Thermotolerant Campylobacter spp., primarily Campylobacter jejuni and Campylobacter coli, are among the most common bacteria causing acute human gastroenteritis throughout the world (Humphrey et al. 2007). Both species colonize the intestinal mucosa of most warm-blooded animals, including food-producing species and humans (Newell 2001). Several avian species are considered the main reservoirs of Campylobacter spp. (Newell and Fearnley 2003). However, current scientific knowledge on the presence of Campylobacter spp. in partridges (Perdix perdix) is limited to one report (Volkheimer and Wuthe 1986). In order to address this lack of information, the present study was undertaken to estimate the prevalence of thermotolerant Campylobacter spp. in commercially reared partridges in southern Italy.
During the period from April to July 2008, 240 living partridges were examined from a game bird farm (total flock size = 11 000 birds) located in the province of Napoli (Southern Italy). The farm was intensive where the young birds were raised in a brooder house and progressively were moved to an aviary until their release for repopulation of protected areas (e.g. National Park, Natural Reserve) and game reserves. To achieve our goal, first, 120 birds between 0 and 1 month of age (young group) were examined. Subsequently, the same flock (additional 120 partridges) was examined when the birds exceeded 1 month of age (adult group). These sample sizes (n = 120 partridges) were calculated using the formula proposed by Thrusfield (1995) for a large (theoretically ‘infinite’) population using the following values: expected prevalence (8%), confidence interval (CI; 95%) and desired absolute precision (5%).
With respect to sex, they were 120 male (60 young and 60 adult individuals) and 120 female (60 young and 60 adults) clinically healthy birds.
Cloacal swabs were collected from all 240 partridges, stored in Amies Transport Medium (Oxoid, Milan, Italy) 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 (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 (42°C for 48 h) under microaerobic conditions, the plates were examined for typical Campylobacter colonies. A loopful of a suspected colony (one suspect colony per plate) was subcultured on sheep blood agar (Oxoid) and finally incubated (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).
The extraction and purification of DNA from isolated colonies on sheep blood agar was performed using a Bactozol kit (Molecular Research Center, Inc., Cincinnati, OH, USA) as described previously (Khan and Edge 2007). The specific detection of the Campylobacter genus was based on PCR amplification of the cadF gene using oligonucleotide primers cadF2B and cadR1B as described by Konkel et al. (1999). All DNA extracts were also examined by a triplex PCR, for the presence of C. jejuni, C. nbsp;coli and Camphylobacter lari species using amplification conditions and oligonucleotide primers ICJ-UP, ICJ-DN, ICC-UP, ICC-DN, and ICL-UP, ICL-DN, respectively, as previously described (Khan and Edge 2007). PCR products were separated by electrophoresis on 1·5% agarose gels (Gibco–BRL, Milan, Italy), stained with ethidium bromide and visualized under UV light. PCR amplified without the DNA template was used as negative control, whereas three reference Campylobacter strains, C. jejuni ATCC 29428, C. coli ATCC 33559 and C. lari ATCC 43675, obtained from LGC Promochem (LGC Promochem, Teddington, UK), were used as positive controls.
The partridge data (age and sex, all categorical) were analysed by univariate (Pearson’s chi-square test for independence) statistical analysis using the Campylobacter spp. status (positive/negative) as dependent variable. Statistical analysis was performed using spss 13 software for Windows (SPSS Inc., Chicago, IL).
Thermotolerant Campylobacter spp. were isolated from 118/240 (49·2%; 95% CI = 42·7–55·7%) of the cloacal swabs examined (Table 1). Regarding the age, young birds showed a prevalence of 3·3% (95% CI = 1·1–8·8%), whereas partridges older than 1 month showed a prevalence of 95·0% (95% CI = 88·9–97·9%); this difference was statistically significant (P = 0·000).
|Partridge data||No. of partridges tested||No. of positive partridges||%||95% CI||P*|
|Young (<1 month)||120||4||3·33||1·07–8·83||0·000|
|Mature (>1 month)||120||114||95·00||88·98–97·95|
In contrast, there was no significant difference related to sex (P = 0·606).
As proved by PCR, 100% of the cloacal samples that were positive for Campylobacter spp. were identified as C. coli (118/118), and 15 of the 118 (12·7%) positive samples were also positive for C. jejuni. In contrast, Campylobacter lari was not identified.
The findings of the present study suggest the very common occurrence of Campylobacter spp. in living partridges in Southern Italy with a prevalence of 49·2%. Camphylobacter coli was the predominant species identified, and this result is noteworthy because C. coli is frequently isolated with a prevalence higher than C. jejuni in game birds (Nebola et al. 2007; Dipineto et al. 2008). Regarding the age, interestingly, partridges older than 1 month showed a significantly higher prevalence than young birds. We can explain this finding taking into account that Campylobacter spp. infection in birds is typically observed from the second to the fourth week of life (Shreeve et al. 2000; Newell and Fearnley 2003) and may be associated with a decline in maternal antibodies. Moreover, adult partridges lived in an aviary, and thus wild birds could act as an important route of infection. In contrast, there was no significant difference related to sex.
To our knowledge, this is the first report of thermotolerant Campylobacter spp. in healthy partridges in Italy. Comparative data on the prevalence of campylobacteriosis in partridges are limited to a single report (Volkheimer and Wuthe 1986). In this latter study, 19 of 20 commercially reared partridges examined were positive for Campylobacter spp. using biochemical tools; because all the positive strains were negative for hippurate hydrolysis, the only species present was probably C. coli. In contrast, in the present study, thermotolerant Campylobacter spp. were isolated with a prevalence of 49·2% identified by PCR assay. This is believed to be the first report of molecular-based identification of Campylobacter spp. in partridges.
In conclusion, our results reinforce the assumption that game birds, as previously reported (Dipineto et al. 2008), may be considered as potential carriers of Campylobacter spp. for humans and other animal species. However, on account of the small sample size, a larger study is required to confirm these birds as potential carriers of Campylobacter spp., and further studies are necessary to evaluate the possible routes of infection to partridges.