808 Journal of Food Protection, Vol. 78, No. 4, 2015, Pages 808-811 doi: 10.4315/0362-028X. JFP-14-494 Copyright © , International Association for Food Protection
Research Note
Zoonotic Species of the Genus Arcobacter in Poultry from Different Regions of Costa Rica ESTEBAN VALVERDE BOGANTES,1 KAROLINA L. FALLAS-PADILLA,1 CARLOS E. RODRIGUEZ-RODRIGUEZ,2 HERIBERTO FERNANDEZ JARAMILLO,3 a n d MARIA LAURA ARIAS ECHANDI1* 1Tropical Disease Research Center and Microbiology Faculty and 2Research Center of Environmental Pollution (CICA), Universidad de Costa Rica, 2060 San Jose, Costa Rica; and 3Institute of Clinical Microbiology, Universidad Austral de Chile, Valdivia, Chile MS 14-494: Received 14 October 2014/Accepted 1 December 2014
ABSTRACT In recent years, emerging pathogens have received special attention due to their consequences for public health. Given that Arcobacter has been isolated in Costa Rica from commercial meat poultry samples, the aim of this research was to determine its isolation frequency from laying hens, broilers, ducks, and geese and to compare two types of samples, namely, cloacal swabs and stool collection. Arcobacter was isolated from 22 (11%) of the 200 samples examined. Fifteen (55%), eight (30%), and four (15%) of the isolated strains were identified as A. butzleri, A. cryareophilus, and Arcobacter spp., respectively. Also, there is a statistically significant difference among the isolation frequencies o f Arcobacter for the types of samples evaluated, yielding more isolates from stool samples than from cloacal swab collection. This work describes the distribution of Arcobacter in farm animals as potential sources for its spread from animal-derived products.
In recent years, interest in emerging pathogens has received special attention due to their consequences for public health, as in the case of Arcobacter, a potential agent of foodbome zoonotic disease (21). The genus Arcobacter was proposed in 1991 to classify organisms initially named “ aerotolerant Campylobacters” ; Arcobacter and the genera Campylobacter, Helicobacter, and Sulfurospirilium now form the family Campylobacteraceae (2, 18). Arcobacter species can be differentiated from Campylobacter because of their ability to grow at lower temperatures (15 to 30°C) and under aerobic conditions, hence, its previous classifi cation as “ aerotolerant Campylobacters” (6). Three species of Arcobacter, namely, A. butzleri, A. cryaerophilus, and A. skirrowii, have been linked to human gastrointestinal diseases, with diarrhea as the main reported symptom (6, 10, 12, 13, 21, 32, 33, 40). The infection can occur through cross-contamination in food handling, consumption of contaminated food of animal origin, drinking contaminated water, or by direct contamination with stools (6, 22). Also, Arcobacter transmission is favored because of its tolerance to high concentrations of sodium hypochlorite and its ability to grow at low temperatures and to adhere to various surfaces (5). The impact of Arcobacter on public health might be underestimated because there is no validated protocol that serves as a gold standard to isolate and identify these bacteria (21, 26, 30, 31). Nevertheless, Arcobacter has been * Author for correspondence. Tel: +506 2511 8643; Fax: +506 2511 4270; E-mail: [email protected].
isolated from poultry, milk, shellfish, and meat products worldwide (2, 4, 8, 17, 24, 28-30, 36, 37), including three reports from Costa Rica (2, 11, 15). Moreover, Arcobacter has also been isolated from stool samples of farm animals, including chickens, cows, and pigs (14, 27, 34, 35). In addition, it has been isolated from other domestic and wild animals, such as dogs, cats, fish, mollusks, turtles, ostriches, monkeys, and raccoons, from surfaces of meat processing plants, and from water, rivers, and seas (1—3, 7, 12, 14). Fecal dispersion of these bacteria has been demonstrat ed in chickens, laying hens, ducks, turkeys, and geese; nevertheless, because in these animals there is no associ ation of the presence of Arcobacter with disease, they have been proposed as natural reservoirs of Arcobacter (3, 25). The high frequency of Arcobacter isolation from intestinal tracts and stool samples from farm animals and from many meat products has led to the hypothesis that transmission through food is the most important transmission route (27). Although Arcobacter was isolated in Costa Rica from poultry meat for the first time in 2011 (2), the source of this contamination has hitherto been unknown. Likewise, it is unknown whether there is any difference in the isolation frequency yielded by the diverse sampling methods employed so far in different studies. The aims of this research were to determine the frequency of isolation of Arcobacter spp. in poultry samples from Costa Rica, particularly from laying hens, broilers, ducks, and geese, and to compare two types of samples for the isolation of Arcobacter, namely cloacal swabs and stool samples.
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TABLE 1. Isolation frequencies and species o/Arcobacter spp. in poultry obtained from two different types o f samples Sample
Duck
Geese
Broiler chicken
Laying hen
Total
Cloacal swab Stool
1/25 9/25
0/25 5/25
0/25 3/25
0/25 4/25
1/100 21/100
10/50
5/50
3/50
4/50
22/200
7 3 1
3 1 1
2 1 0
3 3 2
15 (55%) 8 (30%) 4 (15%)
Total No. of isolations A. butzleri A. cryaerophilus Arcobacter spp.
M A TER IA LS A N D M E TH O D S Sampling. Samples were randomly taken from 25 laying hens, 25 broiler chickens, 25 ducks, and 25 geese from different farms in the country. Farms were randomly distributed in the Metropolitan Area of San Jose, Costa Rica. Laying hens and broiler chickens were sampled from industrialized production farms, geese and ducks from small, nonindustrialized farms. Birds were caged when samples were taken. Two samples from each animal, a cloacal swab and a stool sample, were taken with sterile cotton-tipped swabs. Cloacal swabs were placed in tubes containing 10 ml of H ouf s Arcobacter enrichment broth containing 24 g liter- 1 Arcobacter broth (Oxoid, Basingstoke, UK), and the selective supplement (100 mg liter 1 5-fluorouracil, 10 mg liter 1 amphotericine B, 16 mg liter-1 cefoperazone, 32 mg liter-1 novobiocine, and 64 mg liter-1 trimethoprim) (23) immediately after being taken. Fecal samples were collected less than 5 min after defecation, and approximately 1 g of feces was placed in 9 ml of Arcobacter enrichment broth, as recommended by Houf et al. (22). Samples were transported under refrigeration (2 to 8 C) to the Food Microbiology Laboratory, Faculty of Microbiology, University of Costa Rica, and were analyzed within a maximum period of 24 h after collection. Isolation of Arcobacter. Enrichment media were incubated aerobically at 30°C for 48 h. Then Arcobacter isolation was performed using the membrane filtration technique (3). Briefly, 100 pi of enrichment media was inoculated onto a sterile 0.45-pmpore-size nitrocellulose membrane filter (Millipore, Darmstadt, Germany) placed on the surface of nonselective blood agar plates (Oxoid, Hampshire, UK). The plates were left at room temperature for 1 h, so that mobile Arcobacter cells could pass through the membrane filter. After removing the filter, plates were incubated aerobically at 30UC for up to 5 days. Suspicious colonies (approximately 2 to 3 mm in diameter, round, uncolored) were selected, checked by Gram staining and oxidase test, and purified on blood agar before identification by PCR. Identification of Arcobacter isolates by PCR. DNA extraction was performed using the boiling lysis method. The PCR technique described by Harmon and Wesley (20) was used for identification at genus level. Briefly, PCR amplification was performed in a reaction mixture (50 pi) containing Tris-HCl buffer (pH 7.4), 0.2 mM deoxynucleoside triphosphates, 1.5 mM MgCl2 (Fermentas, Waltham, MA), 1 pM each primer (Fermentas), 1.5 U/ pi Taq polymerase (Oxoid), and 5 pi of template DNA. The Arcobacter genus-specific 16S rRNA fragment was amplified using the forward primer Arco I (5'-AGA GAT TAG CCT GTA TTG TAT C-3') and the reverse primer ArcoII (5'-TAG CAT CCC CGC TTC GAA TGA-3') (20). The thermocycling program was as follows: 94°C for 4 min; 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min (25 cycles); and 72°C for 7 min. Analysis of PCR
products was done by electrophoresis (60 V, 1.5 h) in 1.5% agarose gels (wt/vol) with mass ruler (100 to 1,000 bp; Thermo Scientific, Waltham, MA) and GelRed staining (Biotium, Hay ward, CA). Positive Arcobacter identification was reported when a product of 1,223 bp was obtained. To differentiate Arcobacter isolates at the species level, the multiplex-PCR assay described by Douidah et al. (10) was used. Briefly, amplification of the specific fragments was done with the forward primer Arco F (5'-GCT AGA GGA AGA GAA ATC AA3') and the reverse primers ButR (5'-TCC TGA TAC AAG ATA ATT GTA CG-3'), TherR (5'-GCA ACC TCT TTG GCT TAC GAA-3'), CibR (5'-CGA AC A GGA TTC TCA CCT GT-3'), and SkiR (5'-TCA GGA TAC CAT TAA AGT TAT TGA TG-3') for 4. butzleri (2,061 bp), A. thereius (1,590 bp), A. cibarius (1,125 bp), and A. skirrowii (198 bp), respectively (13). Amplification of the 395-bp fragment from A. cryaerophilus was done with the primers CriF (5'-CAG AGG AAG AGA AAT CAA AT-3') and CriR (5'CCC ACT ATT CCA TCA GTG AG-3') (13). Template and reaction mixture were prepared as described above. Amplification was performed using Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, CA). The thermocycling program was as follows: 94°C for 4 min; 94°C for 45 s, 58°C for 45 s, and 72°C for 2 min (30 cycles); and 72"C for 7 min. PCR products were separated by electrophoresis in 2% agarose gels with mass ruler (100 to 1,000 bp; Thermo Scientific) (wt/vol). Gels were stained using GelRed (Biotium), and UV light was used for visualization. Comparison of sampling methods. A statistical comparison was carried out to determine whether there was statistical difference between the two sampling methods tested, cloacal swabs and stool collection. The chi-square test was applied using the MegaStat complement for Microsoft Excel 2013 (Microsoft Co., Redmond, WA) (a = 0.05). R ESULTS A N D D ISC U SSIO N
This study represents the first evaluation of the isolation frequency of Arcobacter spp. from live poultry in Costa Rica. The results obtained demonstrate the presence of Arcobacter in the birds sampled. Arcobacter was isolated from 22 of the 200 samples examined (11%). Isolation frequencies of Arcobacter spp. by bird and type of sample analyzed are shown in Table 1. In general, the most frequently isolated species was A. butzleri, accounting for 55% of isolates, followed by A. cryaerophilus with 30%. The remaining 15% was identified as Arcobacter spp., and no strain was identified as A. skirrowii. These results are consistent with the literature, where A. butzleri is reported as the most frequently isolated species, followed by A. cryaerophilus (20). The nonrecov-
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VALVERDE BOGANTES ET AL.
ery of A. skirrowii has also been reported by different research groups (21, 27), and this may be due to a low prevalence of these species among poultry or to the use of inadequate isolation methods, where competing bacteria may overgrow A. skirrowii (26, 27). Coinfection with more than one species was detected in four stool samples: one sample with three different species (from a laying hen) and three samples with two different species (one from a duck and the other two from laying hens). Arcobacter isolation frequency from ducks was 4% using cloacal swabs and 36% using stool collection. These results are consistent with those obtained in southern Chile, where the frequency of isolation of Arcobacter from fecal samples of ducks was 40% (14). As to geese, an isolation frequency of 20% was obtained using stool collection, but no isolates were obtained using cloacal swabs. These results contrast with those reported by other authors. For instance, Atabay et al. (3) reported an isolation frequency of 18% using cloacal swabs, whereas Dogan and Atabay (9) reported an isolation frequency of 26%, also from cloacal swabs in the same geographical region. In broiler chickens, an isolation frequency of 12% was obtained when stool collection was performed, and no isolates were recovered when using cloacal swabs. The literature reports diverse results. In Japan (24), an isolation frequency of 14% was obtained using cloacal swabs, whereas in Nigeria a frequency of 1.3% was obtained from the same type of sample (1). Other authors have also reported low isolation frequency in chickens, ranging from 0.1 to 3% (79); this has been controversial in recent years because chicken meat is associated with the highest prevalence of this bacteria. These discrepancies may reflect different methodological aspects, such as type of culture medium, incubation atmosphere, amount of sample analyzed, or even the geographic area where the sampling was performed (16). Finally, in laying hens an isolation frequency of 12% was obtained using feces, and no isolates were recovered using cloacal swabs. This result is consistent with a study from Chile (14), wherein a frequency of isolation of 20% was obtained from feces. Furthermore, a study conducted in the Netherlands (26) also demonstrated that the isolation frequency depends on the geographic area of sampling. The results show a significant difference (P < 0.05) between the numbers of isolates obtained from feces or from the intestinal tract, with a higher isolation frequency when stool collection is performed, compared to the use of cloacal swabs. A possible explanation for this is based on the possibility that bacteria pass through the digestive tract of poultry but are unable to colonize this environment, possibly due to the high corporal temperature of birds (41 °C) (38,39). In this study, the presence of Arcobacter was evaluated in laying hens; nonetheless, the question of whether contamination is transmitted from hens to eggs was not assessed. In this respect, evaluating the isolation frequency of Arcobacter from eggs for human consumption would be of great importance for public health. This work demonstrates the presence of Arcobacter in poultry from different regions of Costa Rica. However,
further research is necessary to better understand the epidemiology of Arcobacter in poultry and to determine the potential risk of transmission in the fami environment or the food chain through poultry-derived products. ACKNOWLEDGMENT The authors are thankful for financial support from Vicerrectorfa de Investigacion of Universidad de Costa Rica through the project 803-B4053.
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Research Note
Zoonotic Species of the Genus Arcobacter in Poultry from Different Regions of Costa Rica ESTEBAN VALVERDE BOGANTES,1 KAROLINA L. FALLAS-PADILLA,1 CARLOS E. RODRIGUEZ-RODRIGUEZ,2 HERIBERTO FERNANDEZ JARAMILLO,3 a n d MARIA LAURA ARIAS ECHANDI1* 1Tropical Disease Research Center and Microbiology Faculty and 2Research Center of Environmental Pollution (CICA), Universidad de Costa Rica, 2060 San Jose, Costa Rica; and 3Institute of Clinical Microbiology, Universidad Austral de Chile, Valdivia, Chile MS 14-494: Received 14 October 2014/Accepted 1 December 2014
ABSTRACT In recent years, emerging pathogens have received special attention due to their consequences for public health. Given that Arcobacter has been isolated in Costa Rica from commercial meat poultry samples, the aim of this research was to determine its isolation frequency from laying hens, broilers, ducks, and geese and to compare two types of samples, namely, cloacal swabs and stool collection. Arcobacter was isolated from 22 (11%) of the 200 samples examined. Fifteen (55%), eight (30%), and four (15%) of the isolated strains were identified as A. butzleri, A. cryareophilus, and Arcobacter spp., respectively. Also, there is a statistically significant difference among the isolation frequencies o f Arcobacter for the types of samples evaluated, yielding more isolates from stool samples than from cloacal swab collection. This work describes the distribution of Arcobacter in farm animals as potential sources for its spread from animal-derived products.
In recent years, interest in emerging pathogens has received special attention due to their consequences for public health, as in the case of Arcobacter, a potential agent of foodbome zoonotic disease (21). The genus Arcobacter was proposed in 1991 to classify organisms initially named “ aerotolerant Campylobacters” ; Arcobacter and the genera Campylobacter, Helicobacter, and Sulfurospirilium now form the family Campylobacteraceae (2, 18). Arcobacter species can be differentiated from Campylobacter because of their ability to grow at lower temperatures (15 to 30°C) and under aerobic conditions, hence, its previous classifi cation as “ aerotolerant Campylobacters” (6). Three species of Arcobacter, namely, A. butzleri, A. cryaerophilus, and A. skirrowii, have been linked to human gastrointestinal diseases, with diarrhea as the main reported symptom (6, 10, 12, 13, 21, 32, 33, 40). The infection can occur through cross-contamination in food handling, consumption of contaminated food of animal origin, drinking contaminated water, or by direct contamination with stools (6, 22). Also, Arcobacter transmission is favored because of its tolerance to high concentrations of sodium hypochlorite and its ability to grow at low temperatures and to adhere to various surfaces (5). The impact of Arcobacter on public health might be underestimated because there is no validated protocol that serves as a gold standard to isolate and identify these bacteria (21, 26, 30, 31). Nevertheless, Arcobacter has been * Author for correspondence. Tel: +506 2511 8643; Fax: +506 2511 4270; E-mail: [email protected].
isolated from poultry, milk, shellfish, and meat products worldwide (2, 4, 8, 17, 24, 28-30, 36, 37), including three reports from Costa Rica (2, 11, 15). Moreover, Arcobacter has also been isolated from stool samples of farm animals, including chickens, cows, and pigs (14, 27, 34, 35). In addition, it has been isolated from other domestic and wild animals, such as dogs, cats, fish, mollusks, turtles, ostriches, monkeys, and raccoons, from surfaces of meat processing plants, and from water, rivers, and seas (1—3, 7, 12, 14). Fecal dispersion of these bacteria has been demonstrat ed in chickens, laying hens, ducks, turkeys, and geese; nevertheless, because in these animals there is no associ ation of the presence of Arcobacter with disease, they have been proposed as natural reservoirs of Arcobacter (3, 25). The high frequency of Arcobacter isolation from intestinal tracts and stool samples from farm animals and from many meat products has led to the hypothesis that transmission through food is the most important transmission route (27). Although Arcobacter was isolated in Costa Rica from poultry meat for the first time in 2011 (2), the source of this contamination has hitherto been unknown. Likewise, it is unknown whether there is any difference in the isolation frequency yielded by the diverse sampling methods employed so far in different studies. The aims of this research were to determine the frequency of isolation of Arcobacter spp. in poultry samples from Costa Rica, particularly from laying hens, broilers, ducks, and geese, and to compare two types of samples for the isolation of Arcobacter, namely cloacal swabs and stool samples.
J. Food Prot., Vol. 78, No. 4
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TABLE 1. Isolation frequencies and species o/Arcobacter spp. in poultry obtained from two different types o f samples Sample
Duck
Geese
Broiler chicken
Laying hen
Total
Cloacal swab Stool
1/25 9/25
0/25 5/25
0/25 3/25
0/25 4/25
1/100 21/100
10/50
5/50
3/50
4/50
22/200
7 3 1
3 1 1
2 1 0
3 3 2
15 (55%) 8 (30%) 4 (15%)
Total No. of isolations A. butzleri A. cryaerophilus Arcobacter spp.
M A TER IA LS A N D M E TH O D S Sampling. Samples were randomly taken from 25 laying hens, 25 broiler chickens, 25 ducks, and 25 geese from different farms in the country. Farms were randomly distributed in the Metropolitan Area of San Jose, Costa Rica. Laying hens and broiler chickens were sampled from industrialized production farms, geese and ducks from small, nonindustrialized farms. Birds were caged when samples were taken. Two samples from each animal, a cloacal swab and a stool sample, were taken with sterile cotton-tipped swabs. Cloacal swabs were placed in tubes containing 10 ml of H ouf s Arcobacter enrichment broth containing 24 g liter- 1 Arcobacter broth (Oxoid, Basingstoke, UK), and the selective supplement (100 mg liter 1 5-fluorouracil, 10 mg liter 1 amphotericine B, 16 mg liter-1 cefoperazone, 32 mg liter-1 novobiocine, and 64 mg liter-1 trimethoprim) (23) immediately after being taken. Fecal samples were collected less than 5 min after defecation, and approximately 1 g of feces was placed in 9 ml of Arcobacter enrichment broth, as recommended by Houf et al. (22). Samples were transported under refrigeration (2 to 8 C) to the Food Microbiology Laboratory, Faculty of Microbiology, University of Costa Rica, and were analyzed within a maximum period of 24 h after collection. Isolation of Arcobacter. Enrichment media were incubated aerobically at 30°C for 48 h. Then Arcobacter isolation was performed using the membrane filtration technique (3). Briefly, 100 pi of enrichment media was inoculated onto a sterile 0.45-pmpore-size nitrocellulose membrane filter (Millipore, Darmstadt, Germany) placed on the surface of nonselective blood agar plates (Oxoid, Hampshire, UK). The plates were left at room temperature for 1 h, so that mobile Arcobacter cells could pass through the membrane filter. After removing the filter, plates were incubated aerobically at 30UC for up to 5 days. Suspicious colonies (approximately 2 to 3 mm in diameter, round, uncolored) were selected, checked by Gram staining and oxidase test, and purified on blood agar before identification by PCR. Identification of Arcobacter isolates by PCR. DNA extraction was performed using the boiling lysis method. The PCR technique described by Harmon and Wesley (20) was used for identification at genus level. Briefly, PCR amplification was performed in a reaction mixture (50 pi) containing Tris-HCl buffer (pH 7.4), 0.2 mM deoxynucleoside triphosphates, 1.5 mM MgCl2 (Fermentas, Waltham, MA), 1 pM each primer (Fermentas), 1.5 U/ pi Taq polymerase (Oxoid), and 5 pi of template DNA. The Arcobacter genus-specific 16S rRNA fragment was amplified using the forward primer Arco I (5'-AGA GAT TAG CCT GTA TTG TAT C-3') and the reverse primer ArcoII (5'-TAG CAT CCC CGC TTC GAA TGA-3') (20). The thermocycling program was as follows: 94°C for 4 min; 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min (25 cycles); and 72°C for 7 min. Analysis of PCR
products was done by electrophoresis (60 V, 1.5 h) in 1.5% agarose gels (wt/vol) with mass ruler (100 to 1,000 bp; Thermo Scientific, Waltham, MA) and GelRed staining (Biotium, Hay ward, CA). Positive Arcobacter identification was reported when a product of 1,223 bp was obtained. To differentiate Arcobacter isolates at the species level, the multiplex-PCR assay described by Douidah et al. (10) was used. Briefly, amplification of the specific fragments was done with the forward primer Arco F (5'-GCT AGA GGA AGA GAA ATC AA3') and the reverse primers ButR (5'-TCC TGA TAC AAG ATA ATT GTA CG-3'), TherR (5'-GCA ACC TCT TTG GCT TAC GAA-3'), CibR (5'-CGA AC A GGA TTC TCA CCT GT-3'), and SkiR (5'-TCA GGA TAC CAT TAA AGT TAT TGA TG-3') for 4. butzleri (2,061 bp), A. thereius (1,590 bp), A. cibarius (1,125 bp), and A. skirrowii (198 bp), respectively (13). Amplification of the 395-bp fragment from A. cryaerophilus was done with the primers CriF (5'-CAG AGG AAG AGA AAT CAA AT-3') and CriR (5'CCC ACT ATT CCA TCA GTG AG-3') (13). Template and reaction mixture were prepared as described above. Amplification was performed using Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, CA). The thermocycling program was as follows: 94°C for 4 min; 94°C for 45 s, 58°C for 45 s, and 72°C for 2 min (30 cycles); and 72"C for 7 min. PCR products were separated by electrophoresis in 2% agarose gels with mass ruler (100 to 1,000 bp; Thermo Scientific) (wt/vol). Gels were stained using GelRed (Biotium), and UV light was used for visualization. Comparison of sampling methods. A statistical comparison was carried out to determine whether there was statistical difference between the two sampling methods tested, cloacal swabs and stool collection. The chi-square test was applied using the MegaStat complement for Microsoft Excel 2013 (Microsoft Co., Redmond, WA) (a = 0.05). R ESULTS A N D D ISC U SSIO N
This study represents the first evaluation of the isolation frequency of Arcobacter spp. from live poultry in Costa Rica. The results obtained demonstrate the presence of Arcobacter in the birds sampled. Arcobacter was isolated from 22 of the 200 samples examined (11%). Isolation frequencies of Arcobacter spp. by bird and type of sample analyzed are shown in Table 1. In general, the most frequently isolated species was A. butzleri, accounting for 55% of isolates, followed by A. cryaerophilus with 30%. The remaining 15% was identified as Arcobacter spp., and no strain was identified as A. skirrowii. These results are consistent with the literature, where A. butzleri is reported as the most frequently isolated species, followed by A. cryaerophilus (20). The nonrecov-
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ery of A. skirrowii has also been reported by different research groups (21, 27), and this may be due to a low prevalence of these species among poultry or to the use of inadequate isolation methods, where competing bacteria may overgrow A. skirrowii (26, 27). Coinfection with more than one species was detected in four stool samples: one sample with three different species (from a laying hen) and three samples with two different species (one from a duck and the other two from laying hens). Arcobacter isolation frequency from ducks was 4% using cloacal swabs and 36% using stool collection. These results are consistent with those obtained in southern Chile, where the frequency of isolation of Arcobacter from fecal samples of ducks was 40% (14). As to geese, an isolation frequency of 20% was obtained using stool collection, but no isolates were obtained using cloacal swabs. These results contrast with those reported by other authors. For instance, Atabay et al. (3) reported an isolation frequency of 18% using cloacal swabs, whereas Dogan and Atabay (9) reported an isolation frequency of 26%, also from cloacal swabs in the same geographical region. In broiler chickens, an isolation frequency of 12% was obtained when stool collection was performed, and no isolates were recovered when using cloacal swabs. The literature reports diverse results. In Japan (24), an isolation frequency of 14% was obtained using cloacal swabs, whereas in Nigeria a frequency of 1.3% was obtained from the same type of sample (1). Other authors have also reported low isolation frequency in chickens, ranging from 0.1 to 3% (79); this has been controversial in recent years because chicken meat is associated with the highest prevalence of this bacteria. These discrepancies may reflect different methodological aspects, such as type of culture medium, incubation atmosphere, amount of sample analyzed, or even the geographic area where the sampling was performed (16). Finally, in laying hens an isolation frequency of 12% was obtained using feces, and no isolates were recovered using cloacal swabs. This result is consistent with a study from Chile (14), wherein a frequency of isolation of 20% was obtained from feces. Furthermore, a study conducted in the Netherlands (26) also demonstrated that the isolation frequency depends on the geographic area of sampling. The results show a significant difference (P < 0.05) between the numbers of isolates obtained from feces or from the intestinal tract, with a higher isolation frequency when stool collection is performed, compared to the use of cloacal swabs. A possible explanation for this is based on the possibility that bacteria pass through the digestive tract of poultry but are unable to colonize this environment, possibly due to the high corporal temperature of birds (41 °C) (38,39). In this study, the presence of Arcobacter was evaluated in laying hens; nonetheless, the question of whether contamination is transmitted from hens to eggs was not assessed. In this respect, evaluating the isolation frequency of Arcobacter from eggs for human consumption would be of great importance for public health. This work demonstrates the presence of Arcobacter in poultry from different regions of Costa Rica. However,
further research is necessary to better understand the epidemiology of Arcobacter in poultry and to determine the potential risk of transmission in the fami environment or the food chain through poultry-derived products. ACKNOWLEDGMENT The authors are thankful for financial support from Vicerrectorfa de Investigacion of Universidad de Costa Rica through the project 803-B4053.
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