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Prevalence of thermotolerant Campylobacter spp. in farmed hares (Lepus europaeus)
The Veterinary Journal 202 (2014) 186–187
Contents lists available at ScienceDirect
The Veterinary Journal j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t v j l
Short Communication
Prevalence of thermotolerant Campylobacter spp. in farmed hares (Lepus europaeus) Antonio Santaniello a,*, Ludovico Dipineto a, Vincenzo Veneziano a, Ugo Mariani b, Alessandro Fioretti a, Lucia Francesca Menna a a b
Department of Veterinary Medicine and Animal Production, University of Naples Federico II, via F. Delpino, 1-80137 Naples, Italy Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici, Contrada San Chirico, 1-82100 Benevento, Italy
A R T I C L E
I N F O
Article history: Accepted 26 June 2014 Keywords: Farmed hares Faeces Thermotolerant Campylobacter spp.
A B S T R A C T
Thermotolerant Campylobacter spp. were isolated from 118/240 (49.2%) rectal swabs from commercially farmed hares (Lepus europaeus) in southern Italy. Using multiplex PCR, Campylobacter coli was identified in 118/118 (100%) positive samples, while 17/118 (14.4%) positive samples were also positive for Campylobacter jejuni. Adult hares had a higher prevalence of infection with Campylobacter spp. than juvenile hares. © 2014 Elsevier Ltd. All rights reserved.
Thermotolerant Campylobacter spp., primarily Campylobacter jejuni and Campylobacter coli, colonise the intestinal mucosa of many food producing animals, as well as human beings. Several avian species are considered to be the main reservoirs of Campylobacter spp. (Newell and Fearnley, 2003). There is little information on the occurrence of Campylobacter spp. in hares (Lepus europaeus). The present study was undertaken to determine the prevalence of thermotolerant Campylobacter spp. in hares farmed for meat production and game in southern Italy. Rectal swabs were collected from 240 clinically healthy hares from February to July 2013 on a breeding farm with 3000 hares located in southern Italy. The hares were reared in outdoor cages, each containing five to six animals. Young hares were moved to a grazing area until release for repopulation of protected areas or for meat consumption. Samples were collected from 120 juvenile hares (preweaning: ≤1 month of age; 60 male, 60 female) and 120 adult hares (≥6 months of age; 60 male, 60 female). The study was approved by the Animal Ethics and Welfare Committee of the University of Naples Federico II (protocol number 10418; date of approval 24 January 2012). Rectal swabs were placed in Amie’s Transport Medium (Oxoid) at 4 °C, then inoculated into Campylobacter selective enrichment broth (Oxoid) within 1 h of collection and incubated at 42 °C for 48 h under microaerophilic conditions (CampyGen; Oxoid). Each sample was then streaked onto Campylobacter blood-free selective agar (Oxoid). Grey, moist, flat, spreading colonies typical of Campylobacter spp. were subcultured on sheep blood agar (Oxoid) and incubated
* Corresponding author. Tel.: +39 81 2536275. E-mail address: [email protected] (A. Santaniello). http://dx.doi.org/10.1016/j.tvjl.2014.06.022 1090-0233/© 2014 Elsevier Ltd. All rights reserved.
for 24 h at 42 °C. Under phase contrast microscopy, colonies with curved or spiral motile rods were presumptively identified as Campylobacter spp. DNA was extracted using a Bactozol kit (Molecular Research Center). Isolates were confirmed as Campylobacter spp. by PCR for the cadF gene using oligonucleotide primers cadF2B and cadR1B (Gargiulo et al., 2008). A triplex PCR was used to speciate C. jejuni, C. coli and Campylobacter lari using oligonucleotide primers (C. jejuni: ICJ-UP and ICJ-DN; C. coli: ICC-UP and ICC-DN; C. lari: ICL-UP and ICL-DN) and amplification conditions according to Khan and Edge (2007). PCR products were separated by electrophoresis on 1.5% agarose gels (Gibco-BRL), stained with ethidium bromide and visualised under ultraviolet light. Three reference strains of Campylobacter spp. (C. jejuni ATCC 29428, C. coli ATCC 33559 and C. lari ATCC 43675; LGC Promochem) were used as positive controls, while PCR without DNA template was used as a negative control. Thermotolerant Campylobacter spp. were recovered from 118/ 240 (49.2%) rectal swabs (Table 1). The prevalence was 4/120 (3.3%) in hares ≤ 1 month of age and 114/120 (95.0%) in hares ≥ 6 months of age; this difference was statistically significant (P < 0.01; Pearson’s χ2; SPSS 13 for Windows). There was no significant difference related to sex (P = 0.606; Pearson’s χ2). C. coli was identified in 118/118 (100%) culture positive samples by multiplex PCR, while 17/118 (14.4%) culture positive samples were also positive by PCR for C. jejuni; C. lari was not identified. The results of this study indicate that thermotolerant Campylobacter spp. are frequent colonisers of the intestinal tract of farmed hares in southern Italy, with an overall prevalence of 49.2%. In contrast, Rosef et al. (1983) reported a prevalence of thermotolerant Campylobacter spp. of 4.3% in 23 wild hares
A. Santaniello et al./The Veterinary Journal 202 (2014) 186–187
Table 1 Detection of thermotolerant Campylobacter spp. by culture in faeces from farmed hares.
Age Juvenile (≤1 month) Adult (≥6 months) Sex Male Female Total a
Number tested
Number positive
% positive
95% Confidence interval
Pa
120 120
4 114
3.3 95.0
1.1–8.8 89.0–98.0
<0.001
120 120 240
61 57 118
50.8 47.5 49.2
41.6–60.0 38.4–56.8 42.7–55.7
0.349
Pearson’s χ2 test.
(Lepus timidus) in Norway, whereas Wahlström et al. (2003) reported a prevalence of 1% in 47 pooled faecal samples from hares in Sweden. In the present study, C. coli was identified more frequently than C. jejuni, similar to findings in pheasants (Phasianus colchicus; Dipineto et al., 2008) and wild red squirrels (Sciurus vulgaris; Dipineto et al., 2009). Hares ≥ 6 months of age had a higher prevalence of thermotolerant Campylobacter spp. than hares ≤ 1 month of age. This difference in prevalence could be explained by the longer period of exposure of adult hares to different sources of infection, such as mowed grass contaminated by wild animal faeces and/or cages contaminated with faeces from wild birds and rodents. In conclusion, the results of this study show that farmed hares may be carriers of thermotolerant Campylobacter spp. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.
187
Acknowledgements This study was supported by the Ministry of Health of the Italian Republic (grant number IZSME 05/10 RC C71J1000012000).
References Dipineto, L., Gargiulo, A., De Luca Bossa, L.M., Rinaldi, L., Borrelli, L., Menna, L.F., Fioretti, A., 2008. Prevalence of thermotolerant Campylobacter in pheasants (Phasianus colchicus). Avian Pathology 37, 507–508. Dipineto, L., Gargiulo, A., Cuomo, A., Santaniello, A., Sensale, M., Borrelli, L., D’Angelo, L., Menna, L.F., Fioretti, A., 2009. Campylobacter jejuni in the red squirrel (Sciurus vulgaris) population of Southern Italy. The Veterinary Journal 179, 149–150. Gargiulo, A., Rinaldi, L., D’Angelo, L., Dipineto, L., Borrelli, L., Fioretti, A., Menna, L.F., 2008. Survey of Campylobacter jejuni in stray cats in Southern Italy. Letters in Applied Microbiology 46, 267–270. Khan, I.U.H., Edge, T.A., 2007. Development of a novel triplex PCR assay for the detection and differentiation of thermophilic species of Campylobacter using 16S–23S rDNA internal transcribed spacer (ITS) region. Journal of Applied Microbiology 103, 2561–2569. Newell, D.G., Fearnley, C., 2003. Sources of Campylobacter colonization in broiler chickens. Applied and Environmental Microbiology 69, 4343–4351. Rosef, O., Gondrosen, B., Kapperud, G., Underdal, B., 1983. Isolation and characterization of Campylobacter jejuni and Campylobacter coli from domestic and wild mammals in Norway. Applied and Environmental Microbiology 46, 855–859. Wahlström, H., Tysén, E., Olsson Engvall, E., Brändström, B., Eriksson, E., Mörner, T., Vågsholm, I., 2003. Survey of Campylobacter species, VTEC O157 and Salmonella species in Swedish wildlife. Veterinary Record 153, 74–80.
Contents lists available at ScienceDirect
The Veterinary Journal j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t v j l
Short Communication
Prevalence of thermotolerant Campylobacter spp. in farmed hares (Lepus europaeus) Antonio Santaniello a,*, Ludovico Dipineto a, Vincenzo Veneziano a, Ugo Mariani b, Alessandro Fioretti a, Lucia Francesca Menna a a b
Department of Veterinary Medicine and Animal Production, University of Naples Federico II, via F. Delpino, 1-80137 Naples, Italy Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici, Contrada San Chirico, 1-82100 Benevento, Italy
A R T I C L E
I N F O
Article history: Accepted 26 June 2014 Keywords: Farmed hares Faeces Thermotolerant Campylobacter spp.
A B S T R A C T
Thermotolerant Campylobacter spp. were isolated from 118/240 (49.2%) rectal swabs from commercially farmed hares (Lepus europaeus) in southern Italy. Using multiplex PCR, Campylobacter coli was identified in 118/118 (100%) positive samples, while 17/118 (14.4%) positive samples were also positive for Campylobacter jejuni. Adult hares had a higher prevalence of infection with Campylobacter spp. than juvenile hares. © 2014 Elsevier Ltd. All rights reserved.
Thermotolerant Campylobacter spp., primarily Campylobacter jejuni and Campylobacter coli, colonise the intestinal mucosa of many food producing animals, as well as human beings. Several avian species are considered to be the main reservoirs of Campylobacter spp. (Newell and Fearnley, 2003). There is little information on the occurrence of Campylobacter spp. in hares (Lepus europaeus). The present study was undertaken to determine the prevalence of thermotolerant Campylobacter spp. in hares farmed for meat production and game in southern Italy. Rectal swabs were collected from 240 clinically healthy hares from February to July 2013 on a breeding farm with 3000 hares located in southern Italy. The hares were reared in outdoor cages, each containing five to six animals. Young hares were moved to a grazing area until release for repopulation of protected areas or for meat consumption. Samples were collected from 120 juvenile hares (preweaning: ≤1 month of age; 60 male, 60 female) and 120 adult hares (≥6 months of age; 60 male, 60 female). The study was approved by the Animal Ethics and Welfare Committee of the University of Naples Federico II (protocol number 10418; date of approval 24 January 2012). Rectal swabs were placed in Amie’s Transport Medium (Oxoid) at 4 °C, then inoculated into Campylobacter selective enrichment broth (Oxoid) within 1 h of collection and incubated at 42 °C for 48 h under microaerophilic conditions (CampyGen; Oxoid). Each sample was then streaked onto Campylobacter blood-free selective agar (Oxoid). Grey, moist, flat, spreading colonies typical of Campylobacter spp. were subcultured on sheep blood agar (Oxoid) and incubated
* Corresponding author. Tel.: +39 81 2536275. E-mail address: [email protected] (A. Santaniello). http://dx.doi.org/10.1016/j.tvjl.2014.06.022 1090-0233/© 2014 Elsevier Ltd. All rights reserved.
for 24 h at 42 °C. Under phase contrast microscopy, colonies with curved or spiral motile rods were presumptively identified as Campylobacter spp. DNA was extracted using a Bactozol kit (Molecular Research Center). Isolates were confirmed as Campylobacter spp. by PCR for the cadF gene using oligonucleotide primers cadF2B and cadR1B (Gargiulo et al., 2008). A triplex PCR was used to speciate C. jejuni, C. coli and Campylobacter lari using oligonucleotide primers (C. jejuni: ICJ-UP and ICJ-DN; C. coli: ICC-UP and ICC-DN; C. lari: ICL-UP and ICL-DN) and amplification conditions according to Khan and Edge (2007). PCR products were separated by electrophoresis on 1.5% agarose gels (Gibco-BRL), stained with ethidium bromide and visualised under ultraviolet light. Three reference strains of Campylobacter spp. (C. jejuni ATCC 29428, C. coli ATCC 33559 and C. lari ATCC 43675; LGC Promochem) were used as positive controls, while PCR without DNA template was used as a negative control. Thermotolerant Campylobacter spp. were recovered from 118/ 240 (49.2%) rectal swabs (Table 1). The prevalence was 4/120 (3.3%) in hares ≤ 1 month of age and 114/120 (95.0%) in hares ≥ 6 months of age; this difference was statistically significant (P < 0.01; Pearson’s χ2; SPSS 13 for Windows). There was no significant difference related to sex (P = 0.606; Pearson’s χ2). C. coli was identified in 118/118 (100%) culture positive samples by multiplex PCR, while 17/118 (14.4%) culture positive samples were also positive by PCR for C. jejuni; C. lari was not identified. The results of this study indicate that thermotolerant Campylobacter spp. are frequent colonisers of the intestinal tract of farmed hares in southern Italy, with an overall prevalence of 49.2%. In contrast, Rosef et al. (1983) reported a prevalence of thermotolerant Campylobacter spp. of 4.3% in 23 wild hares
A. Santaniello et al./The Veterinary Journal 202 (2014) 186–187
Table 1 Detection of thermotolerant Campylobacter spp. by culture in faeces from farmed hares.
Age Juvenile (≤1 month) Adult (≥6 months) Sex Male Female Total a
Number tested
Number positive
% positive
95% Confidence interval
Pa
120 120
4 114
3.3 95.0
1.1–8.8 89.0–98.0
<0.001
120 120 240
61 57 118
50.8 47.5 49.2
41.6–60.0 38.4–56.8 42.7–55.7
0.349
Pearson’s χ2 test.
(Lepus timidus) in Norway, whereas Wahlström et al. (2003) reported a prevalence of 1% in 47 pooled faecal samples from hares in Sweden. In the present study, C. coli was identified more frequently than C. jejuni, similar to findings in pheasants (Phasianus colchicus; Dipineto et al., 2008) and wild red squirrels (Sciurus vulgaris; Dipineto et al., 2009). Hares ≥ 6 months of age had a higher prevalence of thermotolerant Campylobacter spp. than hares ≤ 1 month of age. This difference in prevalence could be explained by the longer period of exposure of adult hares to different sources of infection, such as mowed grass contaminated by wild animal faeces and/or cages contaminated with faeces from wild birds and rodents. In conclusion, the results of this study show that farmed hares may be carriers of thermotolerant Campylobacter spp. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.
187
Acknowledgements This study was supported by the Ministry of Health of the Italian Republic (grant number IZSME 05/10 RC C71J1000012000).
References Dipineto, L., Gargiulo, A., De Luca Bossa, L.M., Rinaldi, L., Borrelli, L., Menna, L.F., Fioretti, A., 2008. Prevalence of thermotolerant Campylobacter in pheasants (Phasianus colchicus). Avian Pathology 37, 507–508. Dipineto, L., Gargiulo, A., Cuomo, A., Santaniello, A., Sensale, M., Borrelli, L., D’Angelo, L., Menna, L.F., Fioretti, A., 2009. Campylobacter jejuni in the red squirrel (Sciurus vulgaris) population of Southern Italy. The Veterinary Journal 179, 149–150. Gargiulo, A., Rinaldi, L., D’Angelo, L., Dipineto, L., Borrelli, L., Fioretti, A., Menna, L.F., 2008. Survey of Campylobacter jejuni in stray cats in Southern Italy. Letters in Applied Microbiology 46, 267–270. Khan, I.U.H., Edge, T.A., 2007. Development of a novel triplex PCR assay for the detection and differentiation of thermophilic species of Campylobacter using 16S–23S rDNA internal transcribed spacer (ITS) region. Journal of Applied Microbiology 103, 2561–2569. Newell, D.G., Fearnley, C., 2003. Sources of Campylobacter colonization in broiler chickens. Applied and Environmental Microbiology 69, 4343–4351. Rosef, O., Gondrosen, B., Kapperud, G., Underdal, B., 1983. Isolation and characterization of Campylobacter jejuni and Campylobacter coli from domestic and wild mammals in Norway. Applied and Environmental Microbiology 46, 855–859. Wahlström, H., Tysén, E., Olsson Engvall, E., Brändström, B., Eriksson, E., Mörner, T., Vågsholm, I., 2003. Survey of Campylobacter species, VTEC O157 and Salmonella species in Swedish wildlife. Veterinary Record 153, 74–80.