Survey of thermotolerant Campylobacter in pheasant (Phasianus colchicus)
Ludovico Dipineto, Antonio Gargiulo, Luigi Maria De Luca Bossa, Alessandra Cuomo, Antonio Santaniello, Mariangela Sensale, Lucia Francesca Menna, Alessandro Fioretti Dipartimento di Patologia e Sanità Animale. Università di Napoli Federico II, Italy Corresponding author: Dr. Ludovico Dipineto. Dipartimento di Patologia e Sanità Animale. Università di Napoli Federico II. Via F. Delpino 1, 80137 Napoli, Italy - Tel. +39 081 2536277 - Fax: +39 081 2536282 Email:
[email protected]
Abstract This study was conducted with the aim to evaluate the prevalence of thermotolerant Campylobacter spp. in pheasant (Phasianus colchicus) in Southern Italy. To achieve this goal, 60 cloacal swabs were collected. Campylobacter spp. were isolated from 52 out of 60 cloacal swabs tested. As proved by PCR, 100% of the strains were identified as C. coli (52/52), and 10 out of the 52 (19.2%) positive samples were also positive to C. jejuni. The pheasant, can be considered, at least theoretically, as potential Campylobacter carriers. Key words: Pheasant, Campylobacter coli, Campylobacter jejuni, Southern Italy.
RIASSUNTO Indagine sulla presenza di Campylobacter termotolleranti nel fagiano (Phasianus colchicus) La presente indagine è stata effettuata con lo scopo di valutare la presenza di Campylobacter termotolleranti nei fagiani (Phasianus colchicus) in Sud Italia. A tal fine sono stati raccolti 60 tamponi cloacali. Campylobacter spp. è stato isolato da 52/60 tamponi analizzati. Come confermato dalla PCR, tutti i ceppi positivi sono stati identificati come C. coli (52/52) e di questi, 10 (19,2%) erano positivi anche a C. jejuni. Dai risultati ottenuti emerge che i fagiani potrebbero essere considerati, almeno teoricamente, come potenziali vettori di Campylobacter. Parole chiave: Fagiani, Campylobacter coli, Campylobacter jejuni, Sud Italia.
Introduction Thermotolerant Campylobacter spp. are among the most common bacteria causing acute human gastroenteritis in industrialized countries (Friedman et al., 2000). C. Ital.J.Anim.Sci.
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jejuni is now recognized as one of the main causes of bacterial foodborne disease in many developed countries, with C. coli less frequently implicated (Moore et al., 2005). Both species colonize the intestinal mucosa of most warm-blooded animals, including all 401
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food-producing species and humans (Newell, 2001). However, the favoured environment appears to be the intestinal tract of all birds where Campylobacter colonize as a commensal organism (Newell and Fearnley, 2003). Little data on the prevalence of Campylobacter spp. in pheasants are available althouth this microorganism has been reported from pheasant (Phasianus colchicus) from Germany (Atanassova and Ring, 1999) with a prevalence of 25.9%. Because no data on the prevalence of campylobacteriosis in pheasant were available for Southern Italy we used molecularbased diagnostics to determine the presence of thermotolerant Campylobacter in pheasant in Southern Italy. Material and methods From January 2007 to March 2007, 60 reared pheasant were examined coming from a game birds farm located in the province of Napoli (Southern Italy). The birds were 60 days old equally shared between female and male individuals. The birds were clinically healthy. Cloacal swabs were collected from all 60 pheasant, stored in Amies Transport Medium (Oxoid, Milan, Italy) at +4°C, transported to the laboratory and analysed within 1h of collection. Samples were inoculated into Campylobacter selective enrichment broth (Oxoid) and incubated at 42°C for 48h under microaerobic conditions provided by CampyGen (Oxoid). Subsequently, each sample was streaked onto Campylobacter blood-free selective agar (CCDA; Oxoid) with the corresponding supplement (SE 155, Oxoid). After incubation at 42°C for 48h under microaerobic conditions, the plates were examined for typical Campylobacter colonies. Suspected colonies were subcultured on sheep blood agar (SBA; Oxoid) and finally incubated for 24h at 42°C. 402
Colonies comprising curved or spiral motile rods, when observed under phase contrast microscopy, were presumptively identified as Campylobacter and submitted to a multiplex polymerase chain reaction (PCR). DNA was extracted from isolated colonies on SBA using the PrepMan sample reagent (PE Applied Biosystems, Foster City, 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 TGT3’, C-4, 5’-GGA TAA GCA TAG CTA GCT GAT-3’ and COL1, 5’-ATG AAA AAA TAT TTA GTT TTT GCA-3’, 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 proposed by Cloak and Fratamico (2002) and the products were separated by electrophoresis on 1.5% agarose gels (GibcoBRL, Milan, Italy), stained with ethidium bromide and visualized under UV light. Results and discussion Thermotolerant Campylobacter spp. were isolated from 52/60 (86.7%) of the cloacal swabs examined. Among these, 52 (100%) were identified as C. coli and 10 out of the 52 (19.2%) positive samples were also positive to C. jejuni. Interestingly, the majority of Campylobacter spp. were isolated from mixed infections. In fact, both C. jejuni and C. coli were frequently recovered, at the same time, from each pheasant examined. Furthermore, the infection status was not related to sex. Ital.J.Anim.Sci.
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Campylobacter
Conclusions To our knowledge this is the first report of thermotolerant Campylobacter spp. isolation in healthy pheasant in Italy. Comparative data on the prevalence of campylobacteriosis in pheasant in Europe is limited (Atanassova and Ring, 1999), and only one negative study is available from Italy. In fact, using PCR, Soncini et al. (2006)
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did not isolate the pathogen from pheasant neck skin. We conclude from our results that Campylobacter may be found in the intestines of apparently healthy pheasant and, as such, may be considered as potential livestock carriers of campylobacteriosis for human being. Further study are necessary to evaluate the role of the pheasant in the epidemiology of campylobacteriosis.
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