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High prevalence of Salmonella enterica subsp. diarizonae in tonsils of sheep at slaughter
Food Research International 45 (2012) 880–884
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Food Research International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s
High prevalence of Salmonella enterica subsp. diarizonae in tonsils of sheep at slaughter R. Bonke a, S. Wacheck a, C. Bumann a, C. Thum a, E. Stüber a, M. König b, R. Stephan c, M. Fredriksson-Ahomaa d,⁎ a
Institute of Food Hygiene, Faculty of Veterinary Medicine, Ludwig-Maximilians University, Munich, Germany Service de la consommation et des affaires vétérinaires, Genève, Switzerland Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland d Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, Finland b c
a r t i c l e
i n f o
Article history: Received 28 November 2010 Received in revised form 17 January 2011 Accepted 20 January 2011 Keywords: Salmonella enterica subsp. diarizonae Small ruminants Tonsils PCR Isolation
a b s t r a c t The presence of Salmonella was studied in tonsils and feces of sheep and goats at slaughter using PCR and culturing. The isolates were further characterized using PFGE to get more information about the genetic diversity of Salmonella strains circulating among sheep and goats. Antimicrobial resistance was studied because resistance to multiple antimicrobial agents among Salmonella is increasing. The prevalence of Salmonella was 43% and 2% in the tonsils of sheep and goat, respectively. Salmonella was not detected in the feces of adult animals and only sporadically in the feces of juveniles (2%). S. enterica subsp. diarizonae 61: k:1,5,(7) was isolated from 20% of the sheep tonsils and 1% of the goat tonsils. In total, 9 genotypes were obtained with PFGE using SpeI, XbaI, NotI and XhoI restriction enzymes; however, one genotype was predominant. All strains were sensitive to most (13/16) of the antimicrobials. Resistance to sulfamethoxazole was high (95%). Three (15%) strains, which were isolated from lambs, were also resistant to colistin. No correlation between the antimicrobial resistance pattern and the genotype was noticed. These results demonstrate that slaughtered sheep are an important reservoir for S. enterica subsp. diarizonae 61:k:1,5,(7) carrying this pathogen frequently in the tonsils. Future studies are needed to elucidate the significance of the tonsils in the contamination of sheep carcasses and meat with Salmonella. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Salmonella enterica is an important human and animal pathogen that may cause severe gastroenteritis, sepsis and occasionally death. Farm animals, especially poultry, pigs and cattle, are major reservoirs of different Salmonella serotypes associated with human salmonellosis (Stevens et al., 2009). Sheep and goats have also shown to be potential carriers and symptomless shedders of Salmonella (Hjartardóttir et al., 2002; Woldemariam et al., 2005). Most cases of human salmonellosis are associated with the consumption of contaminated food products (Tauxe, 1991). Direct contact with infected animals, including sheep, may also serve as a source for Salmonella infections (Baker et al., 2007). S. enterica subspecies diarizonae has frequently been isolated from reptiles and their surroundings (Davies et al., 2001; Bauwens et al., 2006). Several different serotypes of S. enterica subspecies diarizonae including serotype 61:k:1,5,(7) have also been isolated from sheep
⁎ Corresponding author. E-mail address: maria.fredriksson-ahomaa@helsinki.fi (M. Fredriksson-Ahomaa). 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.01.050
(Milnes et al., 2008). S. enterica subspecies diarizonae 61:k:1,5,(7) is considered to be host adapted to sheep like S. Abortusovis, however, it does occasionally occur in other animals and in humans also (Hall and Rowe, 1992). The serotype 61:k:1,5,(7) is one of the most frequently isolated serotype in sheep in Great Britain (Davies et al., 2001; Milnes et al., 2008) and it has also been isolated from sheep in Norway, Switzerland and North America (Pritchard, 1990; Alvseike & Skjerve, 2002; Zweifel et al., 2004). It has been associated with gastroenteritis, abortion and stillbirth (Davies et al., 2001; Alvseike & Skjerve, 2002). Suppurative epididymo-orchitis in ram associated with S. enterica subspecies diarizonae 61:k:1,5,(7) was recently reported in Spain (Ferreras et al., 2007). However, most sheep, adult and juvenile, are asymptomatic carrier (Pritchard, 1990). There is little published information on the carriage of Salmonella in small ruminants, especially in goats (Duffy et al., 2009). Salmonella has mostly been studied in feces samples of small ruminants using culture methods (Milnes et al., 2008). In this work, the presence of Salmonella was studied in the tonsils and feces of sheep and goats at slaughter using PCR and culturing. Furthermore, the isolates were characterized using PFGE to get more information about the genetic diversity of Salmonella strains circulating among sheep and goats. Antimicrobial resistance of the isolates was also studied because
R. Bonke et al. / Food Research International 45 (2012) 880–884
increasingly Salmonella strains were detected that demonstrate resistance to multiple antimicrobial agents (Foley & Lynne, 2008). 2. Materials and methods 2.1. Sampling and sample preparation
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PCR. PCR-positive homogenates were diluted 1:100 with distilled water to get single colonies before inoculation on hektoen-enteric (HE, Merck) agar plates. Single blue to green-blue colonies with and without black centers were inoculated on CASO (Merck) agar plates to get a pure culture for further identification and characterization. 2.4. Phenotypic and genotypic identification of Salmonella
Two hundred small ruminants were sampled at slaughter between October 2008 and April 2009 in Geneva, Switzerland. One to 26 animals were sampled on 22 different days. Tonsils and fecal samples from rectum were investigated from 100 sheep and goats, 50 of each were adult and 50 juvenile (Table 1). Only 2 animals per herd were sampled. The animals originate from 5 cantons (Bern, Fribourg, Geneva, Valais and Vaud) in southeast of Switzerland. Most (62%) of the adult sheep were pregnant. The tonsil and fecal samples were collected after evisceration and placed in sterile plastic bags, which were stored at −18 °C and processed 2–3 weeks later. About a 10-g tonsil and a 1-g fecal sample were homogenized in 90 ml of modified tryptic soy broth (mTSB), which consists of tryptic soy broth (CASO, Merck, Darmstadt, Germany) supplemented with 1.5 g/l bile salt (bile salt no. 3, Oxoid, Basingstoke, UK). The homogenates were enriched overnight for 16 to 18 h at 37 °C. 2.2. Detection of Salmonella using PCR Real-time PCR was used to detect Salmonella in tonsils directly from the overnight enrichment (37 °C, 16–18 h) in mTSB. The DNA was extracted from 100 μl of this enrichment using InstaGene (Bio-Rad, Hercules, CA) based on the chelating properties of Chelex resin. After centrifugation (13000× rpm for 1 min), the supernatant was removed and the pellet was resuspended in 50 μl of InstaGene, which was incubated at 56 °C for 15 min and then at 99 °C for 10 min. After centrifugation at full speed (13000 × rpm) for 3 min, the supernatant was used as template. Real-time PCR based on SYBRGreen was used to detect a 429-bp chromosomal fragment of Salmonella according to Aabo et al. (1993). Briefly, 2 μl of the template was added to 23 μl of the master mix, which contained 1× ready-to-use mix (QuantiTect™SYBRGreen PCR Kit, Qiagen, Hilden, Germany) and 200 nM of primers. A 3-step protocol (denaturation at 95 °C for 10 s, annealing at 58 °C for 30 s and elongation at 72 °C for 10 s) with 40 cycles followed by a melting curve analysis was performed. The PCR fluorescence was detected using the iQ™ Real-Time PCR Detection system (Bio-Rad). A threshold cycle (Ct) under 38 and a specific melting temperature (Tm) indicated a positive result. The Tm for Salmonella was 85.5 °C± 0.5 °C. 2.3. Isolation of Salmonella For direct plating, 100 μl of the homogenates from tonsils and feces were inoculated on xylose-lysine-desoxycholate agar (XLD, Merck, Darmstadt, Germany) before overnight enrichment. All tonsil homogenates were screened with PCR after overnight enrichment (37 °C, 16–18 h) and only PCR-positive samples were inoculated on XLD plates. Fecal homogenates were not screened with PCR but they were all inoculated after overnight enrichment on XLD plates. After incubation of the XLD plates at 37 °C for 18 to 20 h, all kinds of bacterial growth on XLD plates was studied by Table 1 Small ruminants sampled at slaughter between October 2008 and April 2009 in Geneva, Switzerland. Animal species
Number of animals
Number of adults Pregnant
Not pregnant
Sheep Goats
100 100
31 15
19 35
Number of juveniles 50 50
Salmonella was identified by PCR from pure culture. In brief, 2 to 3 colonies from pure culture on CASO agar were suspended in 100 μl of water. DNA was released by boiling the suspension for 10 min. One microlitre of the suspension was used as template and the real-time PCR was done as described above (2.2). PCR-positive isolates were further identified by API 20E. Salmonella isolates were serotyped by the Swiss National Reference Laboratory (NENT). Isolates of serotype 61:k:1,5,(7) were further studied by PCR using primers detecting S. enterica subsp. arizonae and diarizonae according to Hopkins et al. (2009). 2.5. Genotyping of S. enterica subsp. diarizonae 61:k:1,5,(7) by PFGE Genotyping was done using XbaI, SpeI, NotI and XhoI enzymes. DNA was isolated using CHEF Genomic DNA Plug Kits (Bio-Rad). The plugs were lysed for 4 to 6 h at 37 °C in lysozyme solution and over night at 50 °C in proteinase K solution. The plugs were washed 6 times in wash buffer before restriction digestion. The DNA was digested overnight with 20 U of XbaI and XhoI, and with 10 U of SpeI and NotI enzymes according to the manufacturer's instructions (New England Biolabs, Beverly, Mass., USA). The restriction fragments were separated through a 1.0% gel (pulsed-field-certified agarose, BioRad) in 0.5 × TBE with a CHEF Mapper XA system (BioRad). Lambda Ladder PFG marker (New England Biolabs) was used as a size standard. Pulse times were ramped from 2 to 40 s over 24 h for XbaI and SpeI and from 1 to 20 s over 22 h for NotI and XhoI. The gels were stained with ethidium bromide, destained with the running buffer and photographed with a Gel Doc EQ system (BioRad). Isolates were considered to be different when a one-band difference between fragments over 70 kb was observed. 2.6. Antimicrobial resistance of S. enterica subsp. diarizonae 61:k:1,5,(7) by disk diffusion method Antimicrobial resistance analysis was performed with the diskdiffusion test according to Clinical and Laboratory Standards Institute (CLSI, 2008). Salmonella isolates were inoculated into MuellerHinton broth (Oxoid, Basinkstoke, UK) and incubated at 37 °C overnight (16–18 h). The suspensions were adjusted to the turbidity of 0.5 McFarland standards (about 108 cfu/ml) and streaked onto MuellerHinton agar (Oxoid). Antimicrobial disks (Oxoid) were dispensed onto the surface of the inoculated agar plates and the plates were incubated at 37 °C for 16 to 18 h. The following 16 antimicrobials were tested: ampicillin (10 μg), amoxicillin/clavulanic acid (20/10 μg), aztreonam (30 μg), cefotaxim (30 μg), ciprofloxacin (5 μg), chloramphenicol (30 μg), colistin (25 μg), erythromycin (15 μg), furazolidon (50 μg), gentamicin (10 μg), nalidixic acid (30 μg), streptomycin (10 μg), tetracycline (30 μg), trimethoprim (5 μg), trimethoprim/sulfamethoxazole (1.25/23.75 μg) and sulfamethoxazole (25 μg). The results were recorded as susceptible, intermediate or resistant by measuring the inhibition zone diameter according to the interpretative standards of CLSI M31-A3 (2008). The reference strain Escherichia coli ATCC 25922 was used as the quality control organism and included with each batch of isolates tested. Data from all quality-control organisms were within appropriate CLSI quality ranges (CLSI, 2008). 3. Results The prevalence of Salmonella was high (43%) in the tonsils of sheep at slaughter using PCR (Table 2). The prevalence was significantly (pb 0.05,
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X2 test) higher among ewes than lambs. The prevalence was also higher in pregnant ewes (58%) than in non-pregnant (47%), however, without a statistical difference (pN 0.05, X2 test). The detection rate of Salmonella in the tonsils of goats was low (2%). Salmonella was not detected in the feces of adult animals and it was only sporadically detected in the feces of juveniles (2%). The viability of salmonella was confirmed on 33 XLD plates (73%) from 45 PCR-positive tonsil homogenates (Table 2). Salmonella was isolated from 20 of these 33 PCR-positive XLD plates when tonsils were studied but not from the 2 PCR-positive XLD plates when fecal samples were studied. The homogenates (33 from tonsils and 2 from feces), which gave a PCR-positive reaction from XLD plates, were further cultivated on HE plates after 1:100 dilution. In total, 38 Salmonella isolates from tonsils of 20 sheep and one goat were isolated from HE plates. All isolates were identified as salmonella using API 20E test even if some differences in the profiles occurred (Table 3). They all showed positive ONPG reaction due to the β-galactosidase production, which has been reported to be typical for S. enterica subsp. arizonae (IIIa) and diarizonae (IIIb). All isolates were identified as subspecies arizonae or diarizonae by real-time PCR according to Hopkins et al. (2009). After serotyping, the isolates were identified as S. enterica subsp. diarizonae 61:k:1,5,(7). Twenty strains (one isolate per animal), including 19 sheep strains (one was lost during the study) and one goat strain, were further characterized with PFGE. XbaI, SpeI, NotI and XhoI DNA digests classified S. enterica subsp. diarizonae 61:k:1,5,(7) strains into 8, 8, 7, and 6 types, respectively (Table 3). An overall homogeneity was seen among the patterns. Combining the results obtained by SpeI, XbaI, NotI and XhoI enzymes, 9 genotypes (1 to 9) were obtained (Table 3). Genotype 2 was the most frequent type and the only one found in both adult and juvenile sheep and in the juvenile goat. All other genotypes were detected only sporadically (Table 4). Antimicrobial resistance was tested against 16 agents. All 20 strains tested were sensitive to 13 out of 16 antimicrobials. The natural resistance to erythromycin was 100%. Resistance to sulfamethoxazole was high (95%). Three (15%) strains, which were isolated from lambs, were resistant to colistin (Table 4). The same strains showed also resistance to sulfamethoxazole. Small differences in sensitivity to streptomycin and cefotaxim were also observed. Some different antimicrobial patterns were found among strains of genotype 2 (Table 4).
Table 3 Different biochemical profiles and PFGE types of 20 S. enterica subsp. diarizonae 61: k:1,5,(7) strains.
4. Discussion
Table 4 Correlation between the genotype and antimicrobial pattern of 19 S. enterica subsp. diarizonae 61:k:1,5,(7) strains isolated from tonsils of adult and juvenile sheep at slaughter.
The prevalence of salmonella was high in sheep (43%), especially in adults at slaughter. In earlier studies, the reported detection rates in sheep have been clearly lower in Great Britain (1%), Iceland (2%), Ethiopia (3%) and Switzerland (11%) than in our study (Hjartardóttir et al., 2002; Zweifel et al., 2004; Woldemariam et al., 2005; Milnes et al., 2008). Significant regional variation has
Genotype
1 2 3 4 5 6 7 8 9 a
Tonsils b
Sheep
Goat
a b c
a
All (100) Ewes (50) Pregnant (31) Non-pregnant (19) Lamb (50) All (100) Doe (50) Kid (50)
Feces c
mTSB
XLD
43 (43%) 27 (54%) 18 (58%) 9 (47%) 16 (32%) 2 (2%) 0 2 (4%)
32 (32%) 20 (40%) 15 (48%) 5 (26%) 12 (24%) 1 (1%) 0 1 (2%)
Number of animals. Tryptic soy broth supplemented with 1.5 g/l bile salts. Xylose-lysine-desoxycholate agar plate.
XLD 1 (1%) 0 0 0 1 (2%) 1 (1%) 0 1 (2%)
API 20E profile
PFGE types by XbaI
SpeI
NotI
XhoI
1 1 11a 1 1 1 1 1 1 1
7704542 5704502 7704552 7704552 7704552 7704552 7704542 7704552 7704552 7704542
1 2 2 3 4 5 3 6 7 8
1 2 2 3 4 5 6 7 8 2
1 2 2 2 3 4 4 5 6 7
1 2 2 2 2 2 3 4 5 6
The only goat strain belonged to this group.
been reported in Great Britain, Iceland, and Norway (Hjartardóttir et al., 2002; Alvseike & Skjerve, 2002; Milnes et al., 2008). In Norway, the regional prevalence of S. enterica subsp. diarizonae 61: k:1,5,(7) in sheep varied from 0 to 45%, and S. enterica subsp. diarizonae 61:k:1,5,(7) was more prevalent in adults than in lambs. Surprisingly, the prevalence of salmonella was very low in goats (2%) compared to sheep (43%). In Ethiopia, the prevalence of Salmonella at slaughter was higher in goats (9%) than in sheep (3%) (Woldemariam et al., 2005). Several different serotypes were detected among small ruminants in Ethiopia but not S. enterica subsp. diarizonae 61:k:1,5,(7). One explanation can be that sheep are more susceptible to S. enterica subsp. diarizonae 61:k:1,5,(7) than goats. This serotype is the most common type found in sheep but it has also sporadically been isolated from other sources like humans, pigs and dogs, and food (Hall & Rowe, 1992). Sheep have been shown to be asymptomatic shedders of Salmonella in feces (Milnes et al., 2008). However, in our study, all fecal samples of adult sheep and goats were Salmonella negative in one gram and only 2% of the fecal samples taken from young animals were positive, which shows that only a few asymptomatic small ruminants excreted this Salmonella type in their feces. Surprisingly, Zweifel et al. (2004) isolated Salmonella in 11% of the feces samples in Switzerland, which was clearly higher than our results (1%). One
GTa
1 2
Table 2 Detection rate of Salmonella spp. in tonsils and feces of small ruminants at slaughter using PCR after overnight enrichment from homogenates (mTSB) and from agar plates (XLD). Animal species
No. of strains
3 4 5 6 7 8 9 a b c d e f g h
No. of strains
Sheep
1 2 1 3 3 1 1 1 1 1 1 1 1 1
x x
Adult
Antimicrobial pattern Juvenile
x x x x x x x
Genotype, 15 μg. Streptomycin 10 μg. Cefotaxim 30 μg. Sulfamethoxazole 25 μg. Colistin 25 μg. Intermediately resistant. Susceptible. Resistant.
x x x x x
S10b
Ctx30c
RL25d
Ct25e
If S S I I S S S S S S I I I
Sg S S S S S S S S S S S I S
Rh R R R R R R R I R R R R R
I I I I I S R I S I I I R R
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reason can be that we stored the samples at − 18 °C for 2 to 4 weeks before examination. Furthermore, they used a 10-g feces sample instead of 1 g, and two highly-selective enrichment broths instead of low-selective broth used in our study. The viability of Salmonella on XLD plates could not be confirmed from all PCR-positive tonsil homogenates. Twelve (27%) XLD plates, which showed bacterial growth, were PCR negative even if the homogenates were positive. This can be due to a lower number of Salmonella compared to a higher number of other bacteria on the XLD plates, thus giving false-negative results with PCR. It is also possible, that the Salmonella cells were dead and did not grow on the plates. In this case, the number of dead cells in the homogenate has had to be high (N104) to give a positive PCR results. On XLD agar plates, only yellow colonies mostly without black centers were found making the isolation of presumptive Salmonella impossible. Some S. enterica subsp. arizonae and diarizonae bacteria have shown to ferment lactose which can explain the yellow colonies on the XLD (Lapage & Jayaraman, 1964). Black centered colonies were only detected on 3 (9%, 3/35) PCR-positive XLD plates which may be due to the pH being too low for hydrogen sulfide to be produced. Subspecies arizonae and diarizonae bacteria usually show β-galactosidase activity which makes the isolation also very challenging or even impossible using chromogenic agar plates (Pignato et al., 1995; Eigner et al., 2001). Surprisingly we found typical Salmonella colonies, which are blue to blue green with black centers, on 21 (64%) HE agar plates which made it possible to isolate Salmonella from 20 sheep and one goat tonsils. Identification of some strains of S. enterica subsp. arizonae (IIIa) and diarizonae (IIIb) can be challenging with routine biochemical and serological tests. Some members of these subspecies differ from other Salmonella by their ability to ferment lactose (Lapage & Jayaraman, 1964; Chong et al., 1991; Davies et al., 2001). As lactose fermentation is one of the key diagnostic principles on which standard methods for the isolation of Salmonella are based, lactose-positive Salmonella strains represent a special diagnostic problem and may go unnoticed. Furthermore, the antigenic structure is often difficult to determine and the isolates have to be sent to the reference laboratory for identification. S. enterica subsp. arizonae and diarizonae can be differentiated from most other subspecies of S. enterica based on hydrolysis of O-nitrophenyl-ß-D-galactopyranoside (ONPG) after 2 h by ß-galactosidase (encoded by lacZ) (Lapage & Jayaraman, 1964). A rapid and specific identification of both types with real-time PCR based on lacZ has recently been demonstrated (Hopkins et al., 2009). In this study, all S. enterica subsp. diarizonae 61:k:1,5,(7) isolates were successfully identified by this PCR method, also. In the European Union, slaughterhouse operators are required to monitor Salmonella on sheep and goat carcasses (EC No. 2073/2005). For the monitoring, detection of Salmonella using standardized ISO 6579 culture method is recommended. However, in this method, XLD and chromogenic agar plates are used for isolation, and thus lactose and ß-galactosidase-positive Salmonella strains are difficult to identify. To overcome this problem, a validated PCR method could be a good alternative to be used parallel with culturing to detect these untypical strains in naturally contaminated samples like carcass samples. S. enterica subsp. diarizonae 61:k:1,5,(7) was characterized with PFGE using four different restriction enzymes. The genetic diversity was low with all enzymes. Nine different genotypes were obtained among 20 strains when both XbaI and SpeI were used. Genotype 2 was shown to be the most frequent type found in adult and young animals. All other types were only found in one animal. These results are in accordance with the earlier study presented by Zweifel et al. (2004) who identified 22 different XbaI patterns among 64 S. enterica subsp. diarizonae 61:k:1,5,(7) strains of which 17 were found in only one animal. Alvseike et al. (2004) found only 15 distinct XbaI patterns among 54 S. enterica subsp. diarizonae belonging to different serotypes. However, they reported a high genetic diversity among
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serotype 61:k:1,5,(7) strains. These strains originated from different countries and sources and were isolated during different time periods, which may explain the high genetic diversity. S. enterica subsp. diarizonae 61:k:1,5,(7) strains were sensitive to most (13/16) of the antimicrobials tested including ampicillin and tetracyclin. Resistance to these agents has shown to be high among Salmonella strains in the EU which can partly be explained by different serotypes and animal sources tested (EFSA, 2010). The resistance to sulfamethoxazole (95%) was very high among the S. enterica subsp. diarizonae 61:k:1,5,(7) strains. In an earlier study, S. enterica subsp. arizonae and diarizonae strains from human (66 strains) and nonhuman (107 strains) sources demonstrated a very low (b5%) antimicrobial resistance to sulfonamides (Hall & Rowe, 1992). The resistance to sulfonamides is nowadays widespread among Salmonella strains within the EU (EFSA, 2010). Additionally, 3 strains isolated from lambs were also resistant to colistin. In a recent study, S. Indiana was a cause of abortion in ewes and all isolates were sensitive to colistin (Luque et al., 2009). One reason for the resistant strains found in lambs can be that colistin is used for therapy in lambs but not in adult sheep (Gousia et al., 2011). Another reason can be that agar diffusion method gave false resistant results due to low diffusion of colistin into the agar (Boyen et al., 2010). The reason for the sensitive strains found in ewes can be that Luque et al. (2009) used 50 μg/disk instead of 25 μg/disk. Our results demonstrate that especially sheep can carry Salmonella frequently in the tonsils at slaughter. The tonsils colonized with salmonella may play an important role in the contamination of the carcass, the offal and the slaughterhouse environment during slaughter. Several Salmonella outbreaks in Iceland have been traced to consumption of singed sheep heads, a specialty of the country (Hjartardóttir et al., 2002). These outbreaks were explained by insufficient boiling of sheep heads contaminated by Salmonella-positive tonsils. Future studies are needed to elucidate the significance of the tonsils in the contamination of sheep carcasses and meat.
5. Conclusions The prevalence of Salmonella was high in sheep and low in goats at slaughter. The tonsils allow a better estimation of the prevalence of Salmonella in asymptomatic sheep than feces samples. Isolation and identification of S. enterica subsp. diarizonae 61:k:1,5,(7) from asymptomatic small ruminants is very challenging. Several closely related genotypes were distributed among sheep but one genotype was dominant. Most S. enterica subsp. diarizonae 61:k:1,5,(7) strains were sensitive to a panel of the most important antimicrobials used for therapy in sheep and goats.
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Hopkins, K. L., Peters, T. M., Lawson, A. J., & Owen, R. J. (2009). Rapid identification of Salmonella enterica subsp. arizonae and S. enterica subsp. diarizonae by real-time polymerase chain reaction. Diagnostic Microbiology and Infectious Disease, 64, 452−454. Lapage, S. P., & Jayaraman, M. S. (1964). Beta-galactosidase and lactose fermentation in the identification of enterobacteria including salmonellae. Journal of Clinical Pathology, 17, 117−121. Luque, I., Echeita, A., León, J., Herrera-León, S., Tarradas, C., Gonzáles-Sanz, R., Huerta, B., & Astorga, R. J. (2009). Salmonella Indiana as a cause of abortion in ewes: Genetic diversity and resistance patterns. Veterinary Microbiology, 134, 396−399. Milnes, A. S., Stewart, I., Clifton-Hadley, F. A., Davies, R. H., Newell, D. G., Sayers, A. R., Cheasty, T., Cassar, C., Ridley, A., Cook, A. J. C., Evans, S. J., Teale, C. J., Smith, R. P., McNally, A., Toszeghy, M., Futter, R., Kay, A., & Paiba, G. A. (2008). Intestinal carriage of verocytotoxigenic Escherichia coli O157, Salmonella, thermophilic Campylobacter and Yersinia enterocolitica, in cattle, sheep and pigs at slaughter in Great Britain during 2003. Epidemiology and Infection, 136, 739−751. Pignato, S., Giammanco, G., & Giammanco, G. (1995). Rambach agar and SM-ID medium sensitivity for presumptive identification of Salmonella subspecies I-VI. Journal of Medical Microbiology, 43, 68−71. Pritchard, J. (1990). Salmonella arizonae in sheep. Canadian Veterinary Journal, 31, 42. Stevens, M. P., Humphrey, T. J., & Maskell, D. J. (2009). Molecular insights into farm animal and zoonotic Salmonella infections. Philosophical Transactions of the Royal Society B, 364, 2709−2723. Tauxe, R. V. (1991). Salmonella: A postmodern pathogen. Journal of Food Protection, 54, 563−568. Woldemariam, E., Molla, B., Alemayehu, D., & Muckle, A. (2005). Prevalence and distribution of Salmonella in apparently healthy slaughter sheep and goats in Debre Zeit, Ethiopia. Small Ruminant Research, 58, 19−24. Zweifel, C., Zychowska, A., & Stephan, R. (2004). Prevalence and characterisation of Shiga toxin-producing Escherichia coli, Salmonella spp. and Campylobacter spp. isolated from slaughtered sheep in Switzerland. International Journal of Food Microbiology, 92, 45−53.
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High prevalence of Salmonella enterica subsp. diarizonae in tonsils of sheep at slaughter R. Bonke a, S. Wacheck a, C. Bumann a, C. Thum a, E. Stüber a, M. König b, R. Stephan c, M. Fredriksson-Ahomaa d,⁎ a
Institute of Food Hygiene, Faculty of Veterinary Medicine, Ludwig-Maximilians University, Munich, Germany Service de la consommation et des affaires vétérinaires, Genève, Switzerland Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland d Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, Finland b c
a r t i c l e
i n f o
Article history: Received 28 November 2010 Received in revised form 17 January 2011 Accepted 20 January 2011 Keywords: Salmonella enterica subsp. diarizonae Small ruminants Tonsils PCR Isolation
a b s t r a c t The presence of Salmonella was studied in tonsils and feces of sheep and goats at slaughter using PCR and culturing. The isolates were further characterized using PFGE to get more information about the genetic diversity of Salmonella strains circulating among sheep and goats. Antimicrobial resistance was studied because resistance to multiple antimicrobial agents among Salmonella is increasing. The prevalence of Salmonella was 43% and 2% in the tonsils of sheep and goat, respectively. Salmonella was not detected in the feces of adult animals and only sporadically in the feces of juveniles (2%). S. enterica subsp. diarizonae 61: k:1,5,(7) was isolated from 20% of the sheep tonsils and 1% of the goat tonsils. In total, 9 genotypes were obtained with PFGE using SpeI, XbaI, NotI and XhoI restriction enzymes; however, one genotype was predominant. All strains were sensitive to most (13/16) of the antimicrobials. Resistance to sulfamethoxazole was high (95%). Three (15%) strains, which were isolated from lambs, were also resistant to colistin. No correlation between the antimicrobial resistance pattern and the genotype was noticed. These results demonstrate that slaughtered sheep are an important reservoir for S. enterica subsp. diarizonae 61:k:1,5,(7) carrying this pathogen frequently in the tonsils. Future studies are needed to elucidate the significance of the tonsils in the contamination of sheep carcasses and meat with Salmonella. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Salmonella enterica is an important human and animal pathogen that may cause severe gastroenteritis, sepsis and occasionally death. Farm animals, especially poultry, pigs and cattle, are major reservoirs of different Salmonella serotypes associated with human salmonellosis (Stevens et al., 2009). Sheep and goats have also shown to be potential carriers and symptomless shedders of Salmonella (Hjartardóttir et al., 2002; Woldemariam et al., 2005). Most cases of human salmonellosis are associated with the consumption of contaminated food products (Tauxe, 1991). Direct contact with infected animals, including sheep, may also serve as a source for Salmonella infections (Baker et al., 2007). S. enterica subspecies diarizonae has frequently been isolated from reptiles and their surroundings (Davies et al., 2001; Bauwens et al., 2006). Several different serotypes of S. enterica subspecies diarizonae including serotype 61:k:1,5,(7) have also been isolated from sheep
⁎ Corresponding author. E-mail address: maria.fredriksson-ahomaa@helsinki.fi (M. Fredriksson-Ahomaa). 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.01.050
(Milnes et al., 2008). S. enterica subspecies diarizonae 61:k:1,5,(7) is considered to be host adapted to sheep like S. Abortusovis, however, it does occasionally occur in other animals and in humans also (Hall and Rowe, 1992). The serotype 61:k:1,5,(7) is one of the most frequently isolated serotype in sheep in Great Britain (Davies et al., 2001; Milnes et al., 2008) and it has also been isolated from sheep in Norway, Switzerland and North America (Pritchard, 1990; Alvseike & Skjerve, 2002; Zweifel et al., 2004). It has been associated with gastroenteritis, abortion and stillbirth (Davies et al., 2001; Alvseike & Skjerve, 2002). Suppurative epididymo-orchitis in ram associated with S. enterica subspecies diarizonae 61:k:1,5,(7) was recently reported in Spain (Ferreras et al., 2007). However, most sheep, adult and juvenile, are asymptomatic carrier (Pritchard, 1990). There is little published information on the carriage of Salmonella in small ruminants, especially in goats (Duffy et al., 2009). Salmonella has mostly been studied in feces samples of small ruminants using culture methods (Milnes et al., 2008). In this work, the presence of Salmonella was studied in the tonsils and feces of sheep and goats at slaughter using PCR and culturing. Furthermore, the isolates were characterized using PFGE to get more information about the genetic diversity of Salmonella strains circulating among sheep and goats. Antimicrobial resistance of the isolates was also studied because
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increasingly Salmonella strains were detected that demonstrate resistance to multiple antimicrobial agents (Foley & Lynne, 2008). 2. Materials and methods 2.1. Sampling and sample preparation
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PCR. PCR-positive homogenates were diluted 1:100 with distilled water to get single colonies before inoculation on hektoen-enteric (HE, Merck) agar plates. Single blue to green-blue colonies with and without black centers were inoculated on CASO (Merck) agar plates to get a pure culture for further identification and characterization. 2.4. Phenotypic and genotypic identification of Salmonella
Two hundred small ruminants were sampled at slaughter between October 2008 and April 2009 in Geneva, Switzerland. One to 26 animals were sampled on 22 different days. Tonsils and fecal samples from rectum were investigated from 100 sheep and goats, 50 of each were adult and 50 juvenile (Table 1). Only 2 animals per herd were sampled. The animals originate from 5 cantons (Bern, Fribourg, Geneva, Valais and Vaud) in southeast of Switzerland. Most (62%) of the adult sheep were pregnant. The tonsil and fecal samples were collected after evisceration and placed in sterile plastic bags, which were stored at −18 °C and processed 2–3 weeks later. About a 10-g tonsil and a 1-g fecal sample were homogenized in 90 ml of modified tryptic soy broth (mTSB), which consists of tryptic soy broth (CASO, Merck, Darmstadt, Germany) supplemented with 1.5 g/l bile salt (bile salt no. 3, Oxoid, Basingstoke, UK). The homogenates were enriched overnight for 16 to 18 h at 37 °C. 2.2. Detection of Salmonella using PCR Real-time PCR was used to detect Salmonella in tonsils directly from the overnight enrichment (37 °C, 16–18 h) in mTSB. The DNA was extracted from 100 μl of this enrichment using InstaGene (Bio-Rad, Hercules, CA) based on the chelating properties of Chelex resin. After centrifugation (13000× rpm for 1 min), the supernatant was removed and the pellet was resuspended in 50 μl of InstaGene, which was incubated at 56 °C for 15 min and then at 99 °C for 10 min. After centrifugation at full speed (13000 × rpm) for 3 min, the supernatant was used as template. Real-time PCR based on SYBRGreen was used to detect a 429-bp chromosomal fragment of Salmonella according to Aabo et al. (1993). Briefly, 2 μl of the template was added to 23 μl of the master mix, which contained 1× ready-to-use mix (QuantiTect™SYBRGreen PCR Kit, Qiagen, Hilden, Germany) and 200 nM of primers. A 3-step protocol (denaturation at 95 °C for 10 s, annealing at 58 °C for 30 s and elongation at 72 °C for 10 s) with 40 cycles followed by a melting curve analysis was performed. The PCR fluorescence was detected using the iQ™ Real-Time PCR Detection system (Bio-Rad). A threshold cycle (Ct) under 38 and a specific melting temperature (Tm) indicated a positive result. The Tm for Salmonella was 85.5 °C± 0.5 °C. 2.3. Isolation of Salmonella For direct plating, 100 μl of the homogenates from tonsils and feces were inoculated on xylose-lysine-desoxycholate agar (XLD, Merck, Darmstadt, Germany) before overnight enrichment. All tonsil homogenates were screened with PCR after overnight enrichment (37 °C, 16–18 h) and only PCR-positive samples were inoculated on XLD plates. Fecal homogenates were not screened with PCR but they were all inoculated after overnight enrichment on XLD plates. After incubation of the XLD plates at 37 °C for 18 to 20 h, all kinds of bacterial growth on XLD plates was studied by Table 1 Small ruminants sampled at slaughter between October 2008 and April 2009 in Geneva, Switzerland. Animal species
Number of animals
Number of adults Pregnant
Not pregnant
Sheep Goats
100 100
31 15
19 35
Number of juveniles 50 50
Salmonella was identified by PCR from pure culture. In brief, 2 to 3 colonies from pure culture on CASO agar were suspended in 100 μl of water. DNA was released by boiling the suspension for 10 min. One microlitre of the suspension was used as template and the real-time PCR was done as described above (2.2). PCR-positive isolates were further identified by API 20E. Salmonella isolates were serotyped by the Swiss National Reference Laboratory (NENT). Isolates of serotype 61:k:1,5,(7) were further studied by PCR using primers detecting S. enterica subsp. arizonae and diarizonae according to Hopkins et al. (2009). 2.5. Genotyping of S. enterica subsp. diarizonae 61:k:1,5,(7) by PFGE Genotyping was done using XbaI, SpeI, NotI and XhoI enzymes. DNA was isolated using CHEF Genomic DNA Plug Kits (Bio-Rad). The plugs were lysed for 4 to 6 h at 37 °C in lysozyme solution and over night at 50 °C in proteinase K solution. The plugs were washed 6 times in wash buffer before restriction digestion. The DNA was digested overnight with 20 U of XbaI and XhoI, and with 10 U of SpeI and NotI enzymes according to the manufacturer's instructions (New England Biolabs, Beverly, Mass., USA). The restriction fragments were separated through a 1.0% gel (pulsed-field-certified agarose, BioRad) in 0.5 × TBE with a CHEF Mapper XA system (BioRad). Lambda Ladder PFG marker (New England Biolabs) was used as a size standard. Pulse times were ramped from 2 to 40 s over 24 h for XbaI and SpeI and from 1 to 20 s over 22 h for NotI and XhoI. The gels were stained with ethidium bromide, destained with the running buffer and photographed with a Gel Doc EQ system (BioRad). Isolates were considered to be different when a one-band difference between fragments over 70 kb was observed. 2.6. Antimicrobial resistance of S. enterica subsp. diarizonae 61:k:1,5,(7) by disk diffusion method Antimicrobial resistance analysis was performed with the diskdiffusion test according to Clinical and Laboratory Standards Institute (CLSI, 2008). Salmonella isolates were inoculated into MuellerHinton broth (Oxoid, Basinkstoke, UK) and incubated at 37 °C overnight (16–18 h). The suspensions were adjusted to the turbidity of 0.5 McFarland standards (about 108 cfu/ml) and streaked onto MuellerHinton agar (Oxoid). Antimicrobial disks (Oxoid) were dispensed onto the surface of the inoculated agar plates and the plates were incubated at 37 °C for 16 to 18 h. The following 16 antimicrobials were tested: ampicillin (10 μg), amoxicillin/clavulanic acid (20/10 μg), aztreonam (30 μg), cefotaxim (30 μg), ciprofloxacin (5 μg), chloramphenicol (30 μg), colistin (25 μg), erythromycin (15 μg), furazolidon (50 μg), gentamicin (10 μg), nalidixic acid (30 μg), streptomycin (10 μg), tetracycline (30 μg), trimethoprim (5 μg), trimethoprim/sulfamethoxazole (1.25/23.75 μg) and sulfamethoxazole (25 μg). The results were recorded as susceptible, intermediate or resistant by measuring the inhibition zone diameter according to the interpretative standards of CLSI M31-A3 (2008). The reference strain Escherichia coli ATCC 25922 was used as the quality control organism and included with each batch of isolates tested. Data from all quality-control organisms were within appropriate CLSI quality ranges (CLSI, 2008). 3. Results The prevalence of Salmonella was high (43%) in the tonsils of sheep at slaughter using PCR (Table 2). The prevalence was significantly (pb 0.05,
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X2 test) higher among ewes than lambs. The prevalence was also higher in pregnant ewes (58%) than in non-pregnant (47%), however, without a statistical difference (pN 0.05, X2 test). The detection rate of Salmonella in the tonsils of goats was low (2%). Salmonella was not detected in the feces of adult animals and it was only sporadically detected in the feces of juveniles (2%). The viability of salmonella was confirmed on 33 XLD plates (73%) from 45 PCR-positive tonsil homogenates (Table 2). Salmonella was isolated from 20 of these 33 PCR-positive XLD plates when tonsils were studied but not from the 2 PCR-positive XLD plates when fecal samples were studied. The homogenates (33 from tonsils and 2 from feces), which gave a PCR-positive reaction from XLD plates, were further cultivated on HE plates after 1:100 dilution. In total, 38 Salmonella isolates from tonsils of 20 sheep and one goat were isolated from HE plates. All isolates were identified as salmonella using API 20E test even if some differences in the profiles occurred (Table 3). They all showed positive ONPG reaction due to the β-galactosidase production, which has been reported to be typical for S. enterica subsp. arizonae (IIIa) and diarizonae (IIIb). All isolates were identified as subspecies arizonae or diarizonae by real-time PCR according to Hopkins et al. (2009). After serotyping, the isolates were identified as S. enterica subsp. diarizonae 61:k:1,5,(7). Twenty strains (one isolate per animal), including 19 sheep strains (one was lost during the study) and one goat strain, were further characterized with PFGE. XbaI, SpeI, NotI and XhoI DNA digests classified S. enterica subsp. diarizonae 61:k:1,5,(7) strains into 8, 8, 7, and 6 types, respectively (Table 3). An overall homogeneity was seen among the patterns. Combining the results obtained by SpeI, XbaI, NotI and XhoI enzymes, 9 genotypes (1 to 9) were obtained (Table 3). Genotype 2 was the most frequent type and the only one found in both adult and juvenile sheep and in the juvenile goat. All other genotypes were detected only sporadically (Table 4). Antimicrobial resistance was tested against 16 agents. All 20 strains tested were sensitive to 13 out of 16 antimicrobials. The natural resistance to erythromycin was 100%. Resistance to sulfamethoxazole was high (95%). Three (15%) strains, which were isolated from lambs, were resistant to colistin (Table 4). The same strains showed also resistance to sulfamethoxazole. Small differences in sensitivity to streptomycin and cefotaxim were also observed. Some different antimicrobial patterns were found among strains of genotype 2 (Table 4).
Table 3 Different biochemical profiles and PFGE types of 20 S. enterica subsp. diarizonae 61: k:1,5,(7) strains.
4. Discussion
Table 4 Correlation between the genotype and antimicrobial pattern of 19 S. enterica subsp. diarizonae 61:k:1,5,(7) strains isolated from tonsils of adult and juvenile sheep at slaughter.
The prevalence of salmonella was high in sheep (43%), especially in adults at slaughter. In earlier studies, the reported detection rates in sheep have been clearly lower in Great Britain (1%), Iceland (2%), Ethiopia (3%) and Switzerland (11%) than in our study (Hjartardóttir et al., 2002; Zweifel et al., 2004; Woldemariam et al., 2005; Milnes et al., 2008). Significant regional variation has
Genotype
1 2 3 4 5 6 7 8 9 a
Tonsils b
Sheep
Goat
a b c
a
All (100) Ewes (50) Pregnant (31) Non-pregnant (19) Lamb (50) All (100) Doe (50) Kid (50)
Feces c
mTSB
XLD
43 (43%) 27 (54%) 18 (58%) 9 (47%) 16 (32%) 2 (2%) 0 2 (4%)
32 (32%) 20 (40%) 15 (48%) 5 (26%) 12 (24%) 1 (1%) 0 1 (2%)
Number of animals. Tryptic soy broth supplemented with 1.5 g/l bile salts. Xylose-lysine-desoxycholate agar plate.
XLD 1 (1%) 0 0 0 1 (2%) 1 (1%) 0 1 (2%)
API 20E profile
PFGE types by XbaI
SpeI
NotI
XhoI
1 1 11a 1 1 1 1 1 1 1
7704542 5704502 7704552 7704552 7704552 7704552 7704542 7704552 7704552 7704542
1 2 2 3 4 5 3 6 7 8
1 2 2 3 4 5 6 7 8 2
1 2 2 2 3 4 4 5 6 7
1 2 2 2 2 2 3 4 5 6
The only goat strain belonged to this group.
been reported in Great Britain, Iceland, and Norway (Hjartardóttir et al., 2002; Alvseike & Skjerve, 2002; Milnes et al., 2008). In Norway, the regional prevalence of S. enterica subsp. diarizonae 61: k:1,5,(7) in sheep varied from 0 to 45%, and S. enterica subsp. diarizonae 61:k:1,5,(7) was more prevalent in adults than in lambs. Surprisingly, the prevalence of salmonella was very low in goats (2%) compared to sheep (43%). In Ethiopia, the prevalence of Salmonella at slaughter was higher in goats (9%) than in sheep (3%) (Woldemariam et al., 2005). Several different serotypes were detected among small ruminants in Ethiopia but not S. enterica subsp. diarizonae 61:k:1,5,(7). One explanation can be that sheep are more susceptible to S. enterica subsp. diarizonae 61:k:1,5,(7) than goats. This serotype is the most common type found in sheep but it has also sporadically been isolated from other sources like humans, pigs and dogs, and food (Hall & Rowe, 1992). Sheep have been shown to be asymptomatic shedders of Salmonella in feces (Milnes et al., 2008). However, in our study, all fecal samples of adult sheep and goats were Salmonella negative in one gram and only 2% of the fecal samples taken from young animals were positive, which shows that only a few asymptomatic small ruminants excreted this Salmonella type in their feces. Surprisingly, Zweifel et al. (2004) isolated Salmonella in 11% of the feces samples in Switzerland, which was clearly higher than our results (1%). One
GTa
1 2
Table 2 Detection rate of Salmonella spp. in tonsils and feces of small ruminants at slaughter using PCR after overnight enrichment from homogenates (mTSB) and from agar plates (XLD). Animal species
No. of strains
3 4 5 6 7 8 9 a b c d e f g h
No. of strains
Sheep
1 2 1 3 3 1 1 1 1 1 1 1 1 1
x x
Adult
Antimicrobial pattern Juvenile
x x x x x x x
Genotype, 15 μg. Streptomycin 10 μg. Cefotaxim 30 μg. Sulfamethoxazole 25 μg. Colistin 25 μg. Intermediately resistant. Susceptible. Resistant.
x x x x x
S10b
Ctx30c
RL25d
Ct25e
If S S I I S S S S S S I I I
Sg S S S S S S S S S S S I S
Rh R R R R R R R I R R R R R
I I I I I S R I S I I I R R
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reason can be that we stored the samples at − 18 °C for 2 to 4 weeks before examination. Furthermore, they used a 10-g feces sample instead of 1 g, and two highly-selective enrichment broths instead of low-selective broth used in our study. The viability of Salmonella on XLD plates could not be confirmed from all PCR-positive tonsil homogenates. Twelve (27%) XLD plates, which showed bacterial growth, were PCR negative even if the homogenates were positive. This can be due to a lower number of Salmonella compared to a higher number of other bacteria on the XLD plates, thus giving false-negative results with PCR. It is also possible, that the Salmonella cells were dead and did not grow on the plates. In this case, the number of dead cells in the homogenate has had to be high (N104) to give a positive PCR results. On XLD agar plates, only yellow colonies mostly without black centers were found making the isolation of presumptive Salmonella impossible. Some S. enterica subsp. arizonae and diarizonae bacteria have shown to ferment lactose which can explain the yellow colonies on the XLD (Lapage & Jayaraman, 1964). Black centered colonies were only detected on 3 (9%, 3/35) PCR-positive XLD plates which may be due to the pH being too low for hydrogen sulfide to be produced. Subspecies arizonae and diarizonae bacteria usually show β-galactosidase activity which makes the isolation also very challenging or even impossible using chromogenic agar plates (Pignato et al., 1995; Eigner et al., 2001). Surprisingly we found typical Salmonella colonies, which are blue to blue green with black centers, on 21 (64%) HE agar plates which made it possible to isolate Salmonella from 20 sheep and one goat tonsils. Identification of some strains of S. enterica subsp. arizonae (IIIa) and diarizonae (IIIb) can be challenging with routine biochemical and serological tests. Some members of these subspecies differ from other Salmonella by their ability to ferment lactose (Lapage & Jayaraman, 1964; Chong et al., 1991; Davies et al., 2001). As lactose fermentation is one of the key diagnostic principles on which standard methods for the isolation of Salmonella are based, lactose-positive Salmonella strains represent a special diagnostic problem and may go unnoticed. Furthermore, the antigenic structure is often difficult to determine and the isolates have to be sent to the reference laboratory for identification. S. enterica subsp. arizonae and diarizonae can be differentiated from most other subspecies of S. enterica based on hydrolysis of O-nitrophenyl-ß-D-galactopyranoside (ONPG) after 2 h by ß-galactosidase (encoded by lacZ) (Lapage & Jayaraman, 1964). A rapid and specific identification of both types with real-time PCR based on lacZ has recently been demonstrated (Hopkins et al., 2009). In this study, all S. enterica subsp. diarizonae 61:k:1,5,(7) isolates were successfully identified by this PCR method, also. In the European Union, slaughterhouse operators are required to monitor Salmonella on sheep and goat carcasses (EC No. 2073/2005). For the monitoring, detection of Salmonella using standardized ISO 6579 culture method is recommended. However, in this method, XLD and chromogenic agar plates are used for isolation, and thus lactose and ß-galactosidase-positive Salmonella strains are difficult to identify. To overcome this problem, a validated PCR method could be a good alternative to be used parallel with culturing to detect these untypical strains in naturally contaminated samples like carcass samples. S. enterica subsp. diarizonae 61:k:1,5,(7) was characterized with PFGE using four different restriction enzymes. The genetic diversity was low with all enzymes. Nine different genotypes were obtained among 20 strains when both XbaI and SpeI were used. Genotype 2 was shown to be the most frequent type found in adult and young animals. All other types were only found in one animal. These results are in accordance with the earlier study presented by Zweifel et al. (2004) who identified 22 different XbaI patterns among 64 S. enterica subsp. diarizonae 61:k:1,5,(7) strains of which 17 were found in only one animal. Alvseike et al. (2004) found only 15 distinct XbaI patterns among 54 S. enterica subsp. diarizonae belonging to different serotypes. However, they reported a high genetic diversity among
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serotype 61:k:1,5,(7) strains. These strains originated from different countries and sources and were isolated during different time periods, which may explain the high genetic diversity. S. enterica subsp. diarizonae 61:k:1,5,(7) strains were sensitive to most (13/16) of the antimicrobials tested including ampicillin and tetracyclin. Resistance to these agents has shown to be high among Salmonella strains in the EU which can partly be explained by different serotypes and animal sources tested (EFSA, 2010). The resistance to sulfamethoxazole (95%) was very high among the S. enterica subsp. diarizonae 61:k:1,5,(7) strains. In an earlier study, S. enterica subsp. arizonae and diarizonae strains from human (66 strains) and nonhuman (107 strains) sources demonstrated a very low (b5%) antimicrobial resistance to sulfonamides (Hall & Rowe, 1992). The resistance to sulfonamides is nowadays widespread among Salmonella strains within the EU (EFSA, 2010). Additionally, 3 strains isolated from lambs were also resistant to colistin. In a recent study, S. Indiana was a cause of abortion in ewes and all isolates were sensitive to colistin (Luque et al., 2009). One reason for the resistant strains found in lambs can be that colistin is used for therapy in lambs but not in adult sheep (Gousia et al., 2011). Another reason can be that agar diffusion method gave false resistant results due to low diffusion of colistin into the agar (Boyen et al., 2010). The reason for the sensitive strains found in ewes can be that Luque et al. (2009) used 50 μg/disk instead of 25 μg/disk. Our results demonstrate that especially sheep can carry Salmonella frequently in the tonsils at slaughter. The tonsils colonized with salmonella may play an important role in the contamination of the carcass, the offal and the slaughterhouse environment during slaughter. Several Salmonella outbreaks in Iceland have been traced to consumption of singed sheep heads, a specialty of the country (Hjartardóttir et al., 2002). These outbreaks were explained by insufficient boiling of sheep heads contaminated by Salmonella-positive tonsils. Future studies are needed to elucidate the significance of the tonsils in the contamination of sheep carcasses and meat.
5. Conclusions The prevalence of Salmonella was high in sheep and low in goats at slaughter. The tonsils allow a better estimation of the prevalence of Salmonella in asymptomatic sheep than feces samples. Isolation and identification of S. enterica subsp. diarizonae 61:k:1,5,(7) from asymptomatic small ruminants is very challenging. Several closely related genotypes were distributed among sheep but one genotype was dominant. Most S. enterica subsp. diarizonae 61:k:1,5,(7) strains were sensitive to a panel of the most important antimicrobials used for therapy in sheep and goats.
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