- Email: [email protected]
Campylobacter jejuni in the red squirrel (Sciurus vulgaris) population of Southern Italy
Available online at www.sciencedirect.com
The Veterinary Journal The Veterinary Journal 179 (2009) 149–150 www.elsevier.com/locate/tvjl
Short Communication
Campylobacter jejuni in the red squirrel (Sciurus vulgaris) population of Southern Italy Ludovico Dipineto *, Antonio Gargiulo, Alessandra Cuomo, Antonio Santaniello, Mariangela Sensale, Luca Borrelli, Livia D’Angelo, Lucia F. Menna, Alessandro Fioretti Dipartimento di Patologia e Sanita` Animale, Facolta` di Medicina Veterinaria, Universita` di Napoli Federico II, via Delpino, 1, 80137 Napoli, Italy Accepted 19 September 2007
Abstract Rectal swab samples were collected from 60 red squirrels (Sciurus vulgaris) from July 2006 to April 2007 in Southern Italy. Samples were tested for Campylobacter jejuni and C. coli by culture methods and suspected colonies were then confirmed by polymerase chain reaction. C. jejuni was detected in 5/60 (8.3%) samples examined but infection status was not related to age or sex and C. coli was not isolated. This is believed to be the first report of C. jejuni infection in the red squirrel. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Campylobacter jejuni; Campylobacter coli; Italy; Red squirrel; Survey
Campylobacter jejuni is now recognised 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 colonise the intestinal mucosa of most warm-blooded animals, including all food-producing species and humans (Newell, 2001). However, the favoured environment appears to be the intestinal tract of a wide range of birds which Campylobacter colonises as a commensal organism (Newell and Fearnley, 2003). No data on the prevalence of Campylobacter spp. in red squirrels (Sciurus vulgaris) are available, so we undertook this study to evaluate the presence of C. jejuni in this species. Sixty red squirrels were live-trapped from July 2006 to April 2007 in the Cilento and Vallo di Diano National Park located in the province of Salerno (Southern Italy). The corners of collection area were located at 40°06 0 48 0 0 N, 15°14 0 16 0 0 E and 40°15 0 18 0 0 N–15°03 0 17 0 0 E, respectively. The animals were trapped weekly using ground-placed Tomahawk squirrel traps (Tomahawk Live
*
Corresponding author. Tel.: +39 081 2536277; fax: +39 081 2536282. E-mail address: [email protected] (L. Dipineto).
1090-0233/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2007.09.013
Trap) baited with sunflower seeds and hazelnuts. The traps were placed randomly to cover the whole area of sampling. Thirty-four males (24 mature, 10 immature) and 26 females (19 mature, 7 immature) were caught and each was individually sampled using rectal swabs and then released. All squirrels examined were in a healthy body condition (i.e. no diarrhoea and good coat status). Squirrel handling procedures were performed according to the Office of Animal Care and Use Guidelines. Rectal swab samples were stored in Amies Transport Medium (Oxoid) at +4 °C, transported to the laboratory and analysed within 2 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) with the corresponding supplement (SE 155, 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 (SBA, Oxoid) and incubated for a further 24 h at 42 °C. Colonies comprising curved or spiral motile rods, when
150
L. Dipineto et al. / The Veterinary Journal 179 (2009) 149–150
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), 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 0 TTGAAGGTAATTTAGATATG-3 0 and cadR1B, 5 0 CTAATACCTAAAGTTGAAAC-3 0 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 0 -CAAATAAAGTTAGAGGTAG AATGT-3 0 , C-4, 5 0 -GGATAAGCATAGCTAGCTGA T3 0 and COL1, 5 0 -ATGAAAAAATATTTAGTTTTTGC A-3 0 , COL2, 5 0 -ATTTTATTATTTGTAGCAGCG-3 0 , respectively, as described by Winters et al. (1997) and Gonzalez et al. (1997). The PCR conditions were as proposed by Cloak and Fratamico (2002). In brief, amplification reactions were performed in total volumes of 50 lL containing 5 lL of purified template DNA, 10 mM Tris–HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 lM each of the four deoxynucleotide triphosphates, 1.25 U of Taq DNA polymerase (Gibco–BRL), and 0.40 lM (each) primers cadF2B, cadR1B for genus detection, and COL1, COL2, C-1, C-4 for C. jejuni and C. coli identification. A Gene Ruler 100 bp DNA ladder (Fermentas International) was used. Samples were subjected to an initial denaturation step at 94 °C for 4 min, followed by 30 amplification cycles of 94 °C for 1 min, 52 °C for 1 min, and 72 °C for 1 min and a final extension step of 72 °C for 5 min in a Model 9600 thermal cycler (PE Applied Biosystems). The PCR products were separated by electrophoresis on 1.5% agarose gels (Gibco–BRL), stained with ethidium bromide and visualized under UV light. Two reference Campylobacter strains, C. jejuni ATCC 29428 and C. coli ATCC 33559, obtained from LGC Promochem were used as positive controls. Campylobacter was detected from 5/60 (8.3%) squirrels examined. As demonstrated by PCR, all of the strains were identified as C. jejuni. The presence of C. jejuni was similar in males (3/34, 8.8%) and females (2/26, 7.7%) as well as in mature (4/43, 9.3%) and young (1/17, 5.9%) squirrels. In contrast, C. coli was not detected. The results are summarised in Table 1. C. jejuni is one of the most important causes of bacterial food-borne illnesses in humans. Although it was isolated from the normal intestinal flora in a wide range of birds and mammals, no data on the presence of this microorganism in squirrel are available. To our knowledge this is the first report of C. jejuni isolation in squirrels. The role of this rodent in the epidemiology of campylobacteriosis is not clear and cannot be evaluated solely on the basis of this study. In fact, besides the red squirrel, the Cilento and Vallo di Diano National
Table 1 Number (%) of positive samples for Campylobacter jejuni as a function of sex and age of the red squirrels (Sciurus vulgaris) examined Sex and age of squirrels examined
Number (%) of positive samples
Male, young (n = 10) Male, mature (n = 24) Female, young (n = 7) Female, mature (n = 19) Total (n = 60)
1 2 0 2 5
(10%) (8.3%) (0%) (10.5%) (8.3%)
Park houses birds of prey and Passeriformes, wild mammals (e.g. fox, wolf and marten), and reptiles (such as the viper and lizard). A number of potential species in the woodland could act as reservoirs for infection of the squirrel by Campylobacter including other wild mammals or birds. Of these potential sources birds are often described as a reservoir for infection (Huba´lek, 2004) and it would be interesting to determine whether this is the case with the red squirrel. This preliminary study has demonstrated that the presence of C. jejuni in a sampling of the red squirrel population of Southern Italy was relatively low (8.3%). However, on account of the small sample size, a larger study is required to confirm that red squirrels are potential carriers of Campylobacter spp. Moreover, further work is also needed to evaluate the possible routes of infection. References Cloak, O.M., Fratamico, P.M., 2002. A multiplex polymerase chain reaction for the differentiation of Campylobacter jejuni and Campylobacter coli from a swine processing facility and characterization of isolates by pulsed-field gel electrophoresis and antibiotic resistance profiles. Journal of Food Protection 65, 266–273. Gonzalez, I., Grant, K.A., Richardson, P.T., Park, S.F., Collins, M.D., 1997. Specific identification of the enteropathogens Campylobacter jejuni and Campylobacter coli by using a PCR test based on the ceuE gene encoding a putative virulence determinant. Journal of Clinical Microbiology 35, 759–763. Huba´lek, Z., 2004. An annotated checklist of pathogenic microorganisms associated with migratory birds. Journal of Wildlife Diseases 40, 639– 659. Konkel, M.E., Gray, S.A., Kim, B.J., Garvis, S.G., Yoon, J., 1999. Identification of the enteropathogens Campylobacter jejuni and Campylobacter coli based on the cadF virulence gene and its product. Journal of Clinical Microbiology 37, 510–517. Moore, J.E., Corcoran, D., Dooley, J.S., Fanning, S., Lucey, B., Matsuda, M., McDowell, D.A., Me´graud, F., Millar, B.C., O’Mahony, R., O’Riordan, L., O’Rourke, M., Rao, J.R., Rooney, P.J., Sails, A., Whyte, P., 2005. Campylobacter. Veterinary Research 36, 351–382. Newell, D.G., 2001. Animal models of Campylobacter jejuni colonization and disease and the lessons to be learned from similar Helicobacter pylori models. Symposium Series – Society for Applied Microbiology 30, 57S–67S. Newell, D.G., Fearnley, C., 2003. Sources of Campylobacter colonization in broiler chickens. Applied and Environmental Microbiology 69, 4343–4351. Winters, D.K., O’Leary, A.E., Slavik, M.F., 1997. Rapid PCR with nested primers for direct detection of Campylobacter jejuni in chicken washes. Molecular and Cellular Probes 11, 267–271.
The Veterinary Journal The Veterinary Journal 179 (2009) 149–150 www.elsevier.com/locate/tvjl
Short Communication
Campylobacter jejuni in the red squirrel (Sciurus vulgaris) population of Southern Italy Ludovico Dipineto *, Antonio Gargiulo, Alessandra Cuomo, Antonio Santaniello, Mariangela Sensale, Luca Borrelli, Livia D’Angelo, Lucia F. Menna, Alessandro Fioretti Dipartimento di Patologia e Sanita` Animale, Facolta` di Medicina Veterinaria, Universita` di Napoli Federico II, via Delpino, 1, 80137 Napoli, Italy Accepted 19 September 2007
Abstract Rectal swab samples were collected from 60 red squirrels (Sciurus vulgaris) from July 2006 to April 2007 in Southern Italy. Samples were tested for Campylobacter jejuni and C. coli by culture methods and suspected colonies were then confirmed by polymerase chain reaction. C. jejuni was detected in 5/60 (8.3%) samples examined but infection status was not related to age or sex and C. coli was not isolated. This is believed to be the first report of C. jejuni infection in the red squirrel. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Campylobacter jejuni; Campylobacter coli; Italy; Red squirrel; Survey
Campylobacter jejuni is now recognised 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 colonise the intestinal mucosa of most warm-blooded animals, including all food-producing species and humans (Newell, 2001). However, the favoured environment appears to be the intestinal tract of a wide range of birds which Campylobacter colonises as a commensal organism (Newell and Fearnley, 2003). No data on the prevalence of Campylobacter spp. in red squirrels (Sciurus vulgaris) are available, so we undertook this study to evaluate the presence of C. jejuni in this species. Sixty red squirrels were live-trapped from July 2006 to April 2007 in the Cilento and Vallo di Diano National Park located in the province of Salerno (Southern Italy). The corners of collection area were located at 40°06 0 48 0 0 N, 15°14 0 16 0 0 E and 40°15 0 18 0 0 N–15°03 0 17 0 0 E, respectively. The animals were trapped weekly using ground-placed Tomahawk squirrel traps (Tomahawk Live
*
Corresponding author. Tel.: +39 081 2536277; fax: +39 081 2536282. E-mail address: [email protected] (L. Dipineto).
1090-0233/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2007.09.013
Trap) baited with sunflower seeds and hazelnuts. The traps were placed randomly to cover the whole area of sampling. Thirty-four males (24 mature, 10 immature) and 26 females (19 mature, 7 immature) were caught and each was individually sampled using rectal swabs and then released. All squirrels examined were in a healthy body condition (i.e. no diarrhoea and good coat status). Squirrel handling procedures were performed according to the Office of Animal Care and Use Guidelines. Rectal swab samples were stored in Amies Transport Medium (Oxoid) at +4 °C, transported to the laboratory and analysed within 2 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) with the corresponding supplement (SE 155, 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 (SBA, Oxoid) and incubated for a further 24 h at 42 °C. Colonies comprising curved or spiral motile rods, when
150
L. Dipineto et al. / The Veterinary Journal 179 (2009) 149–150
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), 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 0 TTGAAGGTAATTTAGATATG-3 0 and cadR1B, 5 0 CTAATACCTAAAGTTGAAAC-3 0 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 0 -CAAATAAAGTTAGAGGTAG AATGT-3 0 , C-4, 5 0 -GGATAAGCATAGCTAGCTGA T3 0 and COL1, 5 0 -ATGAAAAAATATTTAGTTTTTGC A-3 0 , COL2, 5 0 -ATTTTATTATTTGTAGCAGCG-3 0 , respectively, as described by Winters et al. (1997) and Gonzalez et al. (1997). The PCR conditions were as proposed by Cloak and Fratamico (2002). In brief, amplification reactions were performed in total volumes of 50 lL containing 5 lL of purified template DNA, 10 mM Tris–HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 lM each of the four deoxynucleotide triphosphates, 1.25 U of Taq DNA polymerase (Gibco–BRL), and 0.40 lM (each) primers cadF2B, cadR1B for genus detection, and COL1, COL2, C-1, C-4 for C. jejuni and C. coli identification. A Gene Ruler 100 bp DNA ladder (Fermentas International) was used. Samples were subjected to an initial denaturation step at 94 °C for 4 min, followed by 30 amplification cycles of 94 °C for 1 min, 52 °C for 1 min, and 72 °C for 1 min and a final extension step of 72 °C for 5 min in a Model 9600 thermal cycler (PE Applied Biosystems). The PCR products were separated by electrophoresis on 1.5% agarose gels (Gibco–BRL), stained with ethidium bromide and visualized under UV light. Two reference Campylobacter strains, C. jejuni ATCC 29428 and C. coli ATCC 33559, obtained from LGC Promochem were used as positive controls. Campylobacter was detected from 5/60 (8.3%) squirrels examined. As demonstrated by PCR, all of the strains were identified as C. jejuni. The presence of C. jejuni was similar in males (3/34, 8.8%) and females (2/26, 7.7%) as well as in mature (4/43, 9.3%) and young (1/17, 5.9%) squirrels. In contrast, C. coli was not detected. The results are summarised in Table 1. C. jejuni is one of the most important causes of bacterial food-borne illnesses in humans. Although it was isolated from the normal intestinal flora in a wide range of birds and mammals, no data on the presence of this microorganism in squirrel are available. To our knowledge this is the first report of C. jejuni isolation in squirrels. The role of this rodent in the epidemiology of campylobacteriosis is not clear and cannot be evaluated solely on the basis of this study. In fact, besides the red squirrel, the Cilento and Vallo di Diano National
Table 1 Number (%) of positive samples for Campylobacter jejuni as a function of sex and age of the red squirrels (Sciurus vulgaris) examined Sex and age of squirrels examined
Number (%) of positive samples
Male, young (n = 10) Male, mature (n = 24) Female, young (n = 7) Female, mature (n = 19) Total (n = 60)
1 2 0 2 5
(10%) (8.3%) (0%) (10.5%) (8.3%)
Park houses birds of prey and Passeriformes, wild mammals (e.g. fox, wolf and marten), and reptiles (such as the viper and lizard). A number of potential species in the woodland could act as reservoirs for infection of the squirrel by Campylobacter including other wild mammals or birds. Of these potential sources birds are often described as a reservoir for infection (Huba´lek, 2004) and it would be interesting to determine whether this is the case with the red squirrel. This preliminary study has demonstrated that the presence of C. jejuni in a sampling of the red squirrel population of Southern Italy was relatively low (8.3%). However, on account of the small sample size, a larger study is required to confirm that red squirrels are potential carriers of Campylobacter spp. Moreover, further work is also needed to evaluate the possible routes of infection. References Cloak, O.M., Fratamico, P.M., 2002. A multiplex polymerase chain reaction for the differentiation of Campylobacter jejuni and Campylobacter coli from a swine processing facility and characterization of isolates by pulsed-field gel electrophoresis and antibiotic resistance profiles. Journal of Food Protection 65, 266–273. Gonzalez, I., Grant, K.A., Richardson, P.T., Park, S.F., Collins, M.D., 1997. Specific identification of the enteropathogens Campylobacter jejuni and Campylobacter coli by using a PCR test based on the ceuE gene encoding a putative virulence determinant. Journal of Clinical Microbiology 35, 759–763. Huba´lek, Z., 2004. An annotated checklist of pathogenic microorganisms associated with migratory birds. Journal of Wildlife Diseases 40, 639– 659. Konkel, M.E., Gray, S.A., Kim, B.J., Garvis, S.G., Yoon, J., 1999. Identification of the enteropathogens Campylobacter jejuni and Campylobacter coli based on the cadF virulence gene and its product. Journal of Clinical Microbiology 37, 510–517. Moore, J.E., Corcoran, D., Dooley, J.S., Fanning, S., Lucey, B., Matsuda, M., McDowell, D.A., Me´graud, F., Millar, B.C., O’Mahony, R., O’Riordan, L., O’Rourke, M., Rao, J.R., Rooney, P.J., Sails, A., Whyte, P., 2005. Campylobacter. Veterinary Research 36, 351–382. Newell, D.G., 2001. Animal models of Campylobacter jejuni colonization and disease and the lessons to be learned from similar Helicobacter pylori models. Symposium Series – Society for Applied Microbiology 30, 57S–67S. Newell, D.G., Fearnley, C., 2003. Sources of Campylobacter colonization in broiler chickens. Applied and Environmental Microbiology 69, 4343–4351. Winters, D.K., O’Leary, A.E., Slavik, M.F., 1997. Rapid PCR with nested primers for direct detection of Campylobacter jejuni in chicken washes. Molecular and Cellular Probes 11, 267–271.