TTT GCA-3′) and COL2 (5′-ATT TTA TTA TTT GTA GCA GCG-3′), respectively, as described by Winters et al. (1997) and Gonzalez et al. (1997). The PCR conditions were as described by Cloak & Fratamico (2002) and the products were separated by electrophoresis on 1.5% agarose gels (Gibco–BRL), stained with ethidium bromide and visualized under UV light. A PCR amplified without the DNA was used as negative control, whereas two reference Campylobacter strains (C. jejuni ATCC 29428 and C. coli ATCC 33559, obtained from LGC Promochem) were used as positive controls (LGC Promochem, Teddington, UK).

The pheasant data (age and sex, all categorical) were analysed by univariate statistical analysis (Pearson's chi-square test for independence) using the Campylobacter spp. status (positive/negative) as the dependent variable. Statistical analysis was performed using SPSS 13 for Windows.

Results and Discussion

Thermotolerant Campylobacter spp. were isolated from 104/240 (43.3%; 95% confidence interval = 37.0% to 49.9%) of the cloacal swabs examined (Table 1). Regarding age, young birds showed a prevalence of 3.3% (95% confidence interval = 1.1% to 8.8%) whereas pheasants older than 1 month showed a prevalence of 83.3% (95% confidence interval = 75.2% to 89.3%); this difference was statistically significant (P=0.001). We can explain this finding taking into account that Campylobacter spp. infection in birds is typically observed from the second to the fourth weeks of life (Shreeve et al., 2000; Newell & Fearnley, 2003). In contrast, there was no significant difference related to sex (P=0.602). The results of the chi-square test are summarized in Table 1.

Table 1.  Results and statistical analysis (Pearson's chi-squared test for independence) of the pheasants tested, related to sex and age

Pheasant data	Number of pheasants tested	Number of positive pheasants	Percentage positive	95% confidence interval	P value (chi-square)
Sex					0.602
Male	120	54	45.0	36.0 to 54.3
Female	120	50	41.7	32.9 to 51.0
Age group					0.001
Young (<1 month)	120	4	3.3	1.1 to 8.8
Adult (>1 month)	120	100	83.3	75.2 to 89.3
Total	240	104	43.3	37.0 to 49.9

As proved by PCR, 100% of the strains were identified as C. coli (104/104), and 14 out of the 104 (13.5%) positive samples were also positive to C. jejuni.

The findings of the present survey demonstrate the occurrence of Campylobacter spp. in living pheasants in Southern Italy with a prevalence of 43.3%. C. coli was the predominant species identified, in line with Nebola et al. (2007) who isolated C. coli with a prevalence higher than C. jejuni in pheasants from Czech Republic.

Comparative data on the prevalence of campylobacteriosis in pheasant are limited, although this microorganism has been reported in pheasants from Germany (Atanassova & Ring, 1999), Russia (Stern et al., 2004), and Czech Republic (Nebola et al., 2007) with a prevalence of 25.9%, 26.7%, and 70.2%, respectively. Only one study is available from Italy, in which Soncini et al. (2006) reported negative results from pheasant neck skins.

In conclusion, our results demonstrate that Campylobacter spp. may be found in the intestines of apparently healthy pheasants. Thus, this avian species may be considered a potential carrier of Campylobacter spp. for humans and animals, since pheasants are frequently used for repopulation of protected areas (e.g. National Park, Natural Reserve) as well as game reserves.