Prevalence and characteristics of Salmonella spp. isolated from commercial layer farms in Korea Min Chan Im,∗ So Jeong Jeong,∗ Yong-Kuk Kwon,† Ok-Mi Jeong,† Min-Su Kang,† and Young Ju Lee∗,1 ∗
Department of Public Health, College of Veterinary Medicine, Kyungpook National University, 702-701, Daegu, Republic of Korea; and † Animal and Plant Quarantine Agency, 175 Anyangro, Manan-gu, Anyangsi Gyeonggido, 480-757, Anyang, Republic of Korea (P < 0.001). The most frequently observed Salmonella serovars in the flocks were Salmonella Bareilly (41.2%), Salmonella Mbandaka (32.4%), and Salmonella Rissen (17.6%). Twenty of the flocks revealed multi-serovar contamination, with the isolation of 2 to 4 serovars. Antimicrobial susceptibility testing revealed that 93 out of 101 isolates were susceptible to the 17 tested antimicrobial agents. The remaining isolates displayed resistance to ampicillin (4.0%), nalidixic acid (3.0%), tetracycline (1.0%), cephalothin (1.0%), and gentamicin (1.0%). As human salmonellosis has been repeatedly correlated to the consumption of poultry products worldwide, continuous studies are required to effectively minimize the Salmonella contamination in layer farms and egg products.
Key words: Salmonella, layer farm, prevalence, risk factor, antimicrobial susceptibility 2015 Poultry Science 94:1691–1698 http://dx.doi.org/10.3382/ps/pev137
INTRODUCTION Salmonellosis is one of the most prevalent foodborne diseases, causing diarrhea, fever, vomiting, and sometimes even death. Incidence of salmonellosis ranged between 14.53 and 17.55 cases per 100,000 people in the United States (Johnson et al., 2014), and 95,548 cases in the European Union (EFSA and ECDC, 2013). Salmonella has been reported to be the major pathogen effecting foodborne illnesses in Korea, with a total of 9,472 cases of salmonellosis isolated from among the patients of food- and water-borne illnesses between 1998 and 2007 (Kim, 2010). Outbreaks of salmonellosis in humans have often been associated with the consumption of contaminated raw chicken meat, eggs, beef, pork, seafood, and vegetables (Pires et al., 2014). Among these, chicken meat and eggs are the types of food that cause salmonellosis most often (Jackson et al., 2013; Middleton et al., 2014). In recent times, the incidence of egg-borne salmonel C 2015 Poultry Science Association Inc. Received January 28, 2015. Accepted April 14, 2015. 1 Coressponding author: [email protected]
losis has frequently been reported in many countries. Harker et al. (2013) reported that the human salmonellosis outbreaks between 2000 and 2011 in England and Wales were predominantly caused by the consumption of contaminated eggs. Dominguez et al. (2007) reported that 48% of human salmonellosis were derived from eggs in Spain from 1990 to 2003. In addition, a majority of foodborne salmonellosis cases that occurred between 1998 and 2004 in Japan were associated with eggs (Toyofuku, 2008). The contamination of eggs with Salmonella could have occurred through the laying hens. Such a contamination could occur via horizontal transmission from the feces of the contaminated hen or the environmental dust after oviposition through the eggshell (De Reu et al., 2006), or by vertical transmission through the yolk, albumen, or eggshell membranes before oviposition (Keller et al., 1995). Although the prevalence of Salmonella in raw eggshells has been discovered to be relatively low, salmonellosis continues to be associated with eggs. This is because of the considerable human consumption of eggs, and possible re-contamination resulting from improper egg processing and handling (Hara-Kudo and Takatori, 2009).
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ABSTRACT Salmonellosis is one of the most prevalent foodborne illnesses. The outbreak of this disease is often associated with eggs. In this study, the prevalence and characteristics of Salmonella was surveyed in layer farms in Korea. In addition, the risk factors affecting the prevalence of Salmonella in these farms were also assessed. Of the 32 farms and 67 flocks examined, 19 farms (59.3%) and 34 flocks (50.7%) were observed to be positive for Salmonella contamination. Salmonella was detected in the surrounding environment such as feces (41.8%), dust (40.3%), egg shells (17.2%), as well as the internal egg contents (5.2%). The incidence of Salmonella positives were tended to increase when the flock size is larger (P = 0.021). Differences in the provinces also affected Salmonella prevalence
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There exists a high probability that eggs are contaminated with Salmonella at the layer farms, via contaminated environmental matter, such as the chicken feed, water, feces, and dust. Therefore, the layer farms must be properly managed, in order to diminish prevalence of Salmonella in the hens and eggs. Many studies have attempted to determine the prevalence of Salmonella in layer farms (Barua et al., 2012; Huneau-Sala¨ un et al., 2009; Iwabuchi et al., 2010; Poppe et al., 1991; Snow et al., 2007). However, few studies have examined the occurrence of Salmonella contaminations in Korean layer farms. In this study, we surveyed the prevalence and characteristics of Salmonella in Korean layer farms, and attempted to assess any possible risk factors affecting Salmonella prevalence.
Sample Collection According to Statistics Korea: Livestock statistics in the first quarter 2013 (Statistics Korea, 2013), about 1,200 commercial layer flocks were present and the average number of laying hens in single layer flock was 47,761. Considering our circumstances and the proportion of number of laying hens and total number of layer flocks in different studies, we investigated 32 commercial layer farms (67 flocks) in Korea between 2013 and 2014, collecting dust, feces, and raw shell eggs from each of the flocks. In addition, epidemiological information, such as the province, age, flock size, hen-day egg production, and the form of poultry house, was also obtained. The laying hens in all surveyed layer farms were vaccinated using the Nobilis SG 9R (Intervet International, Netherlands). More than one flock/farm was sampled in farms stocked with multiple flocks of different ages. All flocks tested in this study comprised of over 2,500 birds. Therefore, the dust and feces samples were collected in accordance with the standards set by the National Poultry Improvement Plan (USDA, 2012). Briefly, a sterile surgical gauze swab (12 layers of 10 × 10 cm) was moistened with 12-mL sterile doublestrength skim milk (Fluka, Germany). Fifteen different spots were swabbed per flock in order to collect 10 g each dust sample. Approximately 10 g feces were also sampled from 15 different locations; no individual birds were sampled. Sixty raw shell eggs were also collected per flock. All samples were transported to the laboratory in a cooler and stored at 5◦ C for further use.
Processing of Dust and Feces Samples Fifteen dust and feces samples each per flock were individually weighed. The samples were individually inoculated into buffered peptone water (Difco, United States) at a 1:10 ratio, and incubated for 18 to 24 h at 37◦ C.
Egg surface and contents were processed according to the National Poultry Improvement Plan standards (USDA, 2012) and the Food and Drug Administration Bacteriological Analytical Manual (FDA, 2012), respectively. Twenty eggs were swabbed and pooled for each of egg shell and contents samples (3 swabs and egg pools/flock). The entire external surface of each egg was swabbed with moistened sterile swabs. The swabs were dipped in 90 mL buffered peptone water (Difco, United States) and incubated for 18 to 24 h at 37◦ C. The eggs were then submerged in disinfectants consisting of 3 parts of 70% alcohol to one part iodine (Sigma–Aldrich, Chile) / potassium iodide (Sigma, Germany) solution for 10 s. The eggs were removed from the solution and allowed to air-dry. Each egg was flame-sterilized at the pointed end and cracked open using a sterile scalpel blade. The contents were poured into a sterile WhirlPak stomacher bag (Nasco, United States). The pooled contents of 20 eggs were mixed thoroughly using a sterile tool and gloved hands, until the yolks were mixed completely with the albumen; the gloves were changed between samples. Samples were incubated at room temperature for 96 ± 2 h. Following this, 25 mL from each sample was transferred to 225 mL tryptic soy broth (Dfico, United States) supplemented with 7.8 mg ferrous sulfate (Sigma, United Kingdom) and incubated for 24 ± 2 h at 35◦ C.
Isolation and Identification of Salmonella Pre-enriched buffered peptone water or tryptic soy broth media was transferred to the selective enrichment broth. One hundred μL mixture was transferred to 10 mL Rappaport Vassiliadis medium (Difco, United States). In addition, 1 mL mixture was mixed with 10 mL tetrathionate broth. These 2 media were incubated for 24 ± 2 h at 42◦ C and 24 ± 2 h at 35◦ C, respectively. Each selective broth was streaked onto xylose lysine desoxycholate agar (Difco, United States), bismuth sulfite agar (Difco, United States), and RAMBACH agar (Merek, Germany) using a 3-mm inoculating loop, and incubated for 24 ± 2 h at 35◦ C. The suspected Salmonella colonies observed on the agar plates were confirmed by identification of the invA gene by PCR, using a previously described method (Rahn et al., 1992). One to 3 Salmonella colonies were picked from each agar plates, and serotyped using the commercial Salmonella O and H phase 1 and phase 2 antisera (Difco, United States), as per the Kauffmann– White scheme detailed in a previous study (Grimont and Weill, 2007). If several isolates originating from the same type of samples per flock displayed identical serotypes and antimicrobial susceptibility patterns, only one of these isolates was selected randomly and included in the data analysis.
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MATERIALS AND METHODS
Processing of Shell Eggs
SALMONELLA IN LAYER FARMS IN KOREA
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were observed to be positive for Salmonella. Salmonella was most frequently detected in the feces (41.8%) samples. The contamination was detected at a lower percentage in the dust (40.3%), egg shell (17.2%), and egg content (5.2%) samples.
Risk Factors for Salmonella Prevalence
Figure 1. Prevalence of Salmonella in layer farms and flocks. Farms and flocks were designated as positives when one or more positives were detected from any of feces, dust, eggshell, or egg contents samples.
The antimicrobial susceptibility profiles of the isolates were determined using the Mueller–Hinton agar (Difco, United States) by the disk diffusion method detailed by the Clinical and Laboratory Standards Institute (2012). Seventeen antimicrobial agents (Difco, United States) were tested using the following concentrations: amoxicillin/clavulanic acid (20/10 μg), ampicillin (10 μg), aztreonam (30 μg), cefotaxime (30 μg), cefoxitin (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), cephalothin (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), gentamicin (10 μg), kanamycin (30 μg), meropenam (10 μg), nalidixic acid (30 μg) piperacillin/tazobactam (100/10 μg), tetracycline (30 μg), and trimethoprim/sulfamethoxazole (1.25/23.75 μg). All the results were interpreted according to the Clinical and Laboratory Standards Institute M02 and M07 criteria (CLSI, 2012).
Statistical Analysis The data was analyzed using the Statistical Package for Social Sciences, version 18.0 (IBM, Korea). The chi-squared test and a logistic regression analysis were performed to evaluate the association between many of the risk factors and the prevalence of Salmonella. The chi-squared test was performed for categorically independent variables, such as the province and type of poultry house. Logistic regression analysis was conducted for continuous independent variables, such as the flock size, age of laying hens, and hen-day egg production.
RESULTS Prevalence of Salmonella The prevalence of Salmonella in the layer farms and flocks is displayed in Figure 1. Of the 32 farms and 67 flocks examined, 19 farms (59.3%) and 34 flocks (50.7%)
Distribution of Salmonella Serotypes The Salmonella serotypes identified have been summarized in Table 2. The Salmonella isolates were typed into 15 serovars. The most frequently observed Salmonella serovars in the flocks were S. Bareilly (41.2%), S. Mbandaka (32.4%), S. Rissen (17.6%), S. Heidelberg (11.4%), S. V 1,13,22:i:- (11.4%), S. Agona (11.4%), S. Senftenberg (11.4%), S. Saintpaul (11.4%), and S. Infantis (11.4%).
Multi-Serovar Contamination of the Salmonella-Positive Flocks The distribution of multiple Salmonella serovars in the flocks (tested positive for bacterial infection) is displayed in Table 3. Fourteen flocks were contaminated by only one Salmonella serovar. However, 20 flocks displayed contamination by multiple serovars, with the isolation of 2 to 4 serovars.
Antimicrobial Resistance Profiles The results of the antimicrobial susceptibility analysis of the Salmonella isolates were summarized in Table 4. Ninety-three out of the 101 isolates were observed to be susceptible to the 17 antimicrobial agents tested in this study. The most common antimicrobial agents to which the isolates showed any resistance were ampicillin (4.0%), nalidixic acid (3.0%), tetracycline (1.0%), cephalothin (1.0%), and gentamicin (1.0%). The Salmonella isolates were not resistant to kanamycin, cefotaxime, cefotaxime, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, meropenam, aztreonam, piperacillin/tazobactam, trimethoprim/sulfamethoxazole, and amoxicillin/clavulanic acid. Each isolate of the Agona and Senftenberg serovars of Salmonella displayed resistance to 2 antimicrobial agents, ampicillin and tetracycline, and gentamicin and nalidixic acid, respectively. Five
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Antimicrobial Susceptibility Testing
The relationship between the risk of bacterial prevalence in the flocks and the rates of Salmonella contamination is listed in Table 1. Salmonella contamination was observed to be higher when the flock size was larger (P = 0.021). The difference in provinces was also observed to affect the prevalence of Salmonella (P < 0.001). However, the age, type of poultry house, and hen-day egg production did not cause any significant differences in the prevalence of Salmonella.
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IM ET AL. Table 1. Risk factors for Salmonella at the Korean layer farms. Risk factors
No. flocks
No. samples positive
tested
for Salmonella (%)
Feces
Dust
Egg shells
Egg contents
67
34 (50.7)
28
27
10
3
8 6 15 20 9 9
0 (0) 5 (83.3) 5 (33.3) 9 (45.0) 6 (66.7) 9 (100)
0 5 3 5 6 9
0 5 4 4 6 9
0 0 2 1 3 4
0 3 0 0 0 0
55 12
30 (54.5) 4 (33.3)
25 3
24 3
8 2
3 0
23 19 10 7 8
10 (43.4) 7 (36.8) 4 (40.0) 7 (100) 6 (75.0)
9 4 3 6 6
8 3 3 7 6
2 1 1 4 2
3 0 0 0 0
5 17 14 15 6 10
1 (25.0) 10 (58.8) 7 (50.0) 8 (53.3) 4 (66.7) 4 (40.0)
1 7 6 6 4 4
1 9 4 7 3 3
0 4 1 3 1 1
0 1 1 0 0 1
9 5 11 33 9
4 (44.4) 3 (60.0) 4 (36.3) 17 (63.6) 6 (66.7)
3 2 3 14 6
4 3 3 13 4
0 1 1 6 2
0 1 0 1 1
P value
< 0.001
0.183 0.021
0.868
0.473
1 Laying hens below 18-weeks-old. These flocks were not considered for the construction of logistic regression models assessing the relationship between hen-day egg production and the prevalence of Salmonella.
Table 2. Distribution of Salmonella serotypes observed in isolates obtained from various sources of the 34 Salmonella-positive flocks. Salmonella serotypes
Bareilly Mbandaka Rissen Heidelberg V 1,13,22:i:Agona Senftenberg Infantis Saintpaul Braenderup Thompson Kentucky Livingstone Hato Meleagridis
No. positive flocks (%)1
14 (41.2) 11 (32.4) 6 (17.6) 4 (11.4) 4 (11.4) 4 (11.4) 4 (11.4) 4 (11.4) 4 (11.4) 3 (8.8) 3 (8.8) 1 (2.9) 1 (2.9) 1 (2.9) 1 (2.9)
No. flocks positive for Salmonella contamination per sample type Dust
Feces
Egg shells
Egg contents
11 6 4 1 5 1 4 3 3 2 2 1 1 0 1
12 8 5 1 3 4 0 3 3 1 1 0 0 1 0
5 2 0 2 0 0 0 1 0 1 0 0 0 0 0
0 2 0 1 0 0 0 0 0 0 0 0 0 0 0
1 Flocks were designated as positive when one or more samples from the flocks were determined to be positive for Salmonella contamination, regardless of the sample type.
different resistance patterns were observed among the Salmonella isolates.
DISCUSSION In this study, the prevalence of Salmonella in commercial layer farms in Korea and the constituent flocks was determined to be 59.3 and 50.7%, respectively. The
prevalence was observed to be higher than that observed in recent studies conducted in other nations, such as the United Kingdom (11.7%), France (17.9%), Japan (23.6%), and Bangladesh (18.0%) (Barua et al., 2012; Iwabuchi et al., 2010; Huneau-sala¨ un et al., 2009; Snow et al., 2007). Although the use of different methods for sampling, isolation, and identification could affect the overall results, the prevalence of Salmonella in
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Total Province Jeonbook Jeonnam Kyungbook Kyungnam Kyunggi Chungbook Form of poultry house No windows With windows Flock size (no. layers) Very small (≤20,000) Small (20,001 to 40,000) Medium (40,001 to 60,000) Large (60,001 to 80,000) Very large (≥80,001) Age of laying hens (wk) ≤15 16 to 30 31 to 45 46 to 60 61 to 75 ≥76 Hen-day egg production (%) Pullet1 0–70 71–80 81–90 91–100
No. sample types affected
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SALMONELLA IN LAYER FARMS IN KOREA Table 3. Multi-serovar contamination in 34 Salmonella-positive flocks. Flock code1
1 2 2 4 3 2 1 1 2 1 3 2 2 4 3 2 3 3 2 2 1 1 1 1 2 2 1 1 1 1 1 4 1 3
Serovars isolated per sample type Feces
Dust
Egg shell
Egg contents
Branderup Hato, Heidelberg, Mbandaka Mbandaka, 1,13,22:i:1,13,22:i:Mbandaka Mbandaka Agona Bareilly Bareilly Bareilly Bareilly Mbandaka, Rissen, Saintpaul Mbandaka, Rissen, 1,13,22:i:Bareilly Bareilly Bareilly, Rissen, Saintpaul Bareilly, Rissen Bareilly, Saintpaul Mbandaka Bareilly Infantis Mbandaka Bareilly Agona Agona Agona Bareilly, Infantis, Thompson Infantis -
Bareilly Braenderup, Thompson Bareilly Heidelberg, 1,13,22:i:Mbandaka, 1,13,22:i:Mbandaka, 1,13,22:i:Mbandaka Agona, Meleagridis Bareilly Bareilly Bareilly Bareilly Mbandaka, Saintpaul, 1,13,22:i:Mbandaka, Rissen, 1,13,22:i:Bareilly, Mbandaka Bareilly, Rissen, Senftenberg Bareilly, Rissen, Senftenberg Bareilly, Rissen Bareilly, Saintpaul Thompson Infantis Livingstone Braenderup Kentucky Infantis, Senftenberg Infantis Rissen, Saintpaul, Senftenberg
Braenderup Heidelberg, Mbandaka Heidelberg Mbandaka Bareilly Bareilly Bareilly Bareilly Bareilly Infantis -
Heidelberg Mbandaka Mbandaka -
1 Multiple flocks obtained from the same farm were designated with identical alphabet codes. The number code denotes different flocks.
Table 4. Resistance profiles of Salmonella isolates obtained from the layer farm environment and raw shell eggs to 17 antimicrobials. Salmonella serotypes Mbandaka Agona Braenderup Senftenberg Infantis Livingstone Bareilly 1,13,22:i:Rissen Saintpaul Heidelberg Thompson Kentucky Hato Meleagridis
No. isolates tested (n = 101)
Antimicrobial resistance profiles1
No. resistant isolates (%)
18 5 4 4 7 1 28 8 9 6 5 3 1 1 1
Cephalothin Ampicillin – tetracycline Tetracycline Gentamicin – nalidixic acid Nalidixic acid Nalidixic acid -
1 (5.6) 1 (20.0) 3 (75.0) 1 (25.0) 1 (14.7) 1 (100) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
1 All strains were treated with amoxicillin/clavulanic acid, ampicillin, aztreonam, cefotaxime, cefoxitin, ceftazidime, ceftriaxone, cephalothin, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, meropenam, nalidixic acid, piperacillin/tazobactam, tetracycline, and trimethoprim/sulfamethoxazole.
Korea observed in this study suggested that the Korean layer farms examined in this study exercised poor hygiene management. The classical method for Salmonella detection involves an analysis of the feces (Barua et al., 2012; Li et al., 2007; Poppe et al., 1991) or dust (Iwabuchi et al.,
2010), or both (Huneau-sala¨ un et al., 2009; Sasaki et al., 2012). There have been some controversies regarding the better method for detection of Salmonella contamination. In this study, Salmonella was isolated in 41.8 and 40.3% of the feces and dust samples, respectively. In addition, Salmonella was recovered from only one
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A-1 B-1 C-1 D-1 D-2 D-3 E-1 E-2 F-1 G-1 G-2 G-3 G-4 H-1 H-2 H-3 H-4 H-5 H-6 H-7 I-1 J-1 J-2 K-1 L-1 M-1 N-1 O-1 O-2 P-1 Q-1 R-1 S-1 S-2
No. serovars contaminated
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Li et al., 2007; Poppe et al., 1991; Sasaki et al., 2012). The serovars predominantly-isolated were S. Bareilly, S. Mbandaka and S. Rissen. S. Bareilly was rarely isolated in the poultry farms in Korea (Kim et al., 2013). However, it has been recently isolated from various types of food products, such as bean sprouts, and broiler chicken. Therefore, it is often associated with incidence of human salmonellosis throughout the world (Chiou et al., 2009; Cleary et al., 2010; Kudaka et al., 2006). S. Bareilly has been specifically isolated from many poultry farms and their produce in Japan (Iwabuchi et al., 2010; Kudaka et al., 2006; Sasaki et al., 2012). The second predominant Salmonella serovar identified in this study was S. Mbandaka. This serovar has been frequently isolated from contaminated poultry products throughout the world (Clemente et al., 2014; Iwabuchi et al., 2010; Le Bouquin et al., 2010), and has also been reported in Korea (Kim et al., 2013). S. Rissen, which was also predominantly isolated in our study, was isolated from healthy pigs in Korea (Lim et al., 2009). Although S. Rissen has rarely been isolated from layer farms in other countries, it has been recovered from chicken meat in Thailand (Angkititrakul et al., 2005), suggesting that this serovar could be isolated from poultry products. Notably, S. Enteritidis and S. Gallinarum, serovars that have been frequently isolated in Korea (Kim et al., 2012; Kim et al., 2013; Kwon et al., 2010; Lee et al., 2007), were not isolated in our study. There are several conceivable reasons for this decrease. The vaccination of commercial layer chicken, utilizing the live S. Gallinarum 9R strain, was initiated in Korea since 2001, in order to control fowl typhoid caused by S. Gallinarum. As a result, the number of outbreaks of fowl typhoid in layer chicken has drastically reduced since 2002 (Kwon et al., 2010). Although we do not know exact prevalence about S. Enteritidis from commercial layer farms in Korea because of insufficiency of literature, It is obvious that the S. Enteritidis strains were isolated from layers between 1995 and 1999 (Yang et al., 2002), before vaccination of SG 9R on layer farms. S. Gallinarum and S. Enteritidis belong to the Salmonella serogroup D1, and express the same immune dominance (Ochoa-Rep´araz et al., 2004). Therefore, a reasonable cross-protection between the 2 serovars could be expected (Penha Filho et al., 2009). Feberwee et al. (2001) reported that the prevalence of S. Enteritidis in the group vaccinated with the live S. Gallinarum 9R strain was significantly lower than in the non-vaccinated group. These studies indicate that the occurrence of S. Gallinarum and S. Enteritidis in layer farms may have reduced as a result of the vaccine program in Korea. Antimicrobial susceptibility testing was performed on the 101 isolates obtained in this study. Eight of these isolates showed antimicrobial resistance to at least one antimicrobial agent. The rate of resistance of Salmonella to multiple antimicrobial agents was lower than that previously observed in the Korean broiler
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type of environmental sample of the 32% of the positive flocks. These results demonstrated that the collection of both dust and feces samples was necessary for the accurate detection of Salmonella in layer farms. The frequency of Salmonella incidence was lower in egg shells (17.2%) and egg content (5.2%), compared to the dust and feces samples. Although the rate of isolation of Salmonella from egg shells varied between different studies, the contamination of the egg content was extremely rare (Chemaly et al., 2009; Chousalkar and Roberts, 2012; Garc´ıa et al., 2011; Jones et al., 1995). It has been demonstrated that the Salmonella contaminating the eggshells could migrate to the egg content, under normal conditions of storage and moisture (De Reu et al., 2006). In addition, the eggs could be contaminated by vertical transmission (Keller et al., 1995). We investigated several risk factors that could affect the prevalence of Salmonella in commercial layer farms. In this study, the flock size appeared to affect the prevalence of Salmonella in laying hens. Several studies have demonstrated that the number of hens in each flock is an important risk factor affecting Salmonella infections (Huneau-sala¨ un et al., 2009; Mollenhorst et al., 2005; Snow et al., 2007), and our results corresponded to this theory. In this study, the difference in province was also related to the Salmonella contamination. This may be a result of other complicated factors, the most probable being the relationship between the province and flock size. As Kyunggi and Chungbook provinces are areas with a high population density, the size of the layer farm tends to be larger in these provinces, compared to other provinces for increased supply of eggs (data not shown). A previous study, conducted in Japan, showed that the prevalence of Salmonella was significantly higher in flocks reared in windowless poultry houses than in those reared in poultry houses with windows (Sasaki et al., 2012). However, this study demonstrated that the type of poultry house did not cause any significant impact on the prevalence of Salmonella. The age of the laying hens also did not significantly affect the prevalence of Salmonella in this study. Some studies have demonstrated that the prevalence of Salmonella can be influenced by the age of the laying hens, especially via the feces samples (Gole et al., 2014; Li et al., 2007). However, our study demonstrated similar fecal contamination, regardless of the age of the hens. These results could be a result of endemic contamination in the environment, occurring before the housing of chicks in layer farms in Korea. Therefore, several other risk factors, besides the age of the flocks and types of poultry house, must be investigated to determine their influence on Salmonella contamination. Fifteen different Salmonella serovars were identified in this study. Most of the isolated serovars in this study were not predominantly observed in the broiler supply chains in Korea (Kim et al., 2012; Lee et al., 2007; Yoon et al., 2014). However, these were commonly isolated in the layer farms in other countries (Iwabuchi et al., 2010;
SALMONELLA IN LAYER FARMS IN KOREA
ACKNOWLEDGMENTS This study was supported by a fund (Z-1541781-201315-02) by the Research of Animal and Plant Quarantine Agency, South Korea.
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Clemente, L., I. Correia, P. Themudo, I. Neto, M. Cani¸ca, and F. Bernardo. 2014. Antimicrobial susceptibility of Salmonella enterica isolates from healthy breeder and broiler flocks in Portugal. Vet. J. 200:276–281. Clinical and Laboratory Standards Institute (CLSI). 2012. Performance standards for antimicrobial susceptibility testing, M100S22. Clinical and Laboratory Standards Institute, Wayne, PA. De Reu, K., K. Grijspeerdt, W. Messens, M. Heyndrickx, M. Uyttendaele, J. Debevere, and L. Herman. 2006. Eggshell factors influencing eggshell penetration and whole egg contamination by different bacteria, including Salmonella enteritidis. Int. J. Food Microbiol. 112:253–260. Dominguez, A., N. Torner, L. Ruiz, A. Martinez, R. Bartolome, E. Sulleiro, A. Teixid´ o, and A. Plasencia. 2007. Foodborne Salmonella-caused outbreaks in Catalonia (Spain), 1990 to 2003. J. Food Prot. 70:209–213. European Food Safety Authority (EFSA), European Center for Disease Prevention and Control (ECDC). 2013. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2011. EFSA J. 11:e3129. Feberwee, A., T. S. De Vries, E. G. Hartman, J. J. De Wit, A. R. W. Elbers, and W. A. De Jong. 2001. Vaccination against Salmonella enteritidis in Dutch commercial layer flocks with a vaccine based on a live Salmonella gallinarum 9R strain: Evaluation of efficacy, safety, and performance of serologic Salmonella tests. Avian Dis. 45:83–91. Filho, R. A. C. P., J. B. de Paiva, Y. M. S. Arg¨ uello, M. D. da Silva, Y. Gardin, F. Resende, A. B. Berchieri Jr, and L. Sesti. 2009. Efficacy of several vaccination programmes in commercial layer and broiler breeder hens against experimental challenge with Salmonella enterica serovar Enteritidis. Avian Pathol. 38:367– 375. Food and Drug Administration. 2012. FDA’s bacteriological analytical manual (BAM). Chapter 5: Salmonella, Washington, DC. Garc´ıa, C., J. M. Soriano, V. Ben´ıtez, and P. Catal´ a-Gregori. 2011. Assessment of Salmonella spp. in feces, cloacal swabs, and eggs (eggshell and content separately) from a laying hen farm. Poult Sci. 90:1581–1585. Gole, V. C., C. G. Caraguel, M. Sexton, C. Fowler, and K. K. Chousalkar. 2014. Shedding of Salmonella in single age caged commercial layer flock at an early stage of lay. Int. J. Food Microbiol. 189:61–66. Grimont, P. A., and F. X. Weill. 2007. Antigenic formulae of the Salmonella serovars. WHO Collaborating Center for Reference and Research on Salmonella, Institut Pasteur, Paris, France. Hara-Kudo, Y., and K. Takatori. 2009. Microbial quality of liquid egg and Salmonella infection status in Japan. Shokuhin Eiseigaku Zasshi. 50:34–40. Harker, K. S., C. Lane, F. J. Gormley, and G. K. Adak. 2013. National outbreaks of Salmonella infection in the UK, 2000–2011. Epidemiol. Infect. 142:601–607. Huneau-Sala¨ un, A., C. Marianne, L. B. Sophie, L. Fran¸coise, P. Isabelle, R. Sandra, M. Virginie, F. Philippe, and R. Nicolas. 2009. Risk factors for Salmonella enterica subsp. Enterica contamination in 519 French laying hen flocks at the end of the laying period. Prev. Vet. Med. 89:51–58. Iwabuchi, E., N. Maruyama, A. Hara, M. Nishimura, M. Muramatsu, T. Ochiai, and K. Hirai. 2010. Nationwide survey of Salmonella prevalence in environmental dust from layer farms in Japan. J. Food Prot. 73:1993–2000. Iwabuchi, E., S. Yamamoto, Y. Endo, T. Ochiai, and K. Hirai. 2011. Prevalence of Salmonella isolates and antimicrobial resistance patterns in chicken meat throughout Japan. J. Food Prot. 74:270– 273. Jackson, B. R., P. M. Griffin, D. Cole, K. A. Walsh, and S. J. Chai. 2013. Outbreak-associated Salmonella enterica serotypes and food commodities, United States, 1998–2008. Emerg. Infect. Dis. 19:1239–1244. Johnson, N. B., L. D. Hayes, K. Brown, E. C. Hoo, and K. A. Ethier. 2014. CDC national health report: Leading causes of morbidity and mortality and associated behavioral risk and protective factors–United States, 2005–2013. MMWR Surveill. Summ. 63:3– 27.
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supply chains (Bae et al., 2013; Kim et al., 2012; Lee et al., 2007). Generally, Salmonella isolated from the broilers demonstrated greater multi-drug resistance compared to those isolated from layers and eggs (Asai et al., 2006; Iwabuchi et al., 2010; Iwabuchi et al., 2011; Kudaka et al., 2006). In addition, different Salmonella serovars tended to display different types of antimicrobial resistance (Clemente et al., 2014; Lai et al., 2014). Although most of the isolates displayed a high level of susceptibility to most of the antimicrobial agents tested in this study, the antimicrobial susceptibility must be evaluated continually because of the possibility of multi-drug resistant Salmonella outbreaks in the future. In conclusion, this study attempted to analyze the various characteristics of Salmonella contamination, such as the prevalence, antimicrobial resistance profiles, and risk factor assessment, in layer farms in Korea. As human salmonellosis has been repeatedly related to the consumption of poultry products worldwide, continuous research must be conducted to minimize the Salmonella contamination in layer farms and egg products.
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Jones, F. T., D. V. Rives, and J. B. Carey. 1995. Salmonella contamination in commercial eggs and an egg production facility. Poult Sci. 74:753–757. Keller, L. H., C. E. Benson, K. Krotec, and R. J. Eckroade. 1995. Salmonella enteritidis colonization of the reproductive tract and forming and freshly laid eggs of chickens. Infect. Immun. 63:2443– 2449. Kim, J. H., J. K. Cho, and K. S. Kim. 2013. Prevalence and characterization of plasmid-mediated quinolone resistance genes in Salmonella isolated from poultry in Korea. Avian Pathol. 42:221– 229. Kim, M. S., T. H. Lim, J. H. Jang, D. H. Lee, B. Y. Kim, J. H. Kwon, S. W. Choi, J. Y. Noh, Y. H. Hong, S. B. Lee, S. Y. Yang, H. J. Lee, J. B. Lee, S. Y. Park, I. S. Choi, and C. S. Song. 2012. Prevalence and antimicrobial resistance of Salmonella species isolated from chicken meats produced by different integrated broiler operations in Korea. Poult. Sci. 91:2370–2375. Kim, S. 2010. Salmonella serovars from foodborne and waterborne diseases in Korea, 1998–2007: Total isolates decreasing versus rare serovars emerging. J. Korean Med. Sci. 25:1693–1699. Kudaka, J., K. Itokazu, K. Taira, A. Iwai, M. Kondo, T. Susa, and M. Iwanaga. 2006. Characterization of Salmonella isolated in Okinawa, Japan. Jpn. J. Infect. Dis. 59:15–19. Kwon, Y. K., A. Kim, M. S. Kang, M. Her, B. Y. Jung, K. M. Lee, W. Jeong, B. K. An, and J. H. Kwon. 2010. Prevalence and characterization of Salmonella Gallinarum in the chicken in Korea during 2000 to 2008. Poult. Sci. 89:236–242. Lai, J., C. Wu, C. Wu, J. Qi, Y. Wang, H. Wang, Y. Liu, and J. Shen. 2014. Serotype distribution and antibiotic resistance of Salmonella in food-producing animals in Shandong province of China, 2009 and 2012. Int. J. Food Microbiol. 180:30–38. Le Bouquin, S., V. Allain, S. Rouxel, I. Petetin, M. Picherot, V. Michel, and M. Chemaly. 2010. Prevalence and risk factors for Salmonella spp. contamination in French broiler-chicken flocks at the end of the rearing period. Prev. Vet. Med. 97:245–251. Lee, Y. J., H. J. Kim, C. K. Park, K. S. Kim, D. H. Bae, M. S. Kang, J. K. Cho, A. R. Kim, J. W. Kim, and B. H. Kim. 2007. Characterization of Salmonella spp. isolated from an integrated broiler chicken operation in Korea. J. Vet. Med. Sci. 69:399–404. Li, X., J. B. Payne, F. B. Santos, J. F. Levine, K. E. Anderson, and B. W. Sheldon. 2007. Salmonella populations and prevalence in layer feces from commercial high-rise houses and characterization of the Salmonella isolates by serotyping, antibiotic resistance analysis, and pulsed field gel electrophoresis. Poult Sci. 86:591–597. Lim, S. K., H. S. Lee, H. M. Nam, S. C. Jung, H. B. Koh, and I. S. Roh. 2009. Antimicrobial resistance and phage types of Salmonella isolates from healthy and diarrheic pigs in Korea. Foodborne Pathog. Dis. 6:981–987. Middleton, D., R. Savage, M. K. Tighe, L. Vrbova, R. Walton, Y. Whitfield, C. Varga, B. Lee, L. Rosella, B. Dhar, C. Johnson, R. Ahmed, V. G. Allen, and N. S. Crowcroft. 2014. Risk factors
Department of Public Health, College of Veterinary Medicine, Kyungpook National University, 702-701, Daegu, Republic of Korea; and † Animal and Plant Quarantine Agency, 175 Anyangro, Manan-gu, Anyangsi Gyeonggido, 480-757, Anyang, Republic of Korea (P < 0.001). The most frequently observed Salmonella serovars in the flocks were Salmonella Bareilly (41.2%), Salmonella Mbandaka (32.4%), and Salmonella Rissen (17.6%). Twenty of the flocks revealed multi-serovar contamination, with the isolation of 2 to 4 serovars. Antimicrobial susceptibility testing revealed that 93 out of 101 isolates were susceptible to the 17 tested antimicrobial agents. The remaining isolates displayed resistance to ampicillin (4.0%), nalidixic acid (3.0%), tetracycline (1.0%), cephalothin (1.0%), and gentamicin (1.0%). As human salmonellosis has been repeatedly correlated to the consumption of poultry products worldwide, continuous studies are required to effectively minimize the Salmonella contamination in layer farms and egg products.
Key words: Salmonella, layer farm, prevalence, risk factor, antimicrobial susceptibility 2015 Poultry Science 94:1691–1698 http://dx.doi.org/10.3382/ps/pev137
INTRODUCTION Salmonellosis is one of the most prevalent foodborne diseases, causing diarrhea, fever, vomiting, and sometimes even death. Incidence of salmonellosis ranged between 14.53 and 17.55 cases per 100,000 people in the United States (Johnson et al., 2014), and 95,548 cases in the European Union (EFSA and ECDC, 2013). Salmonella has been reported to be the major pathogen effecting foodborne illnesses in Korea, with a total of 9,472 cases of salmonellosis isolated from among the patients of food- and water-borne illnesses between 1998 and 2007 (Kim, 2010). Outbreaks of salmonellosis in humans have often been associated with the consumption of contaminated raw chicken meat, eggs, beef, pork, seafood, and vegetables (Pires et al., 2014). Among these, chicken meat and eggs are the types of food that cause salmonellosis most often (Jackson et al., 2013; Middleton et al., 2014). In recent times, the incidence of egg-borne salmonel C 2015 Poultry Science Association Inc. Received January 28, 2015. Accepted April 14, 2015. 1 Coressponding author: [email protected]
losis has frequently been reported in many countries. Harker et al. (2013) reported that the human salmonellosis outbreaks between 2000 and 2011 in England and Wales were predominantly caused by the consumption of contaminated eggs. Dominguez et al. (2007) reported that 48% of human salmonellosis were derived from eggs in Spain from 1990 to 2003. In addition, a majority of foodborne salmonellosis cases that occurred between 1998 and 2004 in Japan were associated with eggs (Toyofuku, 2008). The contamination of eggs with Salmonella could have occurred through the laying hens. Such a contamination could occur via horizontal transmission from the feces of the contaminated hen or the environmental dust after oviposition through the eggshell (De Reu et al., 2006), or by vertical transmission through the yolk, albumen, or eggshell membranes before oviposition (Keller et al., 1995). Although the prevalence of Salmonella in raw eggshells has been discovered to be relatively low, salmonellosis continues to be associated with eggs. This is because of the considerable human consumption of eggs, and possible re-contamination resulting from improper egg processing and handling (Hara-Kudo and Takatori, 2009).
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ABSTRACT Salmonellosis is one of the most prevalent foodborne illnesses. The outbreak of this disease is often associated with eggs. In this study, the prevalence and characteristics of Salmonella was surveyed in layer farms in Korea. In addition, the risk factors affecting the prevalence of Salmonella in these farms were also assessed. Of the 32 farms and 67 flocks examined, 19 farms (59.3%) and 34 flocks (50.7%) were observed to be positive for Salmonella contamination. Salmonella was detected in the surrounding environment such as feces (41.8%), dust (40.3%), egg shells (17.2%), as well as the internal egg contents (5.2%). The incidence of Salmonella positives were tended to increase when the flock size is larger (P = 0.021). Differences in the provinces also affected Salmonella prevalence
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There exists a high probability that eggs are contaminated with Salmonella at the layer farms, via contaminated environmental matter, such as the chicken feed, water, feces, and dust. Therefore, the layer farms must be properly managed, in order to diminish prevalence of Salmonella in the hens and eggs. Many studies have attempted to determine the prevalence of Salmonella in layer farms (Barua et al., 2012; Huneau-Sala¨ un et al., 2009; Iwabuchi et al., 2010; Poppe et al., 1991; Snow et al., 2007). However, few studies have examined the occurrence of Salmonella contaminations in Korean layer farms. In this study, we surveyed the prevalence and characteristics of Salmonella in Korean layer farms, and attempted to assess any possible risk factors affecting Salmonella prevalence.
Sample Collection According to Statistics Korea: Livestock statistics in the first quarter 2013 (Statistics Korea, 2013), about 1,200 commercial layer flocks were present and the average number of laying hens in single layer flock was 47,761. Considering our circumstances and the proportion of number of laying hens and total number of layer flocks in different studies, we investigated 32 commercial layer farms (67 flocks) in Korea between 2013 and 2014, collecting dust, feces, and raw shell eggs from each of the flocks. In addition, epidemiological information, such as the province, age, flock size, hen-day egg production, and the form of poultry house, was also obtained. The laying hens in all surveyed layer farms were vaccinated using the Nobilis SG 9R (Intervet International, Netherlands). More than one flock/farm was sampled in farms stocked with multiple flocks of different ages. All flocks tested in this study comprised of over 2,500 birds. Therefore, the dust and feces samples were collected in accordance with the standards set by the National Poultry Improvement Plan (USDA, 2012). Briefly, a sterile surgical gauze swab (12 layers of 10 × 10 cm) was moistened with 12-mL sterile doublestrength skim milk (Fluka, Germany). Fifteen different spots were swabbed per flock in order to collect 10 g each dust sample. Approximately 10 g feces were also sampled from 15 different locations; no individual birds were sampled. Sixty raw shell eggs were also collected per flock. All samples were transported to the laboratory in a cooler and stored at 5◦ C for further use.
Processing of Dust and Feces Samples Fifteen dust and feces samples each per flock were individually weighed. The samples were individually inoculated into buffered peptone water (Difco, United States) at a 1:10 ratio, and incubated for 18 to 24 h at 37◦ C.
Egg surface and contents were processed according to the National Poultry Improvement Plan standards (USDA, 2012) and the Food and Drug Administration Bacteriological Analytical Manual (FDA, 2012), respectively. Twenty eggs were swabbed and pooled for each of egg shell and contents samples (3 swabs and egg pools/flock). The entire external surface of each egg was swabbed with moistened sterile swabs. The swabs were dipped in 90 mL buffered peptone water (Difco, United States) and incubated for 18 to 24 h at 37◦ C. The eggs were then submerged in disinfectants consisting of 3 parts of 70% alcohol to one part iodine (Sigma–Aldrich, Chile) / potassium iodide (Sigma, Germany) solution for 10 s. The eggs were removed from the solution and allowed to air-dry. Each egg was flame-sterilized at the pointed end and cracked open using a sterile scalpel blade. The contents were poured into a sterile WhirlPak stomacher bag (Nasco, United States). The pooled contents of 20 eggs were mixed thoroughly using a sterile tool and gloved hands, until the yolks were mixed completely with the albumen; the gloves were changed between samples. Samples were incubated at room temperature for 96 ± 2 h. Following this, 25 mL from each sample was transferred to 225 mL tryptic soy broth (Dfico, United States) supplemented with 7.8 mg ferrous sulfate (Sigma, United Kingdom) and incubated for 24 ± 2 h at 35◦ C.
Isolation and Identification of Salmonella Pre-enriched buffered peptone water or tryptic soy broth media was transferred to the selective enrichment broth. One hundred μL mixture was transferred to 10 mL Rappaport Vassiliadis medium (Difco, United States). In addition, 1 mL mixture was mixed with 10 mL tetrathionate broth. These 2 media were incubated for 24 ± 2 h at 42◦ C and 24 ± 2 h at 35◦ C, respectively. Each selective broth was streaked onto xylose lysine desoxycholate agar (Difco, United States), bismuth sulfite agar (Difco, United States), and RAMBACH agar (Merek, Germany) using a 3-mm inoculating loop, and incubated for 24 ± 2 h at 35◦ C. The suspected Salmonella colonies observed on the agar plates were confirmed by identification of the invA gene by PCR, using a previously described method (Rahn et al., 1992). One to 3 Salmonella colonies were picked from each agar plates, and serotyped using the commercial Salmonella O and H phase 1 and phase 2 antisera (Difco, United States), as per the Kauffmann– White scheme detailed in a previous study (Grimont and Weill, 2007). If several isolates originating from the same type of samples per flock displayed identical serotypes and antimicrobial susceptibility patterns, only one of these isolates was selected randomly and included in the data analysis.
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MATERIALS AND METHODS
Processing of Shell Eggs
SALMONELLA IN LAYER FARMS IN KOREA
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were observed to be positive for Salmonella. Salmonella was most frequently detected in the feces (41.8%) samples. The contamination was detected at a lower percentage in the dust (40.3%), egg shell (17.2%), and egg content (5.2%) samples.
Risk Factors for Salmonella Prevalence
Figure 1. Prevalence of Salmonella in layer farms and flocks. Farms and flocks were designated as positives when one or more positives were detected from any of feces, dust, eggshell, or egg contents samples.
The antimicrobial susceptibility profiles of the isolates were determined using the Mueller–Hinton agar (Difco, United States) by the disk diffusion method detailed by the Clinical and Laboratory Standards Institute (2012). Seventeen antimicrobial agents (Difco, United States) were tested using the following concentrations: amoxicillin/clavulanic acid (20/10 μg), ampicillin (10 μg), aztreonam (30 μg), cefotaxime (30 μg), cefoxitin (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), cephalothin (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), gentamicin (10 μg), kanamycin (30 μg), meropenam (10 μg), nalidixic acid (30 μg) piperacillin/tazobactam (100/10 μg), tetracycline (30 μg), and trimethoprim/sulfamethoxazole (1.25/23.75 μg). All the results were interpreted according to the Clinical and Laboratory Standards Institute M02 and M07 criteria (CLSI, 2012).
Statistical Analysis The data was analyzed using the Statistical Package for Social Sciences, version 18.0 (IBM, Korea). The chi-squared test and a logistic regression analysis were performed to evaluate the association between many of the risk factors and the prevalence of Salmonella. The chi-squared test was performed for categorically independent variables, such as the province and type of poultry house. Logistic regression analysis was conducted for continuous independent variables, such as the flock size, age of laying hens, and hen-day egg production.
RESULTS Prevalence of Salmonella The prevalence of Salmonella in the layer farms and flocks is displayed in Figure 1. Of the 32 farms and 67 flocks examined, 19 farms (59.3%) and 34 flocks (50.7%)
Distribution of Salmonella Serotypes The Salmonella serotypes identified have been summarized in Table 2. The Salmonella isolates were typed into 15 serovars. The most frequently observed Salmonella serovars in the flocks were S. Bareilly (41.2%), S. Mbandaka (32.4%), S. Rissen (17.6%), S. Heidelberg (11.4%), S. V 1,13,22:i:- (11.4%), S. Agona (11.4%), S. Senftenberg (11.4%), S. Saintpaul (11.4%), and S. Infantis (11.4%).
Multi-Serovar Contamination of the Salmonella-Positive Flocks The distribution of multiple Salmonella serovars in the flocks (tested positive for bacterial infection) is displayed in Table 3. Fourteen flocks were contaminated by only one Salmonella serovar. However, 20 flocks displayed contamination by multiple serovars, with the isolation of 2 to 4 serovars.
Antimicrobial Resistance Profiles The results of the antimicrobial susceptibility analysis of the Salmonella isolates were summarized in Table 4. Ninety-three out of the 101 isolates were observed to be susceptible to the 17 antimicrobial agents tested in this study. The most common antimicrobial agents to which the isolates showed any resistance were ampicillin (4.0%), nalidixic acid (3.0%), tetracycline (1.0%), cephalothin (1.0%), and gentamicin (1.0%). The Salmonella isolates were not resistant to kanamycin, cefotaxime, cefotaxime, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, meropenam, aztreonam, piperacillin/tazobactam, trimethoprim/sulfamethoxazole, and amoxicillin/clavulanic acid. Each isolate of the Agona and Senftenberg serovars of Salmonella displayed resistance to 2 antimicrobial agents, ampicillin and tetracycline, and gentamicin and nalidixic acid, respectively. Five
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Antimicrobial Susceptibility Testing
The relationship between the risk of bacterial prevalence in the flocks and the rates of Salmonella contamination is listed in Table 1. Salmonella contamination was observed to be higher when the flock size was larger (P = 0.021). The difference in provinces was also observed to affect the prevalence of Salmonella (P < 0.001). However, the age, type of poultry house, and hen-day egg production did not cause any significant differences in the prevalence of Salmonella.
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IM ET AL. Table 1. Risk factors for Salmonella at the Korean layer farms. Risk factors
No. flocks
No. samples positive
tested
for Salmonella (%)
Feces
Dust
Egg shells
Egg contents
67
34 (50.7)
28
27
10
3
8 6 15 20 9 9
0 (0) 5 (83.3) 5 (33.3) 9 (45.0) 6 (66.7) 9 (100)
0 5 3 5 6 9
0 5 4 4 6 9
0 0 2 1 3 4
0 3 0 0 0 0
55 12
30 (54.5) 4 (33.3)
25 3
24 3
8 2
3 0
23 19 10 7 8
10 (43.4) 7 (36.8) 4 (40.0) 7 (100) 6 (75.0)
9 4 3 6 6
8 3 3 7 6
2 1 1 4 2
3 0 0 0 0
5 17 14 15 6 10
1 (25.0) 10 (58.8) 7 (50.0) 8 (53.3) 4 (66.7) 4 (40.0)
1 7 6 6 4 4
1 9 4 7 3 3
0 4 1 3 1 1
0 1 1 0 0 1
9 5 11 33 9
4 (44.4) 3 (60.0) 4 (36.3) 17 (63.6) 6 (66.7)
3 2 3 14 6
4 3 3 13 4
0 1 1 6 2
0 1 0 1 1
P value
< 0.001
0.183 0.021
0.868
0.473
1 Laying hens below 18-weeks-old. These flocks were not considered for the construction of logistic regression models assessing the relationship between hen-day egg production and the prevalence of Salmonella.
Table 2. Distribution of Salmonella serotypes observed in isolates obtained from various sources of the 34 Salmonella-positive flocks. Salmonella serotypes
Bareilly Mbandaka Rissen Heidelberg V 1,13,22:i:Agona Senftenberg Infantis Saintpaul Braenderup Thompson Kentucky Livingstone Hato Meleagridis
No. positive flocks (%)1
14 (41.2) 11 (32.4) 6 (17.6) 4 (11.4) 4 (11.4) 4 (11.4) 4 (11.4) 4 (11.4) 4 (11.4) 3 (8.8) 3 (8.8) 1 (2.9) 1 (2.9) 1 (2.9) 1 (2.9)
No. flocks positive for Salmonella contamination per sample type Dust
Feces
Egg shells
Egg contents
11 6 4 1 5 1 4 3 3 2 2 1 1 0 1
12 8 5 1 3 4 0 3 3 1 1 0 0 1 0
5 2 0 2 0 0 0 1 0 1 0 0 0 0 0
0 2 0 1 0 0 0 0 0 0 0 0 0 0 0
1 Flocks were designated as positive when one or more samples from the flocks were determined to be positive for Salmonella contamination, regardless of the sample type.
different resistance patterns were observed among the Salmonella isolates.
DISCUSSION In this study, the prevalence of Salmonella in commercial layer farms in Korea and the constituent flocks was determined to be 59.3 and 50.7%, respectively. The
prevalence was observed to be higher than that observed in recent studies conducted in other nations, such as the United Kingdom (11.7%), France (17.9%), Japan (23.6%), and Bangladesh (18.0%) (Barua et al., 2012; Iwabuchi et al., 2010; Huneau-sala¨ un et al., 2009; Snow et al., 2007). Although the use of different methods for sampling, isolation, and identification could affect the overall results, the prevalence of Salmonella in
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Total Province Jeonbook Jeonnam Kyungbook Kyungnam Kyunggi Chungbook Form of poultry house No windows With windows Flock size (no. layers) Very small (≤20,000) Small (20,001 to 40,000) Medium (40,001 to 60,000) Large (60,001 to 80,000) Very large (≥80,001) Age of laying hens (wk) ≤15 16 to 30 31 to 45 46 to 60 61 to 75 ≥76 Hen-day egg production (%) Pullet1 0–70 71–80 81–90 91–100
No. sample types affected
1695
SALMONELLA IN LAYER FARMS IN KOREA Table 3. Multi-serovar contamination in 34 Salmonella-positive flocks. Flock code1
1 2 2 4 3 2 1 1 2 1 3 2 2 4 3 2 3 3 2 2 1 1 1 1 2 2 1 1 1 1 1 4 1 3
Serovars isolated per sample type Feces
Dust
Egg shell
Egg contents
Branderup Hato, Heidelberg, Mbandaka Mbandaka, 1,13,22:i:1,13,22:i:Mbandaka Mbandaka Agona Bareilly Bareilly Bareilly Bareilly Mbandaka, Rissen, Saintpaul Mbandaka, Rissen, 1,13,22:i:Bareilly Bareilly Bareilly, Rissen, Saintpaul Bareilly, Rissen Bareilly, Saintpaul Mbandaka Bareilly Infantis Mbandaka Bareilly Agona Agona Agona Bareilly, Infantis, Thompson Infantis -
Bareilly Braenderup, Thompson Bareilly Heidelberg, 1,13,22:i:Mbandaka, 1,13,22:i:Mbandaka, 1,13,22:i:Mbandaka Agona, Meleagridis Bareilly Bareilly Bareilly Bareilly Mbandaka, Saintpaul, 1,13,22:i:Mbandaka, Rissen, 1,13,22:i:Bareilly, Mbandaka Bareilly, Rissen, Senftenberg Bareilly, Rissen, Senftenberg Bareilly, Rissen Bareilly, Saintpaul Thompson Infantis Livingstone Braenderup Kentucky Infantis, Senftenberg Infantis Rissen, Saintpaul, Senftenberg
Braenderup Heidelberg, Mbandaka Heidelberg Mbandaka Bareilly Bareilly Bareilly Bareilly Bareilly Infantis -
Heidelberg Mbandaka Mbandaka -
1 Multiple flocks obtained from the same farm were designated with identical alphabet codes. The number code denotes different flocks.
Table 4. Resistance profiles of Salmonella isolates obtained from the layer farm environment and raw shell eggs to 17 antimicrobials. Salmonella serotypes Mbandaka Agona Braenderup Senftenberg Infantis Livingstone Bareilly 1,13,22:i:Rissen Saintpaul Heidelberg Thompson Kentucky Hato Meleagridis
No. isolates tested (n = 101)
Antimicrobial resistance profiles1
No. resistant isolates (%)
18 5 4 4 7 1 28 8 9 6 5 3 1 1 1
Cephalothin Ampicillin – tetracycline Tetracycline Gentamicin – nalidixic acid Nalidixic acid Nalidixic acid -
1 (5.6) 1 (20.0) 3 (75.0) 1 (25.0) 1 (14.7) 1 (100) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
1 All strains were treated with amoxicillin/clavulanic acid, ampicillin, aztreonam, cefotaxime, cefoxitin, ceftazidime, ceftriaxone, cephalothin, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, meropenam, nalidixic acid, piperacillin/tazobactam, tetracycline, and trimethoprim/sulfamethoxazole.
Korea observed in this study suggested that the Korean layer farms examined in this study exercised poor hygiene management. The classical method for Salmonella detection involves an analysis of the feces (Barua et al., 2012; Li et al., 2007; Poppe et al., 1991) or dust (Iwabuchi et al.,
2010), or both (Huneau-sala¨ un et al., 2009; Sasaki et al., 2012). There have been some controversies regarding the better method for detection of Salmonella contamination. In this study, Salmonella was isolated in 41.8 and 40.3% of the feces and dust samples, respectively. In addition, Salmonella was recovered from only one
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A-1 B-1 C-1 D-1 D-2 D-3 E-1 E-2 F-1 G-1 G-2 G-3 G-4 H-1 H-2 H-3 H-4 H-5 H-6 H-7 I-1 J-1 J-2 K-1 L-1 M-1 N-1 O-1 O-2 P-1 Q-1 R-1 S-1 S-2
No. serovars contaminated
1696
IM ET AL.
Li et al., 2007; Poppe et al., 1991; Sasaki et al., 2012). The serovars predominantly-isolated were S. Bareilly, S. Mbandaka and S. Rissen. S. Bareilly was rarely isolated in the poultry farms in Korea (Kim et al., 2013). However, it has been recently isolated from various types of food products, such as bean sprouts, and broiler chicken. Therefore, it is often associated with incidence of human salmonellosis throughout the world (Chiou et al., 2009; Cleary et al., 2010; Kudaka et al., 2006). S. Bareilly has been specifically isolated from many poultry farms and their produce in Japan (Iwabuchi et al., 2010; Kudaka et al., 2006; Sasaki et al., 2012). The second predominant Salmonella serovar identified in this study was S. Mbandaka. This serovar has been frequently isolated from contaminated poultry products throughout the world (Clemente et al., 2014; Iwabuchi et al., 2010; Le Bouquin et al., 2010), and has also been reported in Korea (Kim et al., 2013). S. Rissen, which was also predominantly isolated in our study, was isolated from healthy pigs in Korea (Lim et al., 2009). Although S. Rissen has rarely been isolated from layer farms in other countries, it has been recovered from chicken meat in Thailand (Angkititrakul et al., 2005), suggesting that this serovar could be isolated from poultry products. Notably, S. Enteritidis and S. Gallinarum, serovars that have been frequently isolated in Korea (Kim et al., 2012; Kim et al., 2013; Kwon et al., 2010; Lee et al., 2007), were not isolated in our study. There are several conceivable reasons for this decrease. The vaccination of commercial layer chicken, utilizing the live S. Gallinarum 9R strain, was initiated in Korea since 2001, in order to control fowl typhoid caused by S. Gallinarum. As a result, the number of outbreaks of fowl typhoid in layer chicken has drastically reduced since 2002 (Kwon et al., 2010). Although we do not know exact prevalence about S. Enteritidis from commercial layer farms in Korea because of insufficiency of literature, It is obvious that the S. Enteritidis strains were isolated from layers between 1995 and 1999 (Yang et al., 2002), before vaccination of SG 9R on layer farms. S. Gallinarum and S. Enteritidis belong to the Salmonella serogroup D1, and express the same immune dominance (Ochoa-Rep´araz et al., 2004). Therefore, a reasonable cross-protection between the 2 serovars could be expected (Penha Filho et al., 2009). Feberwee et al. (2001) reported that the prevalence of S. Enteritidis in the group vaccinated with the live S. Gallinarum 9R strain was significantly lower than in the non-vaccinated group. These studies indicate that the occurrence of S. Gallinarum and S. Enteritidis in layer farms may have reduced as a result of the vaccine program in Korea. Antimicrobial susceptibility testing was performed on the 101 isolates obtained in this study. Eight of these isolates showed antimicrobial resistance to at least one antimicrobial agent. The rate of resistance of Salmonella to multiple antimicrobial agents was lower than that previously observed in the Korean broiler
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type of environmental sample of the 32% of the positive flocks. These results demonstrated that the collection of both dust and feces samples was necessary for the accurate detection of Salmonella in layer farms. The frequency of Salmonella incidence was lower in egg shells (17.2%) and egg content (5.2%), compared to the dust and feces samples. Although the rate of isolation of Salmonella from egg shells varied between different studies, the contamination of the egg content was extremely rare (Chemaly et al., 2009; Chousalkar and Roberts, 2012; Garc´ıa et al., 2011; Jones et al., 1995). It has been demonstrated that the Salmonella contaminating the eggshells could migrate to the egg content, under normal conditions of storage and moisture (De Reu et al., 2006). In addition, the eggs could be contaminated by vertical transmission (Keller et al., 1995). We investigated several risk factors that could affect the prevalence of Salmonella in commercial layer farms. In this study, the flock size appeared to affect the prevalence of Salmonella in laying hens. Several studies have demonstrated that the number of hens in each flock is an important risk factor affecting Salmonella infections (Huneau-sala¨ un et al., 2009; Mollenhorst et al., 2005; Snow et al., 2007), and our results corresponded to this theory. In this study, the difference in province was also related to the Salmonella contamination. This may be a result of other complicated factors, the most probable being the relationship between the province and flock size. As Kyunggi and Chungbook provinces are areas with a high population density, the size of the layer farm tends to be larger in these provinces, compared to other provinces for increased supply of eggs (data not shown). A previous study, conducted in Japan, showed that the prevalence of Salmonella was significantly higher in flocks reared in windowless poultry houses than in those reared in poultry houses with windows (Sasaki et al., 2012). However, this study demonstrated that the type of poultry house did not cause any significant impact on the prevalence of Salmonella. The age of the laying hens also did not significantly affect the prevalence of Salmonella in this study. Some studies have demonstrated that the prevalence of Salmonella can be influenced by the age of the laying hens, especially via the feces samples (Gole et al., 2014; Li et al., 2007). However, our study demonstrated similar fecal contamination, regardless of the age of the hens. These results could be a result of endemic contamination in the environment, occurring before the housing of chicks in layer farms in Korea. Therefore, several other risk factors, besides the age of the flocks and types of poultry house, must be investigated to determine their influence on Salmonella contamination. Fifteen different Salmonella serovars were identified in this study. Most of the isolated serovars in this study were not predominantly observed in the broiler supply chains in Korea (Kim et al., 2012; Lee et al., 2007; Yoon et al., 2014). However, these were commonly isolated in the layer farms in other countries (Iwabuchi et al., 2010;
SALMONELLA IN LAYER FARMS IN KOREA
ACKNOWLEDGMENTS This study was supported by a fund (Z-1541781-201315-02) by the Research of Animal and Plant Quarantine Agency, South Korea.
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