2093 Journal of Food Protection, Vol. 78, No. 11, 2015, Pages 2093–2098 doi:10.4315/0362-028X.JFP-15-121 Copyright Q, International Association for Food Protection
Research Note
Prevalence of Foodborne Pathogens in Freshwater Fish in Latvia MARGARITA TERENTJEVA,1* INGA EIZENBERGA,1,2 OLGA VALCIN A,2 ALEKSANDR NOVOSLAVSKIJ,3 ¯ VITA STRAZDIN A,2 AND AIVARS BERZIN Sˇ 1,2 1Institute of Food and Environmental Hygiene, Faculty of Veterinary Medicine, Latvia University of Agriculture, K. Helman a iela 8, LV-3004, Jelgava, Latvia; 2Institute of Food Safety, Animal Health and Environment, BIOR, Lejupes iela 3, R¯ıga, LV-1076, Latvia; and 3Lithuanian University of Health Sciences, Veterinary Academy, Tilˇze˙ s Street 18, 47181, Kaunas, Lithuania
MS 15-121: Received 16 March 2015/Accepted 30 May 2015
ABSTRACT The aim of this study was to detect the prevalence of Salmonella spp., Listeria monocytogenes, and Yersinia enterocolitica in freshwater fish in Latvia. In total, 235 samples, including freshly caught fish from fives lakes (n ¼ 129) and fish from retail markets (n ¼ 106), were collected from April 2014 to December 2014 in Latvia. Samples were tested according to International Organization for Standardization methods. No Salmonella spp. were found in fresh fish from lakes or in commercially available fish. In contrast, the overall prevalence of L. monocytogenes and Y. enterocolitica in freshwater fish was 13% (30 of 235) and 14% (34 of 235), respectively, and no significant difference between the prevalence of L. monocytogenes and Y. enterocolitica was observed (P . 0.05). All Y. enterocolitica isolates belonged to the nonpathogenic 1A biotype. Molecular serotyping of L. monocytogenes revealed that the most distributed serogroup was 1/2a-3a (65%), followed by 1/2c-3c (25%), 1/2b-3b (5%), and 4b, 4d, 4e (5%). The prevalence of L. monocytogenes and Y. enterocolitica in freshwater lake fish was 2% (2 of 129) and 3% (4 of 129), respectively. In contrast, the prevalence of L. monocytogenes and Y. enterocolitica in fish at retail markets was 26% (28 of 106) and 28% (30 of 106), respectively. In retail samples, 9 of 58 positive fish contained both L. monocytogenes and Y. enterocolitica. In general, differences in the prevalences of L. monocytogenes and Y. enterocolitica in retail samples were significantly higher than those in freshly caught fish (P , 0.05). The results of this study indicate that freshwater fish could be an important source of Y. enterocolitica and L. monocytogenes for consumers in Latvia.
Fish meat is an important part of the diet in Latvia, and the average consumption of fish per capita was 6.6 kg in 2013 (9). Freshwater fish are widespread in the inland waters of Latvia and comprise a significant part of the total fish and fish products available in markets (1). Along with commercial fishing, recreational fishing is very common in Latvia, and the amount of freshwater fish obtained by fishermen comprises 61% of total fish caught in the inland waters of Latvia. This reflects the specific pattern of fish consumption in Latvia; therefore, the microbiological quality of freshwater fish is crucial to ensuring the quality and safety of food products intended for human consumption (5). Various factors, such as runoff from agricultural areas, and human and animal sources may affect the microbiological safety of fish and derived products intended for human consumption. Wildlife and domestic animals may excrete bacteria of zoonotic significance and thereby contaminate water sources and fish with foodborne pathogens. This may especially affect the safety of inland waters and freshwater fish because of limited water circulation. Fish and fish products have been reported in previous studies as potential vehicles for the transmission of pathogens to consumers (11, * Author for correspondence. Tel: þ371-6-3024663; Fax: þ371-63027344; E-mail: [email protected].
12, 23, 25). The consumption of contaminated fish may lead to foodborne infections, raising public health concerns, especially regarding the spread of zoonotic foodborne pathogens, such as Salmonella spp., Listeria monocytogenes, and Yersinia enterocolitica (12, 33). Therefore, the possible contamination of fish with pathogens of public health concern, including freshly caught and retailed fish, should be considered if food safety aspects are to be properly evaluated. The microbiological assessment of freshwater fish is necessary to estimate the safety of fish intended for local and domestic consumption and offered for sale at retail markets. Consumer behavior and poor hygiene at home during the skinning and gutting of fish during meal preparation may facilitate the spread of pathogens, affecting the safety of food products and posing public health concerns in case the fish is contaminated with pathogens. Previous studies on the prevalence of foodborne pathogens covered limited geographical regions and a small number of fish species, especially regarding freshwater fish, and mostly did not address the microbiological safety of fish immediately after angling and placement on market. Bacterial contamination of fish with zoonotic pathogens in Latvia had not been studied; therefore, the present study was conducted to increase the awareness of bacterial contamination of fish among consumers, fisherman, and the Latvian
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TABLE 1. Targeted genes and nucleotide sequence of the primers applied for serogrouping of Listeria monocytogenes Gene
Target
Nucleotide sequence of the primera
Product size (bp)
lmo0737
LMO0737 1 and LMO0737 2
691
13
lmo1118
LMO1118 1 and LMO1118 2
906
13
ORF2819
ORF2819 1 and ORF2819 2
471
13
ORF2110
ORF2110 1 and ORF2110 2
597
13
prs
PRS 1 and PRS 2
370
13
prfA
LIP 1 and LIP 2a
274
10, 20
flaA
FlaA-F and FlaA-R
F: AGG GCT TCA AGG ACT TAC CC R: ACG ATT TCT GCT TGC CAT TC F: AGG GGT CTT AAA TCC TGG AA R: CGG CTT GTT CGG CAT ACT TA F: AGC AAA ATG CCA AAA CTC GT R: CAT CAC TAA AGC CTC CCA TTG F: AGT GGA CAA TTG ATT GGT GAA R: CAT CCA TCC CTT ACT TTG GAC F: GCT GAA GAG ATT GCG AAA GAA G R: CAA AGA AAC CTT GGA TTT GCG G F: GAT ACA GAA ACA TCG GTT GGC R: GTG TAA TCT TGA TGC CAT CAG G F: TTA CTA GAT CAA ACT GCT CC R: AAG AAA AGC CCC TCG TCC
538
7
a
Reference(s)
F, forward; R, reverse.
state authorities. This study covers the prevalence of the most important pathogens of public health significance in freshwater fish, with the aim of detecting the prevalence of the foodborne pathogens Salmonella spp., L. monocytogenes, and Y. enterocolitica in freshly caught freshwater fish and retailed freshwater fish in Latvia.
lysine deoxycholate and brilliant green agars were screened for colonies resembling Salmonella spp. by morphology, and suspect colonies were transferred into triple sugar iron agar (Biolife) for further confirmation. The inoculated triple sugar iron agar was incubated for 24 h at 378C and confirmed with API 20E test kit (bioM´erieux, Inc., Mancy l’Etoile, France).
MATERIALS AND METHODS
Isolation and molecular serotyping of L. monocytogenes. For isolation of L. monocytogenes, 25 g of the sample was added to 225 ml of Half-Fraser broth (Biolife) for incubation for 24 h at 308C. A 0.1-ml aliquot of suspension after primary enrichment was transferred into 10 ml of Fraser broth, and at the same time a loopful with 10 ll of suspension in Half-Fraser broth was plated out on agar Listeria according to Ottaviani and Agosti (ALOA, Biolife) and Oxford agar (Biolife). The inoculated Fraser broth was incubated for 48 h at 378C, whereas the ALOA and Oxford agars were incubated for 24 to 48 h at 378C. After incubation, a loopful with 10 ll of enriched suspension in Fraser broth was plated on ALOA and Oxford agars with subsequent incubation for 24 to 48 h at 378C. The ALOA and Oxford agars after incubation were checked for typical colonies with morphology resembling L. monocytogenes, and three suspected colonies—green-blue colonies surrounded by an opaque halo from ALOA agar and browncolored colonies with signs of aesculin hydrolysis—were selected for confirmation. These colonies were transferred on blood agar (defibrinated sheep agar, Biolife) and incubated for 24 h at 378C. After incubation, Gram staining, b-hemolysis, motility, and catalase activity were detected. This confirmation was followed by biochemical identification with the API Listeria system (bioM´erieux, Inc.). For molecular serotyping of L. monocytogenes, multiplex PCR and PCR for detection of the flaA gene encoding flagellar protein were applied (Table 1) (7, 10, 13, 20). For multiplex PCR, a 25-ll amount of amplification mix consisted of 1 ll of tested DNA; 15.6 ll of RNase-free water (QIAGEN, Hilden, Germany); 13 FastStart buffer without MgCl2; 2 mM MgCl2 (Thermo Scientific, Lithuania); 0.2 mM deoxynucleoside triphosphate (dNTP) mix (QIAGEN); 0.4 lM of each of the primers lmo0737-1, lmo0737-2, lmo1118-1, lmo1118-2, orf2110-1, orf2110-2, orf2819-1, and orf2819-2; 0.1 lM prs-1 and prs-2; 0.2 lM lip-1 and lip-2; and 1 U of recombinant Taq polymerase (Thermo Scientific). Amplification was performed under the following conditions: first denaturation for 3 min at 948C, continued by forty 30-s cycles at 948C, 40 s at 618C, and 1 min
Freshwater fish sampling and sample preparation. In total, 235 samples of freshly caught freshwater fish (n ¼ 129) and retailed freshwater fish (n ¼ 106) were collected between April and December in 2014. Altogether, 129 samples of freshly caught freshwater fish were obtained from five lakes located in different parts of Latvia and included eel (Anguilla anguilla, n ¼ 56), European perch (Perca fluvialitis, n ¼ 48), silver bream (Blicca bjoerkna, n ¼ 24), and bream (Abramis brama, n ¼ 1). Fish were transported on ice to the laboratory immediately after sampling. In addition, 106 freshwater fish from different retailers were purchased at the R¯ıga Central Market. The selected species of fish were those more frequently used for human consumption in Latvia and were caught in the inland waters of Latvia. Samples of European perch (n ¼ 11), silver bream (n ¼ 20), bream (n ¼ 16), roach (Rutilus rutilus, n ¼ 25), crucian carp (Carassius carassius, n ¼ 10), tench (Tinca tinca, n ¼ 1), carp (Cyprinus carpio, n ¼ 2), vimba (Vimba vimba, n ¼ 4), and rudd (Scardinius erythrophtalmus, n ¼ 6) were included in this study. The fish at the sampling point were stored on ice in refrigeration facilities and transported on ice to the laboratory. Testing was initiated immediately after arrival at the laboratory. For preparation of samples, a 25-g portion of pooled skin, muscle, and internal organs was used for the detection of each pathogen. Isolation of Salmonella spp. For detection of Salmonella spp., a 25-g portion of the sample was transferred into 225 ml of buffered peptone water (Biolife Italiana S.r.l., Milan, Italy) and incubated for 16 to 20 h at 378C. After incubation, 1 ml of the preenriched suspension was transferred to Rappaport-Vassiliadis soya broth (Biolife) and M¨uller-Kauffmann tetrathionate broth (Biolife) and incubated for 24 h at 41.5 and 378C, respectively. A 10-ll aliquot of enriched broth was plated out on xylose lysine deoxycholate agar (Biolife) and brilliant green agar (Biolife), with subsequent incubation for 24 h at 378C. After incubation, xylose
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TABLE 2. Prevalence of Salmonella, Listeria monocytogenes, and Yersinia enterocolitica in freshwater fish originating from lakes and retail market No. of positive samples/no. of samples (%) Salmonella Fish
Eels (Anguilla anguilla) European perch (Perca fluvialitis) Silver bream (Blicca bjoerkna) Bream (Abramis brama) Roach (Rutilus rutilus) Crucian carp (Carassius carassius) Tench (Tinca tinca) Carp (Cyprinus carpio) Rudd (Scardinius erythrophtalmus) Vimba (Vimba vimba) Total
No. of samples
56 59 44 17 25 10 1 2 6 15
L. monocytogenes
Y. enterocolitica
Lake
Market
Lake
Market
Lake
Market
0/56 (0) 0/48 (0) 0/24 (0) 0/1 (0) NA NA NA NA NA NA 0/129 (0)
NAa 0/11 (0) 0/20 (0) 0/16 (0) 0/25 (0) 0/10 (0) 0/1 (0) 0/2 (0) 0/6 (0) 0/15 (0) 0/106 (0)
0/56 (0) 2/48 (4) 0/24 (0) 0/1 (0) NA NA NA NA NA NA 2/129 (2)e
NA 4/11 (36)b 8/20 (40)c 3/16 (19)b 5/25 (20)b 4/10 (40)b 0/1 (0) 0/2 (0) 0/6 (0) 4/15 (27)d 28/106 (26)f
1/56 (2) 3/48 (6) 0/24 (0) 0/1 (0) NA NA NA NA NA NA 4/129 (3)
NA 5/11 (45) 9/20 (45) 4/16 (25) 1/25 (4) 0/10 (0) 0/1 (0) 2/2 (100) 0/6 (0) 9/15 (60) 30/106 (28)
a
NA, samples were not available. L. monocytogenes serogroups IIa (serovars 1/2a-3a) and IIc (1/2c-3c) were identified. c L. monocytogenes serogroups IIa (1/2a-3a), IIb (1/2b-3b-7), and IIc (1/2c-3c) were identified. d L. monocytogenes serogroups IIa (1/2a-3a) and IVb (4ab-4b-4d-4e) were identified. e The prevalence of both L. monocytogenes and Y. enterocolitica in retail fish was significantly higher than the prevalence in freshly caught fish (P , 0.05). f There were no differences between the prevalence of L. monocytogenes and Y. enterocolitica in retail samples of freshwater fish (P . 0.05). b
at 728C, followed by a final extension for 7 min at 728C. For the detection of the flaA gene, the applied master mix consisted of 2 ll of the tested DNA, 12.1 ll of RNase-free water (QIAGEN), 13 FastStart buffer without MgCl2, 4 mM MgCl2 (Thermo Scientific), 0.2 mM dNTP mix (QIAGEN), 0.8 lM of each of the primers flaA-F and flaA-R, and 1 U of recombinant Taq polymerase (Thermo Scientific). The cycling program consisted of first denaturation for 3 min at 948C, continued by forty 30-s cycles at 948C, 40 s at 618C, and 1 min at 728C, followed by a final extension for 7 min at 728C. The results of amplification of PCR products were read by QIAxcel Advanced capillary electrophoresis system (QIAGEN). Based on the results, L. monocytogenes serogroups were differentiated. Isolation of Y. enterocolitica. For culturing of Y. enterocolitica, a 25-g amount of sample was transferred into 225 ml of peptone-sorbitol-bile (PSB) broth (Biolife) and homogenized. After sample preparation, 1 ml of PSB broth was transferred into 9 ml of Irgasan-ticarcillin-potassium chlorate (ITC) broth (Biolife), and inoculated ITC broth was incubated for 48 h at 258C, but sample suspension in PSB was incubated at 258C for 5 days. After incubation, a loopful of 10 ll of enriched suspension from ITC and PSB was plated out on cefsulodin-Irgasan-novobiocin (CIN) agar (Biolife) and incubated for 48 h at 308C. An alkali treatment with 0.25% KOH solution was used before plating on CIN agar after incubation of PSB for 5 days. The CIN agar plates were examined for the presence of typical colonies with a red center surrounded by a transparent zone. Presumptive colonies were confirmed with API 20E kit (bioM´erieux, Inc.). All Y. enterocolitica isolates were biotypes according to Wauters et al. (36). Statistical analyses. The chi-square test was used to detect differences between the prevalence of L. monocytogenes and Y. enterocolitica in freshly caught and retailed freshwater fish in Latvia.
RESULTS Overall, the prevalence of L. monocytogenes in freshwater fish was 13% (30 of 235), whereas Y. enterocolitica was found in 14% (34 of 235) of the samples; there were no significant differences between the prevalence of L. monocytogenes and Y. enterocolitica in freshwater fish in Latvia (P . 0.05) (Table 2). Salmonella spp. were not found in freshwater fish from either lake (0%, 0 of 129) or retail market (0%, 0 of 106) samples. L. monocytogenes was isolated from European perch from lake samples and from silver bream, perch, bream, roach, crucian carp, and vimba from retail market samples. Eel, silver bream, and bream from lake samples, as well as tench, carp, and rudd obtained from retail markets, were L. monocytogenes negative. The highest prevalence of L. monocytogenes at retail markets was observed in silver bream and crucian carp, 40% (8 of 20) and 40% (4 of 10) of samples, whereas the lowest prevalence was observed in bream, 19% (3 of 16). According to molecular serotyping of L. monocytogenes, the most widespread serogroup was 1/2a-3a (65%), followed by 1/2c-3c (25%), 1/2c-3c (5%), and 4b, 4d, 4e (5%). Y. enterocolitica was found in eel and European perch from lakes and in European perch, silver bream, bream, roach, vimba, and carp obtained from retail market. All silver bream and bream from lakes, as well as crucian carp, tench, and rudd samples from retail market, were negative for Y. enterocolitica. The highest prevalence of Y. enterocolitica was identified in European perch, 38% (3 of 8), whereas the lowest prevalence was identified in silver bream, 14% (1 of 7), among the freshwater fish collected from lakes. The highest prevalence of Y. enterocolitica was found in carp, 100% (2 of 2), whereas the lowest prevalence
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TABLE 3. Prevalence of Salmonella spp., Listeria monocytogenes, and Yersinia enterocolitica in Latvian lakes No. of positive samplse/no. of samples (%) Salmonella spp.
L. monocytogenes
Y. enterocolitica
Eel (Anguilla anguilla) Silver bream (Blicca bjoerkna)
0/20 (0) 0/7 (0) 0/27 (0)
0/20 (0) 0/7 (0) 0/27 (0)
0/20 (0) 0/7 (0) 0/27 (0)
Eel European perch (Perca fluvialitis) Silver bream
0/11 0/24 0/7 0/42
(0) (0) (0) (0)
0/11 1/24 0/7 1/42
(0) (4) (0) (2)
1/11 0/24 0/7 1/42
(9) (0) (0) (2)
Eel European perch Silver bream
0/16 0/12 0/9 0/37
(0) (0) (0) (0)
0/16 1/12 0/9 1/37
(0) (8) (0) (3)
0/16 3/12 0/9 3/37
(0) (25) (0) (8)
European perch Silver bream Bream (Abramis brama)
0/12 0/1 0/1 0/14
(0) (0) (0) (0)
0/12 0/1 0/1 0/14
(0) (0) (0) (0)
0/12 0/1 0/1 0/14
(0) (0) (0) (0)
Eel
0/9 (0) 0/9 (0) 0/129 (0)
Lake
Al¯uksne
Fish
Subtotal Usma
Subtotal S¯ıvers
Subtotal K ¯ısˇ ezers
Subtotal Daugavpils Subtotal Total
was in roach, 4% (1 of 25) (Table 2), in retail market samples. All Y. enterocolitica isolates were confirmed as the nonpathogenic biotype 1A. Altogether, the prevalence of L. monocytogenes and Y. enterocolitica in freshwater fish from retail markets was 46% (49 of 106), a value that was higher compared with fish caught by fishermen, 5% (6 of 129), and the prevalence of both L. monocytogenes and Y. enterocolitica from retail markets was significantly higher than the prevalence in fish caught by fishermen (P , 0.05) (Table 2). In retail samples, 9 of 58 positive samples contained both L. monocytogenes and Y. enterocolitica. In general, the differences in the prevalence of Y. enterocolitica (28%, 30 of 106) and L. monocytogenes (26%, 28 of 106) in retail samples were not significant (P . 0.05). The prevalence of L. monocytogenes and Y. enterocolitica in freshwater fish from lakes was 2% (2 of 129) and 3% (4 of 129), respectively. All the tested samples were Salmonella spp. negative (0%, 0 of 129). L. monocytogenes was isolated from perch (4%, 2 of 48), whereas all eel, silver bream, and bream samples were negative. Y. enterocolitica was found in eel (2%, 1 of 56) and perch (6%, 3 of 48), but not in silver bream and bream (Table 3). Y. enterocolitica and L. monocytogenes were recovered from fish caught in Usma and S¯ıvers lakes, whereas all tested samples from Al¯uksne, K ¯ısˇ ezers, and Daugavpils lakes were found to be negative.
DISCUSSION Salmonella was not isolated from the freshwater fish in the present study, whereas Y. enterocolitica and L. monocytogenes were found both in freshly caught and retailed fish samples. Salmonella is mainly transmitted by wildlife and domestic animals excreting this pathogen with
0/9 (0) 0/9 (0) 2/129 (2)
0/9 (0) 0/9 (0) 4/129 (3)
feces and contaminating the environment. Freshwater sources could be affected by stream water and groundwater contaminated with Salmonella (8, 21). Poor sanitation and incorrect disposal of human and animal waste also may lead to the contamination of water environments, and fish may harbor Salmonella even after placement in clean water (2, 6). Salmonella-negative results in our study are in agreement with those of previous reports for France, Great Britain, Portugal, Czech Republic, Slovakia, and the United States for marine and freshwater fish (3, 12, 18, 29). But, the presence of Salmonella in fish was detected in several countries of Asia and Africa: India (14.25%), Iran (10.4%), Kenya (31.7%), Nigeria (11.5%), Malaysia (28.1% in catfish and 43.8% in tilapia), and Egypt (3.9%) (11, 17, 30, 32, 37). In previous reports, Salmonella was isolated both from ponds and market samples (8, 12). The prevalence varied from 0% in freshly caught fish in Iran to 41.3% in fish at wet markets in Malaysia; the reason for high prevalence of Salmonella in marketed fish in India was poor hygienic quality of material used for the prevention of overheating of products, e.g., ice, water, and sand (4, 8, 17, 31). L. monocytogenes was isolated from fish samples obtained from fishermen and from retail markets, and the overall prevalence was 13% (30 of 235). L. monocytogenes is a foodborne pathogen that is widely distributed in the environment and can cause severe illness in patients with suppressed immunity after consumption of contaminated food. This pathogen can be isolated from freshwater and from seawater in coastal areas, and the presence of L. monocytogenes may arise as a result of pollution from industrial, human, and animal sources (14). Good water management practices in indoor aquaculture could be an effective tool to avoid the contamination of the fish with L. monocytogenes (29). Because of the widespread occurrence
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of pathogens in the environment, it is necessary to control and monitor for the presence of L. monocytogenes in raw materials and final products of fish processing. The prevalence of L. monocytogenes from marine and freshwater fish in previous studies varied from 0% in the United States and Greece to 77% in the United Kingdom (12, 27, 29), and our results are comparable with the prevalences in Finland (14.6%), Denmark (8.6%), and the United States (7.8%), but they are lower than prevalences reported in France (65%), the United Kingdom (77%), and Portugal (25%) (12, 24, 26, 34). Our findings on molecular serotyping of L. monocytogenes are important since the isolated serogroups 1/2a-3a (65%) followed by 1/2c-3c (25%) and 1/2b-3b are commonly present in food and are responsible for listeriosis cases among consumers. Also, the confirmation of serogroup 4b, 4d, 4e isolates (potential serotype 4b) in fish, responsible for the majority of human outbreaks, indicates that freshwater fish may serve as a vehicle of L. monocytogenes to consumers (35). Y. enterocolitica had been isolated from freshwater fish in Latvia, and there were no significant differences between the prevalence of L. monocytogenes and Y. enterocolitica in fish (P . 0.05). The prevalence of Y. enterocolitica was 14% (34 of 235), a relatively high value compared with those of other studies (12, 19, 22). Y. enterocolitica is a psychrotropic microorganism that is capable of growth at low temperatures and can cause foodborne infection characterized by gastrointestinal disorders. Y. enterocolitica could reach and contaminate inland waters from animal, agricultural, and human sources, with subsequent contamination of fish. The principal reservoirs of Y. enterocolitica are suggested to be wildlife and farm animals; therefore, the prevalence of Y. enterocolitica in fish has been not studied extensively (15). Our results indicate that freshwater fish could be a significant source of Y. enterocolitica and are in line with previously reported data showing the prevalence of Y. enterocolitica ranging from 0 to 23% (12, 19). However, in the Hudson et al. (19) and Davies et al. (12) studies, the prevalence of Y. enterocolitica in fish was significantly lower than the prevalence of L. monocytogenes. The present study findings confirm that freshwater fish could be an important source of both microorganisms, and our results show that nine retail market samples contained both Y. enterocolitica and L. monocytogenes. The prevalence of L. monocytogenes and Y. enterocolitica was higher in fish from retail markets than in freshly caught fish (P , 0.05). The contamination routes of freshwater fish during recreational fishing are limited, and the main source of introduction of pathogens is the contaminated aquatic environment. Regardless, the microbiological safety of fish from retail sources could be affected during primary processing and marketing by various factors, such as personnel, fish storage conditions, hygiene, and quality of ice, and pathogens could be transferred to fish as a result of cross-contamination. The high prevalence of L. monocytogenes due to poor sanitation was reported by Y¨ucel and Balci (38) in Turkey. Our results confirm the findings of a previous study that freshly caught fish from a clean environment were mostly free from pathogenic microorgan-
isms (29). The findings on the prevalence of pathogens during the production of cold-smoked fish in Denmark revealed that the level of contamination with L. monocytogenes increased with human activity (4, 16, 23, 25). In markets, where the samples were collected, hazard analysis and critical control point procedures are well established; however, our on-site observations showed that retailers are occasionally mixing fish from different lots and that the fish ice counter is usually overwhelmed with different fish species. Such fish layout is believed by retailers to be visually more attractive for consumers, enhancing the sales of the products. This practice could facilitate the spread of the microorganisms between fish of different origins as the result of cross-contamination. The prevalence of L. monocytogenes and Y. enterocolitica in freshly caught fish in Latvia was significantly lower than that in fish from retail markets; however, 6 (5%) of 129 samples obtained from fishermen were positive. This represents public health implications for consumption of fish caught via recreational fishing. As angling is very common in Latvia, information on the possible hazards to fishermen and consumers was published by the Food and Veterinary Service authority in Latvia (28). However, the material mostly addresses chemical hazards and nutritive values of fish meat and contains no information regarding the microbiological contamination of fish, especially with pathogens of public health concern. Our results showed that freshly caught fish could be a significant vector for the transmission of pathogenic microorganisms, even in inland waters of good quality without apparent source of contamination, and this problem is underestimated in Latvia. In conclusion, this study revealed that the prevalence of L. monocytogenes and Y. enterocolitica significantly increased in freshwater fish from retail markets in comparison with freshly caught fish. This is the first study on the prevalence of bacterial foodborne pathogens in fish from Latvia, confirming that freshly caught and retailed freshwater fish can be an important source of L. monocytogenes and Y. enterocolitica.
ACKNOWLEDGMENT This study was conducted within the European Social Fund project 2013/0016/1DP/1.1.1.2.0/13/APIA/VIAA/055, Iekˇse¯ jo u¯ denu zivju resursu k¯ımisk¯a un biolog isk¯a pies¯arnojuma p¯etniec¯ıbas grupas izveide.
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22. Lyhs, U., M. Hatakka, N. Maki- Pet¨ays, E. Hyyti¨a, and H. Korkeala. 1998. Microbiological quality of Finnish vacuum-packaged fishery products at retail level. Arch. Lebensmittelhyg. 49:146–150. 23. Markkula, A., T. Autio, J. Lund´en, and H. Korkeala. 2005. Raw and processed fish show identical Listeria monocytogenes genotypes with pulsed-field gel electrophoresis. J. Food Prot. 68:1228–1231. 24. Miettinen, H., and G. Wirtanen. 2005. Prevalence and location of Listeria monocytogenes in farmed rainbow trout. Int. J. Food Microbiol. 104:135–143. 25. Miettinen, H., and G. Wirtanen. 2006. Ecology of Listeria spp. in a fish farm and molecular typing of Listeria monocytogenes from fish farming and processing companies. Int. J. Food Microbiol. 112:138–146. 26. Norton, D. M., M. A. McCamey, K. Gall, J. M. Scarlet, K. J. Boor, and M. Wiedman. 2001. Molecular studies on the ecology of Listeria monocytogenes in the smoking fish processing industry. Appl. Environ. Microbiol. 67:198–205. 27. Papadopoulos, T., A. Abrahim, D. Sergelidis, I. Kirkoudis, and K. Bitchava. 2010. Prevalence of Listeria spp. in freshwater fish (Oncorhynchus mykiss and Carassius gibelio) and the environment of fish markets in northern Greece. J. Hell. Vet. Med. Soc. 61:15–22. 28. P¯artikas un veterin¯arais dienests [Food and Veterinary Service]. 2014. P¯artikas un veterin¯ar¯a dienesta ieteikumi, lietojot uztur¯a zivis [Recommendation of the Food and Veterinary Service for consumption of fish]. Available at: http://www.pvd.gov.lv/lat/augj_izvlne/ iedzvotjiem_un_uzmjiem/informativie_materiali/iedzvotjiem_un_ patrtjiem. Accessed 1 December 2014. 29. Pullela, S., C. F. Fernandes, G. J. Flick, G. S. Libey, S. A. Smith, and C. W. Coale. 1998. Indicative and pathogenic microbiological quality of aquacultured finfish grown in different production systems. J. Food Prot. 61:205–210. 30. Raufu, A. I., F. A. Lawan, H. S. Bello, A. S. Musa, J. A. Ameh, and A. G. Ambali. 2014. Occurrence and antimicrobial susceptibility profiles of Salmonella serovars from fish in Maiduguri, sub-Sahara, Nigeria. Egypt. J. Aquat. Res. 40:59–63. 31. Razavilar, V., M. R. Khani, and A. A. Motallebi. 2012. Bacteriological study of cultured silver carp (Hypophthalmichthys molitrix) in Gilan province, Iran. Iran. J. Fish. Sci. 12:689–701. 32. Shabarinath, S., H. S. Kumar, R. Khushiramani, I. Karunasagar, and I. Karunasagar. 2007. Detection and characterization of Salmonella associated with tropical seafood. Int. J. Food Microbiol. 114:227– 233. 33. Viau, E. J., K. D. Goodwin, K. M. Yamahara, B. A. Layton, L. M. Sassoubre, S. L. Burns, H.-I. Tong, S. H. C. Wong, Y. Lu, and A. B. Boehm. 2011. Bacterial pathogens in Hawaiian coastal streams— associations with fecal indicators, land cover, and water quality. Water Res. 4511:3279–3290. 34. Vogel, B. F., H. H. Huss, B. Ojeniji, P. Ahrens, and L. Gram. 2001. Elucidation of Listeria monocytogenes contamination routes in coldsmoked salmon processing plants detected by DNA-based typing methods. Appl. Environ. Microbiol. 67:2586–2595. 35. Ward, T. J., T. Usgaard, and P. Evans. 2010. A targeted multilocus genotyping assay for lineage, serogroup, and epidemic clone typing of Listeria monocytogenes. Appl. Environ. Microbiol. 76:6680– 6684. 36. Wauters, G., K. Kandolo, and M. Janssens. 1987. Revised biogrouping scheme of Yersinia enterocolitica. Contrib. Microbiol. Immunol. 9:14–21. 37. Youssef, H., A. K. El-Timawy, and S. Ahmed. 1992. Role of aerobic intestinal pathogens of fresh water fish in transmission of human diseases. Food Control 4:34–40. 38. Y¨ucel, N., and S . Balci. 2010. Prevalence of Listeria, Aeromonas, and Vibrio species in fish used for human consumption in Turkey. J. Food Prot. 73:380–384.
Research Note
Prevalence of Foodborne Pathogens in Freshwater Fish in Latvia MARGARITA TERENTJEVA,1* INGA EIZENBERGA,1,2 OLGA VALCIN A,2 ALEKSANDR NOVOSLAVSKIJ,3 ¯ VITA STRAZDIN A,2 AND AIVARS BERZIN Sˇ 1,2 1Institute of Food and Environmental Hygiene, Faculty of Veterinary Medicine, Latvia University of Agriculture, K. Helman a iela 8, LV-3004, Jelgava, Latvia; 2Institute of Food Safety, Animal Health and Environment, BIOR, Lejupes iela 3, R¯ıga, LV-1076, Latvia; and 3Lithuanian University of Health Sciences, Veterinary Academy, Tilˇze˙ s Street 18, 47181, Kaunas, Lithuania
MS 15-121: Received 16 March 2015/Accepted 30 May 2015
ABSTRACT The aim of this study was to detect the prevalence of Salmonella spp., Listeria monocytogenes, and Yersinia enterocolitica in freshwater fish in Latvia. In total, 235 samples, including freshly caught fish from fives lakes (n ¼ 129) and fish from retail markets (n ¼ 106), were collected from April 2014 to December 2014 in Latvia. Samples were tested according to International Organization for Standardization methods. No Salmonella spp. were found in fresh fish from lakes or in commercially available fish. In contrast, the overall prevalence of L. monocytogenes and Y. enterocolitica in freshwater fish was 13% (30 of 235) and 14% (34 of 235), respectively, and no significant difference between the prevalence of L. monocytogenes and Y. enterocolitica was observed (P . 0.05). All Y. enterocolitica isolates belonged to the nonpathogenic 1A biotype. Molecular serotyping of L. monocytogenes revealed that the most distributed serogroup was 1/2a-3a (65%), followed by 1/2c-3c (25%), 1/2b-3b (5%), and 4b, 4d, 4e (5%). The prevalence of L. monocytogenes and Y. enterocolitica in freshwater lake fish was 2% (2 of 129) and 3% (4 of 129), respectively. In contrast, the prevalence of L. monocytogenes and Y. enterocolitica in fish at retail markets was 26% (28 of 106) and 28% (30 of 106), respectively. In retail samples, 9 of 58 positive fish contained both L. monocytogenes and Y. enterocolitica. In general, differences in the prevalences of L. monocytogenes and Y. enterocolitica in retail samples were significantly higher than those in freshly caught fish (P , 0.05). The results of this study indicate that freshwater fish could be an important source of Y. enterocolitica and L. monocytogenes for consumers in Latvia.
Fish meat is an important part of the diet in Latvia, and the average consumption of fish per capita was 6.6 kg in 2013 (9). Freshwater fish are widespread in the inland waters of Latvia and comprise a significant part of the total fish and fish products available in markets (1). Along with commercial fishing, recreational fishing is very common in Latvia, and the amount of freshwater fish obtained by fishermen comprises 61% of total fish caught in the inland waters of Latvia. This reflects the specific pattern of fish consumption in Latvia; therefore, the microbiological quality of freshwater fish is crucial to ensuring the quality and safety of food products intended for human consumption (5). Various factors, such as runoff from agricultural areas, and human and animal sources may affect the microbiological safety of fish and derived products intended for human consumption. Wildlife and domestic animals may excrete bacteria of zoonotic significance and thereby contaminate water sources and fish with foodborne pathogens. This may especially affect the safety of inland waters and freshwater fish because of limited water circulation. Fish and fish products have been reported in previous studies as potential vehicles for the transmission of pathogens to consumers (11, * Author for correspondence. Tel: þ371-6-3024663; Fax: þ371-63027344; E-mail: [email protected].
12, 23, 25). The consumption of contaminated fish may lead to foodborne infections, raising public health concerns, especially regarding the spread of zoonotic foodborne pathogens, such as Salmonella spp., Listeria monocytogenes, and Yersinia enterocolitica (12, 33). Therefore, the possible contamination of fish with pathogens of public health concern, including freshly caught and retailed fish, should be considered if food safety aspects are to be properly evaluated. The microbiological assessment of freshwater fish is necessary to estimate the safety of fish intended for local and domestic consumption and offered for sale at retail markets. Consumer behavior and poor hygiene at home during the skinning and gutting of fish during meal preparation may facilitate the spread of pathogens, affecting the safety of food products and posing public health concerns in case the fish is contaminated with pathogens. Previous studies on the prevalence of foodborne pathogens covered limited geographical regions and a small number of fish species, especially regarding freshwater fish, and mostly did not address the microbiological safety of fish immediately after angling and placement on market. Bacterial contamination of fish with zoonotic pathogens in Latvia had not been studied; therefore, the present study was conducted to increase the awareness of bacterial contamination of fish among consumers, fisherman, and the Latvian
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TABLE 1. Targeted genes and nucleotide sequence of the primers applied for serogrouping of Listeria monocytogenes Gene
Target
Nucleotide sequence of the primera
Product size (bp)
lmo0737
LMO0737 1 and LMO0737 2
691
13
lmo1118
LMO1118 1 and LMO1118 2
906
13
ORF2819
ORF2819 1 and ORF2819 2
471
13
ORF2110
ORF2110 1 and ORF2110 2
597
13
prs
PRS 1 and PRS 2
370
13
prfA
LIP 1 and LIP 2a
274
10, 20
flaA
FlaA-F and FlaA-R
F: AGG GCT TCA AGG ACT TAC CC R: ACG ATT TCT GCT TGC CAT TC F: AGG GGT CTT AAA TCC TGG AA R: CGG CTT GTT CGG CAT ACT TA F: AGC AAA ATG CCA AAA CTC GT R: CAT CAC TAA AGC CTC CCA TTG F: AGT GGA CAA TTG ATT GGT GAA R: CAT CCA TCC CTT ACT TTG GAC F: GCT GAA GAG ATT GCG AAA GAA G R: CAA AGA AAC CTT GGA TTT GCG G F: GAT ACA GAA ACA TCG GTT GGC R: GTG TAA TCT TGA TGC CAT CAG G F: TTA CTA GAT CAA ACT GCT CC R: AAG AAA AGC CCC TCG TCC
538
7
a
Reference(s)
F, forward; R, reverse.
state authorities. This study covers the prevalence of the most important pathogens of public health significance in freshwater fish, with the aim of detecting the prevalence of the foodborne pathogens Salmonella spp., L. monocytogenes, and Y. enterocolitica in freshly caught freshwater fish and retailed freshwater fish in Latvia.
lysine deoxycholate and brilliant green agars were screened for colonies resembling Salmonella spp. by morphology, and suspect colonies were transferred into triple sugar iron agar (Biolife) for further confirmation. The inoculated triple sugar iron agar was incubated for 24 h at 378C and confirmed with API 20E test kit (bioM´erieux, Inc., Mancy l’Etoile, France).
MATERIALS AND METHODS
Isolation and molecular serotyping of L. monocytogenes. For isolation of L. monocytogenes, 25 g of the sample was added to 225 ml of Half-Fraser broth (Biolife) for incubation for 24 h at 308C. A 0.1-ml aliquot of suspension after primary enrichment was transferred into 10 ml of Fraser broth, and at the same time a loopful with 10 ll of suspension in Half-Fraser broth was plated out on agar Listeria according to Ottaviani and Agosti (ALOA, Biolife) and Oxford agar (Biolife). The inoculated Fraser broth was incubated for 48 h at 378C, whereas the ALOA and Oxford agars were incubated for 24 to 48 h at 378C. After incubation, a loopful with 10 ll of enriched suspension in Fraser broth was plated on ALOA and Oxford agars with subsequent incubation for 24 to 48 h at 378C. The ALOA and Oxford agars after incubation were checked for typical colonies with morphology resembling L. monocytogenes, and three suspected colonies—green-blue colonies surrounded by an opaque halo from ALOA agar and browncolored colonies with signs of aesculin hydrolysis—were selected for confirmation. These colonies were transferred on blood agar (defibrinated sheep agar, Biolife) and incubated for 24 h at 378C. After incubation, Gram staining, b-hemolysis, motility, and catalase activity were detected. This confirmation was followed by biochemical identification with the API Listeria system (bioM´erieux, Inc.). For molecular serotyping of L. monocytogenes, multiplex PCR and PCR for detection of the flaA gene encoding flagellar protein were applied (Table 1) (7, 10, 13, 20). For multiplex PCR, a 25-ll amount of amplification mix consisted of 1 ll of tested DNA; 15.6 ll of RNase-free water (QIAGEN, Hilden, Germany); 13 FastStart buffer without MgCl2; 2 mM MgCl2 (Thermo Scientific, Lithuania); 0.2 mM deoxynucleoside triphosphate (dNTP) mix (QIAGEN); 0.4 lM of each of the primers lmo0737-1, lmo0737-2, lmo1118-1, lmo1118-2, orf2110-1, orf2110-2, orf2819-1, and orf2819-2; 0.1 lM prs-1 and prs-2; 0.2 lM lip-1 and lip-2; and 1 U of recombinant Taq polymerase (Thermo Scientific). Amplification was performed under the following conditions: first denaturation for 3 min at 948C, continued by forty 30-s cycles at 948C, 40 s at 618C, and 1 min
Freshwater fish sampling and sample preparation. In total, 235 samples of freshly caught freshwater fish (n ¼ 129) and retailed freshwater fish (n ¼ 106) were collected between April and December in 2014. Altogether, 129 samples of freshly caught freshwater fish were obtained from five lakes located in different parts of Latvia and included eel (Anguilla anguilla, n ¼ 56), European perch (Perca fluvialitis, n ¼ 48), silver bream (Blicca bjoerkna, n ¼ 24), and bream (Abramis brama, n ¼ 1). Fish were transported on ice to the laboratory immediately after sampling. In addition, 106 freshwater fish from different retailers were purchased at the R¯ıga Central Market. The selected species of fish were those more frequently used for human consumption in Latvia and were caught in the inland waters of Latvia. Samples of European perch (n ¼ 11), silver bream (n ¼ 20), bream (n ¼ 16), roach (Rutilus rutilus, n ¼ 25), crucian carp (Carassius carassius, n ¼ 10), tench (Tinca tinca, n ¼ 1), carp (Cyprinus carpio, n ¼ 2), vimba (Vimba vimba, n ¼ 4), and rudd (Scardinius erythrophtalmus, n ¼ 6) were included in this study. The fish at the sampling point were stored on ice in refrigeration facilities and transported on ice to the laboratory. Testing was initiated immediately after arrival at the laboratory. For preparation of samples, a 25-g portion of pooled skin, muscle, and internal organs was used for the detection of each pathogen. Isolation of Salmonella spp. For detection of Salmonella spp., a 25-g portion of the sample was transferred into 225 ml of buffered peptone water (Biolife Italiana S.r.l., Milan, Italy) and incubated for 16 to 20 h at 378C. After incubation, 1 ml of the preenriched suspension was transferred to Rappaport-Vassiliadis soya broth (Biolife) and M¨uller-Kauffmann tetrathionate broth (Biolife) and incubated for 24 h at 41.5 and 378C, respectively. A 10-ll aliquot of enriched broth was plated out on xylose lysine deoxycholate agar (Biolife) and brilliant green agar (Biolife), with subsequent incubation for 24 h at 378C. After incubation, xylose
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TABLE 2. Prevalence of Salmonella, Listeria monocytogenes, and Yersinia enterocolitica in freshwater fish originating from lakes and retail market No. of positive samples/no. of samples (%) Salmonella Fish
Eels (Anguilla anguilla) European perch (Perca fluvialitis) Silver bream (Blicca bjoerkna) Bream (Abramis brama) Roach (Rutilus rutilus) Crucian carp (Carassius carassius) Tench (Tinca tinca) Carp (Cyprinus carpio) Rudd (Scardinius erythrophtalmus) Vimba (Vimba vimba) Total
No. of samples
56 59 44 17 25 10 1 2 6 15
L. monocytogenes
Y. enterocolitica
Lake
Market
Lake
Market
Lake
Market
0/56 (0) 0/48 (0) 0/24 (0) 0/1 (0) NA NA NA NA NA NA 0/129 (0)
NAa 0/11 (0) 0/20 (0) 0/16 (0) 0/25 (0) 0/10 (0) 0/1 (0) 0/2 (0) 0/6 (0) 0/15 (0) 0/106 (0)
0/56 (0) 2/48 (4) 0/24 (0) 0/1 (0) NA NA NA NA NA NA 2/129 (2)e
NA 4/11 (36)b 8/20 (40)c 3/16 (19)b 5/25 (20)b 4/10 (40)b 0/1 (0) 0/2 (0) 0/6 (0) 4/15 (27)d 28/106 (26)f
1/56 (2) 3/48 (6) 0/24 (0) 0/1 (0) NA NA NA NA NA NA 4/129 (3)
NA 5/11 (45) 9/20 (45) 4/16 (25) 1/25 (4) 0/10 (0) 0/1 (0) 2/2 (100) 0/6 (0) 9/15 (60) 30/106 (28)
a
NA, samples were not available. L. monocytogenes serogroups IIa (serovars 1/2a-3a) and IIc (1/2c-3c) were identified. c L. monocytogenes serogroups IIa (1/2a-3a), IIb (1/2b-3b-7), and IIc (1/2c-3c) were identified. d L. monocytogenes serogroups IIa (1/2a-3a) and IVb (4ab-4b-4d-4e) were identified. e The prevalence of both L. monocytogenes and Y. enterocolitica in retail fish was significantly higher than the prevalence in freshly caught fish (P , 0.05). f There were no differences between the prevalence of L. monocytogenes and Y. enterocolitica in retail samples of freshwater fish (P . 0.05). b
at 728C, followed by a final extension for 7 min at 728C. For the detection of the flaA gene, the applied master mix consisted of 2 ll of the tested DNA, 12.1 ll of RNase-free water (QIAGEN), 13 FastStart buffer without MgCl2, 4 mM MgCl2 (Thermo Scientific), 0.2 mM dNTP mix (QIAGEN), 0.8 lM of each of the primers flaA-F and flaA-R, and 1 U of recombinant Taq polymerase (Thermo Scientific). The cycling program consisted of first denaturation for 3 min at 948C, continued by forty 30-s cycles at 948C, 40 s at 618C, and 1 min at 728C, followed by a final extension for 7 min at 728C. The results of amplification of PCR products were read by QIAxcel Advanced capillary electrophoresis system (QIAGEN). Based on the results, L. monocytogenes serogroups were differentiated. Isolation of Y. enterocolitica. For culturing of Y. enterocolitica, a 25-g amount of sample was transferred into 225 ml of peptone-sorbitol-bile (PSB) broth (Biolife) and homogenized. After sample preparation, 1 ml of PSB broth was transferred into 9 ml of Irgasan-ticarcillin-potassium chlorate (ITC) broth (Biolife), and inoculated ITC broth was incubated for 48 h at 258C, but sample suspension in PSB was incubated at 258C for 5 days. After incubation, a loopful of 10 ll of enriched suspension from ITC and PSB was plated out on cefsulodin-Irgasan-novobiocin (CIN) agar (Biolife) and incubated for 48 h at 308C. An alkali treatment with 0.25% KOH solution was used before plating on CIN agar after incubation of PSB for 5 days. The CIN agar plates were examined for the presence of typical colonies with a red center surrounded by a transparent zone. Presumptive colonies were confirmed with API 20E kit (bioM´erieux, Inc.). All Y. enterocolitica isolates were biotypes according to Wauters et al. (36). Statistical analyses. The chi-square test was used to detect differences between the prevalence of L. monocytogenes and Y. enterocolitica in freshly caught and retailed freshwater fish in Latvia.
RESULTS Overall, the prevalence of L. monocytogenes in freshwater fish was 13% (30 of 235), whereas Y. enterocolitica was found in 14% (34 of 235) of the samples; there were no significant differences between the prevalence of L. monocytogenes and Y. enterocolitica in freshwater fish in Latvia (P . 0.05) (Table 2). Salmonella spp. were not found in freshwater fish from either lake (0%, 0 of 129) or retail market (0%, 0 of 106) samples. L. monocytogenes was isolated from European perch from lake samples and from silver bream, perch, bream, roach, crucian carp, and vimba from retail market samples. Eel, silver bream, and bream from lake samples, as well as tench, carp, and rudd obtained from retail markets, were L. monocytogenes negative. The highest prevalence of L. monocytogenes at retail markets was observed in silver bream and crucian carp, 40% (8 of 20) and 40% (4 of 10) of samples, whereas the lowest prevalence was observed in bream, 19% (3 of 16). According to molecular serotyping of L. monocytogenes, the most widespread serogroup was 1/2a-3a (65%), followed by 1/2c-3c (25%), 1/2c-3c (5%), and 4b, 4d, 4e (5%). Y. enterocolitica was found in eel and European perch from lakes and in European perch, silver bream, bream, roach, vimba, and carp obtained from retail market. All silver bream and bream from lakes, as well as crucian carp, tench, and rudd samples from retail market, were negative for Y. enterocolitica. The highest prevalence of Y. enterocolitica was identified in European perch, 38% (3 of 8), whereas the lowest prevalence was identified in silver bream, 14% (1 of 7), among the freshwater fish collected from lakes. The highest prevalence of Y. enterocolitica was found in carp, 100% (2 of 2), whereas the lowest prevalence
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TABLE 3. Prevalence of Salmonella spp., Listeria monocytogenes, and Yersinia enterocolitica in Latvian lakes No. of positive samplse/no. of samples (%) Salmonella spp.
L. monocytogenes
Y. enterocolitica
Eel (Anguilla anguilla) Silver bream (Blicca bjoerkna)
0/20 (0) 0/7 (0) 0/27 (0)
0/20 (0) 0/7 (0) 0/27 (0)
0/20 (0) 0/7 (0) 0/27 (0)
Eel European perch (Perca fluvialitis) Silver bream
0/11 0/24 0/7 0/42
(0) (0) (0) (0)
0/11 1/24 0/7 1/42
(0) (4) (0) (2)
1/11 0/24 0/7 1/42
(9) (0) (0) (2)
Eel European perch Silver bream
0/16 0/12 0/9 0/37
(0) (0) (0) (0)
0/16 1/12 0/9 1/37
(0) (8) (0) (3)
0/16 3/12 0/9 3/37
(0) (25) (0) (8)
European perch Silver bream Bream (Abramis brama)
0/12 0/1 0/1 0/14
(0) (0) (0) (0)
0/12 0/1 0/1 0/14
(0) (0) (0) (0)
0/12 0/1 0/1 0/14
(0) (0) (0) (0)
Eel
0/9 (0) 0/9 (0) 0/129 (0)
Lake
Al¯uksne
Fish
Subtotal Usma
Subtotal S¯ıvers
Subtotal K ¯ısˇ ezers
Subtotal Daugavpils Subtotal Total
was in roach, 4% (1 of 25) (Table 2), in retail market samples. All Y. enterocolitica isolates were confirmed as the nonpathogenic biotype 1A. Altogether, the prevalence of L. monocytogenes and Y. enterocolitica in freshwater fish from retail markets was 46% (49 of 106), a value that was higher compared with fish caught by fishermen, 5% (6 of 129), and the prevalence of both L. monocytogenes and Y. enterocolitica from retail markets was significantly higher than the prevalence in fish caught by fishermen (P , 0.05) (Table 2). In retail samples, 9 of 58 positive samples contained both L. monocytogenes and Y. enterocolitica. In general, the differences in the prevalence of Y. enterocolitica (28%, 30 of 106) and L. monocytogenes (26%, 28 of 106) in retail samples were not significant (P . 0.05). The prevalence of L. monocytogenes and Y. enterocolitica in freshwater fish from lakes was 2% (2 of 129) and 3% (4 of 129), respectively. All the tested samples were Salmonella spp. negative (0%, 0 of 129). L. monocytogenes was isolated from perch (4%, 2 of 48), whereas all eel, silver bream, and bream samples were negative. Y. enterocolitica was found in eel (2%, 1 of 56) and perch (6%, 3 of 48), but not in silver bream and bream (Table 3). Y. enterocolitica and L. monocytogenes were recovered from fish caught in Usma and S¯ıvers lakes, whereas all tested samples from Al¯uksne, K ¯ısˇ ezers, and Daugavpils lakes were found to be negative.
DISCUSSION Salmonella was not isolated from the freshwater fish in the present study, whereas Y. enterocolitica and L. monocytogenes were found both in freshly caught and retailed fish samples. Salmonella is mainly transmitted by wildlife and domestic animals excreting this pathogen with
0/9 (0) 0/9 (0) 2/129 (2)
0/9 (0) 0/9 (0) 4/129 (3)
feces and contaminating the environment. Freshwater sources could be affected by stream water and groundwater contaminated with Salmonella (8, 21). Poor sanitation and incorrect disposal of human and animal waste also may lead to the contamination of water environments, and fish may harbor Salmonella even after placement in clean water (2, 6). Salmonella-negative results in our study are in agreement with those of previous reports for France, Great Britain, Portugal, Czech Republic, Slovakia, and the United States for marine and freshwater fish (3, 12, 18, 29). But, the presence of Salmonella in fish was detected in several countries of Asia and Africa: India (14.25%), Iran (10.4%), Kenya (31.7%), Nigeria (11.5%), Malaysia (28.1% in catfish and 43.8% in tilapia), and Egypt (3.9%) (11, 17, 30, 32, 37). In previous reports, Salmonella was isolated both from ponds and market samples (8, 12). The prevalence varied from 0% in freshly caught fish in Iran to 41.3% in fish at wet markets in Malaysia; the reason for high prevalence of Salmonella in marketed fish in India was poor hygienic quality of material used for the prevention of overheating of products, e.g., ice, water, and sand (4, 8, 17, 31). L. monocytogenes was isolated from fish samples obtained from fishermen and from retail markets, and the overall prevalence was 13% (30 of 235). L. monocytogenes is a foodborne pathogen that is widely distributed in the environment and can cause severe illness in patients with suppressed immunity after consumption of contaminated food. This pathogen can be isolated from freshwater and from seawater in coastal areas, and the presence of L. monocytogenes may arise as a result of pollution from industrial, human, and animal sources (14). Good water management practices in indoor aquaculture could be an effective tool to avoid the contamination of the fish with L. monocytogenes (29). Because of the widespread occurrence
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of pathogens in the environment, it is necessary to control and monitor for the presence of L. monocytogenes in raw materials and final products of fish processing. The prevalence of L. monocytogenes from marine and freshwater fish in previous studies varied from 0% in the United States and Greece to 77% in the United Kingdom (12, 27, 29), and our results are comparable with the prevalences in Finland (14.6%), Denmark (8.6%), and the United States (7.8%), but they are lower than prevalences reported in France (65%), the United Kingdom (77%), and Portugal (25%) (12, 24, 26, 34). Our findings on molecular serotyping of L. monocytogenes are important since the isolated serogroups 1/2a-3a (65%) followed by 1/2c-3c (25%) and 1/2b-3b are commonly present in food and are responsible for listeriosis cases among consumers. Also, the confirmation of serogroup 4b, 4d, 4e isolates (potential serotype 4b) in fish, responsible for the majority of human outbreaks, indicates that freshwater fish may serve as a vehicle of L. monocytogenes to consumers (35). Y. enterocolitica had been isolated from freshwater fish in Latvia, and there were no significant differences between the prevalence of L. monocytogenes and Y. enterocolitica in fish (P . 0.05). The prevalence of Y. enterocolitica was 14% (34 of 235), a relatively high value compared with those of other studies (12, 19, 22). Y. enterocolitica is a psychrotropic microorganism that is capable of growth at low temperatures and can cause foodborne infection characterized by gastrointestinal disorders. Y. enterocolitica could reach and contaminate inland waters from animal, agricultural, and human sources, with subsequent contamination of fish. The principal reservoirs of Y. enterocolitica are suggested to be wildlife and farm animals; therefore, the prevalence of Y. enterocolitica in fish has been not studied extensively (15). Our results indicate that freshwater fish could be a significant source of Y. enterocolitica and are in line with previously reported data showing the prevalence of Y. enterocolitica ranging from 0 to 23% (12, 19). However, in the Hudson et al. (19) and Davies et al. (12) studies, the prevalence of Y. enterocolitica in fish was significantly lower than the prevalence of L. monocytogenes. The present study findings confirm that freshwater fish could be an important source of both microorganisms, and our results show that nine retail market samples contained both Y. enterocolitica and L. monocytogenes. The prevalence of L. monocytogenes and Y. enterocolitica was higher in fish from retail markets than in freshly caught fish (P , 0.05). The contamination routes of freshwater fish during recreational fishing are limited, and the main source of introduction of pathogens is the contaminated aquatic environment. Regardless, the microbiological safety of fish from retail sources could be affected during primary processing and marketing by various factors, such as personnel, fish storage conditions, hygiene, and quality of ice, and pathogens could be transferred to fish as a result of cross-contamination. The high prevalence of L. monocytogenes due to poor sanitation was reported by Y¨ucel and Balci (38) in Turkey. Our results confirm the findings of a previous study that freshly caught fish from a clean environment were mostly free from pathogenic microorgan-
isms (29). The findings on the prevalence of pathogens during the production of cold-smoked fish in Denmark revealed that the level of contamination with L. monocytogenes increased with human activity (4, 16, 23, 25). In markets, where the samples were collected, hazard analysis and critical control point procedures are well established; however, our on-site observations showed that retailers are occasionally mixing fish from different lots and that the fish ice counter is usually overwhelmed with different fish species. Such fish layout is believed by retailers to be visually more attractive for consumers, enhancing the sales of the products. This practice could facilitate the spread of the microorganisms between fish of different origins as the result of cross-contamination. The prevalence of L. monocytogenes and Y. enterocolitica in freshly caught fish in Latvia was significantly lower than that in fish from retail markets; however, 6 (5%) of 129 samples obtained from fishermen were positive. This represents public health implications for consumption of fish caught via recreational fishing. As angling is very common in Latvia, information on the possible hazards to fishermen and consumers was published by the Food and Veterinary Service authority in Latvia (28). However, the material mostly addresses chemical hazards and nutritive values of fish meat and contains no information regarding the microbiological contamination of fish, especially with pathogens of public health concern. Our results showed that freshly caught fish could be a significant vector for the transmission of pathogenic microorganisms, even in inland waters of good quality without apparent source of contamination, and this problem is underestimated in Latvia. In conclusion, this study revealed that the prevalence of L. monocytogenes and Y. enterocolitica significantly increased in freshwater fish from retail markets in comparison with freshly caught fish. This is the first study on the prevalence of bacterial foodborne pathogens in fish from Latvia, confirming that freshly caught and retailed freshwater fish can be an important source of L. monocytogenes and Y. enterocolitica.
ACKNOWLEDGMENT This study was conducted within the European Social Fund project 2013/0016/1DP/1.1.1.2.0/13/APIA/VIAA/055, Iekˇse¯ jo u¯ denu zivju resursu k¯ımisk¯a un biolog isk¯a pies¯arnojuma p¯etniec¯ıbas grupas izveide.
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