International Journal of Food Microbiology 235 (2016) 125–132

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Detection, seroprevalence and antimicrobial resistance of Yersinia

enterocolitica and Yersinia pseudotuberculosis in pig tonsils in
Northern Italy
S. Bonardi a,⁎, I. Bruini a, M. D'Incau b, I. Van Damme c, E. Carniel d, S. Brémont d, P. Cavallini e,
S. Tagliabue b, F. Brindani a
a
Department of Veterinary Science, Unit of Food Hygiene, University of Parma, Via del Taglio 10, 43126 Parma, Italy
b
Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna, Sezione di Brescia, Via Bianchi, 9, 25124 Brescia, Italy
c
Department Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
d
Centre National de Référence des Yersinia, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris, France
e
National Veterinary Service, Local Unit of Parma, Via Vasari 13/A, 43126 Parma, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Yersiniosis is the third most common reported zoonoses in Europe, with Y. enterocolitica and Y. pseudotuberculosis
Received 31 May 2016 responsible for 98.66% and 0.94% of the confirmed human cases in 2013. From June 2013 to October 2014, 201
Received in revised form 6 July 2016 pigs at slaughter belonging to 67 batches were tested for Y. enterocolitica and Y. pseudotuberculosis in tonsils. Di-
Accepted 27 July 2016
aphragm muscle samples were tested for antibodies against Yersinia by a commercially available ELISA test. Y.
Available online 28 July 2016
enterocolitica 4/O:3 was detected in 55/201 pig tonsils (27.4%; 95% CI 23.1–37.1). The positive pigs came from
Keywords:
38/67 batches (56.7%) and were reared in 36/61 farms (59.0%). There was no statistical difference between far-
Yersinia enterocolitica row-to-finish and finishing farms. The mean count of Y. enterocolitica was 3.56 ± 0.85 log10 CFU/g with a min-
Yersinia pseudotuberculosis imum of 2.0 log10 CFU/g and a maximum of 4.78 log10 CFU/g. Y. pseudotuberculosis was isolated from 4/201 pig
Pig tonsils (2.0%; 95% CI 0.0–4.5). Three isolates belonged to serotype O:3 and one to serotype O:1. The positive pigs
Slaughterhouse belonged to 4/67 batches (6.0%) and came from finishing farms only. Y. pseudotuberculosis could be enumerated
Seroprevalence in one sample only (4.27 log10 CFU/g). The ELISA test demonstrated that 56.1% of the meat juice samples were
Antimicrobial resistance positive for Yersinia antibodies. Serological positivity was found in 67.9% (36/53) of the Y. enterocolitica- and
75.0% (3/4) of the Y. pseudotuberculosis positive pigs. A significant association was found between serological re-
sults and the presence of Y. enterocolitica in tonsils (OR = 1.97, p = 0.044).
All the Y. enterocolitica 4/O:3 isolates were susceptible to amoxicillin-clavulanic acid, gentamicin, ceftazidime,
ertapenem and meropenem, 94.5% to cefotaxime, 89.1% to kanamycin and 78.2% to tetracycline. The highest re-
sistance rates were observed for ampicillin (100%), sulphonamides (98.2%) and streptomycin (78.2%). Y. pseudo-
tuberculosis strains were sensitive to all the antimicrobials tested, i.e. amoxicillin, amoxicillin/clavulanic acid,
azithromycin, cephalothin, cefoxitin, ceftriaxone, ciprofloxacin, nalidixic acid, sulphonamide, tetracycline and
ticarcillin.
The study shows that Italian fattening pigs are frequently infected with human pathogenic Y. enterocolitica 4/O:3.
Although the isolation rate is slightly lower than in other European countries, the serological test demonstrates
that the infection is widespread among pig population. In fact, seroprevalence is similar to other EU countries.
The detection of Y. pseudotuberculosis serotypes O:1 and O:3 in pig tonsils is of concern.
Since tonsils may represent a contamination source for pig meat at slaughter, further studies regarding human
infections by both microbial species are strongly recommended.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction

Yersinia enterocolitica and Yersinia pseudotuberculosis are food-borne

⁎ Corresponding author at: Department of Animal Health, Faculty of Veterinary
Medicine, University of Parma, Via del Taglio, 10, 43126 Parma, Italy.
E-mail address: silvia.bonardi@unipr.it (S. Bonardi).
pathogens that can cause serious disease in humans (Bottone, 2015;
Jalava et al., 2006). They belong to the family Enterobacteriaceae. The
genus Yersinia comprises three pathogenic species which share tropism
for lymphatic tissue: Y. enterocolitica, Y. pseudotuberculosis and Y. pestis,
whose genomes are 97% identical. Nevertheless, severity of the diseases

http://dx.doi.org/10.1016/j.ijfoodmicro.2016.07.033
0168-1605/© 2016 Elsevier B.V. All rights reserved.
126 S. Bonardi et al. / International Journal of Food Microbiology 235 (2016) 125–132
they cause in humans is vastly different (Bergsbaken and Cookson,
2009). Y. enterocolitica is responsible for acute gastroenteritis in
humans, but more invasive syndromes as terminal ileitis, mesenteric
lymphadenitis mimicking appendicitis and septicemia may occur
(Bottone, 1999; Bottone, 2015). Y. pseudotuberculosis causes acute gas-
troenteritis and mesenteric lymphadenitis, whose symptoms of fever
and acute abdominal pain are clinically indistinguishable from those
of an acute appendicitis (Long et al., 2010; Tertti et al., 1984; Tertti et
al., 1989). Y. enterocolitica and Y. pseudotuberculosis postinfection se-
quelae include reactive arthritis, erythema nodosum and glomerulone-
phritis (Bottone, 1999; Hannu et al., 2003; Jalava et al., 2006).
Yersiniosis is the third most common reported zoonoses in the Euro-
pean Union (EU) and European Economic Area (EEA) with 6471 con-
firmed cases in 2013 and a notification rate of 1.92 cases per 100,000
population. Y. enterocolitica was isolated from 98.66% and Y. pseudotu-
berculosis from the 0.94% of the confirmed human cases (EFSA and
ECDC, 2015).
Y. enterocolitica shows large heterogeneity, as it is characterized by
six biotypes (1 A, 1B, 2, 3, 4, 5) (Skurnik et al., 2009) and different sero-
types, with the invasive strains predominantly belonging to serotypes
O:3, O:9, O:5,27 and O:8 (Kirjavainen et al., 2008). The biotypes are
characterized according to their pathogenicity: nonpathogenic biotype
1A, weakly pathogenic biotypes 2–5 and highly pathogenic biotype
1B. Only 1B and 2–5 carry the Yersinia virulence plasmid (pYV)
(Cornelis et al., 1989) and several chromosomal genes that have been
implicated in virulence, such as ail (adhesion and invasion locus), inv
(invasion), and ystA (Yersinia stable toxin A), which allow the bacteria
to penetrate the epithelial layer, elicit an inflammatory response and
evade phagocytosis by neutrophils and macrophages (Revell and
Miller, 2001). Only the biotype 1B harbors the chromosomal high-path-
ogenicity island (HPI), as do almost all European isolates of Y. pseudotu-
berculosis serotype O:1 (Carniel, 1999). The biotype 1A lacks the pYV
plasmid and is generally regarded as avirulent, even if there is some
clinical evidence that some strains can cause food-borne gastroenteritis
(Tennant et al., 2003). Further, Y. enterocolitica biotype 1A may repre-
sent more than one genetic group, with different pathogenic potential
(Sihvonen et al., 2012). Most strains causing yersiniosis in Europe be-
long to the bio-serotypes 4/O:3 and 2/O:9 (EFSA, 2007). Y. enterocolitica
is recovered from farm animals and domestic pets to wild animals
(Wang et al., 2009). Among livestock animals, pigs are regarded to be
the main reservoir of the microorganism, which finds a niche in the
lymphatic tissue and may be shed by the faecal route (Bonardi et al.,
2013; Fredriksson-Ahomaa et al., 2007). Since pig carcass contamina-
tion at slaughter occurs, Yersinia-carrier pigs and Yersinia-positive
farms can be identified before slaughter by serological monitoring of
pigs, which could be a useful, simple and time-saving tool compared
to microbiological examinations (Van Damme et al., 2014).
Y. pseudotuberculosis can be classified into 15 serotypes (O:1–O:15)
and 10 subtypes (O:1a–O:1c, O:2a–O2:c, O:4a–O:4b, O:5a–O:5b)
(Bogdanovich et al., 2003). In Europe most strains belong to the serotypes
O:1–O:3, while serotypes O:4–O:15 are common in Asia (Niskanen et al.,
2009). Most strains are considered pathogenic, containing the 70 kb vir-
ulence plasmid (pYV) and chromosomal genes encoding virulence fac-
tors. Among them, the invasion protein (Inv) encoded by the inv gene
is the most important factor in the early phases of intestinal infection
(Marra and Isberg, 1997), promoting binding to and invasion of the intes-
tinal epithelial cells (Simonet and Falkow, 1992).
Y. pseudotuberculosis has a worldwide distribution and is primarily an
animal pathogen which infects humans rarely (Bergman et al., 2010;
Schiemann, 1989). It can infect many animal species, including pigs,
sheep, goats (Slee and Button, 1990; Toma, 1986), cattle (Toma, 1986;
Welsh and Stair, 1993), deer (Toma, 1986), horses (Czernomysy-
Furowicz, 1997), buffalos (Hum et al., 1997), birds (Wallner-Pendleton
and Cooper, 1983), rabbits and hares (Percy and Barthold, 2007), ham-
sters, guinea pigs and beavers (Percy and Barthold, 2007; Toma, 1986),
bats (Childs-Sanford et al., 2009) and primates (Buhles et al., 1981).
The aims of this study were to estimate the prevalence of Y.
enterocolitica and Y. pseudotuberculosis in tonsils of pigs at slaughter
and to determine the association between cultural detection and posi-
tive Yersinia serology. The ISO 10273:2003 method was compared to di-
rect culture and cold enrichment to evaluate its performance. Virulence
of the isolates was studied, in order to identify pathogenicity of the
strains circulating among pigs in Italy. The antimicrobial resistance of
the isolates was tested in accordance with Directive 2003/99/EC on zoo-
nosis and zoonotic agents (Anonymous, 2003).
2. Materials and methods
From June 2013 to October 2014, 201 pigs were randomly selected at
slaughter to be tested for Y. enterocolitica and Y. pseudotuberculosis in
tonsils. Tonsils were aseptically excised after carcass evisceration and
placed in sterile bags. From each carcass, a 10 g sample of diaphragm
muscle was collected and placed in sterile containers. The samples
were collected from 67 batches of pigs (three pigs per batch) at one
slaughter plant processing 280 pigs/h during 33 sampling visits. The
batch definition used was a group of pigs coming from a single farm in
a given day. The pigs were reared in 61 farms located in four regions
of Northern Italy (Lombardy, Emilia-Romagna, Piedmont and Veneto re-
gions); 12 farms were farrow-to-finish and 49 were finishing farms.
Sampled pigs were minimum nine months old with an average live
weight of 160 kg (Landrace, Large White, Duroc and their hybrids) as re-
quired by DOP (Protected Designation of Origin) Italian pork products,
such as Parma Ham and traditional dry-cured salami. All samples were
transported to the laboratory at refrigeration conditions and tested on
the day of collection.
2.1. Yersinia enterocolitica detection, enumeration and typing
Tonsils were tested for Y. enterocolitica following the ISO
10273:2003 (International Organization for Standardization, 2003)
method and by direct plating. Enumeration of the microorganism was
performed by the direct plating method.
Tonsils were washed by pouring sterile water before being aseptical-
ly cut into small pieces. A10 g aliquot of tonsils was suspended 1:10 in
Phosphate Buffered Saline added with 2% sorbitol and 1.5% bile salts
(PSB; Biolife Italiana, Milan, Italy) and homogenized for 4 min in a Stom-
acher blender (Van Damme et al., 2010). From the 1:10 PSB initial sus-
pension, testing was carried out as follows.
i) Enrichment following ISO 10273:2003: a) The 1:10 PSB suspen-
sion was incubated at 25 ± 1 °C for 2 days. Thereafter, 10 μl were
streaked onto CIN agar plates, which were incubated at 30 ± 1 °C
for 48 h. In parallel, the enriched PSB cultures were treated with
alkali before plating onto CIN agar, mixing 0.5 ml of the broth cul-
ture with 4.5 ml of 0.5% potassium hydroxide (KOH) solution for
20 s. After mixing, 10 μl of the alkali treated cultures were plated
onto CIN agar plates, incubated at 30 ± 1 °C for 48 h. b) 10 ml of
the initial 1:10 PSB suspension were transferred to 90 ml of
Irgasan-Ticarcillin-Potassium Chlorate broth (ITC broth; Biolife)
and incubated at 25 ± 1 °C for 48 h. Thereafter, 10 μl were streaked
onto CIN agar plates, which were incubated at 30 ± 1 °C for 48 h.
CIN agar was the medium of choice, instead of Salmonella – Shigella
agar with sodium desoxicholate and calcium chloride (SSDC) rec-
ommended by the ISO method, because of its higher sensitivity
(Bonardi et al., 2014). Flat, not mould colonies with entire edge
having a red centre (“bull's-eye”) surrounded by a translucent,
transparent or milk-white zone were considered suspect Y.
enterocolitica colonies. Five to ten characteristic colonies were se-
lected for biochemical confirmation, or all colonies if less than
five were present.
 S. Bonardi et al. / International Journal of Food Microbiology 235 (2016) 125–132
ii) Direct plating and enumeration: after resuscitation for 2 h at room
temperature, 50 μl aliquots of the initial 1:10 PSB suspension were
plated onto two plates of Cefsulodin-Irgasan-Novobiocin Yersinia-
Selective agar (CIN agar; Oxoid, Basingstoke, UK) and incubated at
30 ± 1 °C for 48 h. The number of suspect colonies was counted
and five colonies per plate were selected for biochemical confirma-
tion, or all colonies if less than five were present.
Before biochemical tests, the suspect colonies were streaked onto
Tryptone Soy Agar (TSA, Oxoid) to obtain pure cultures and incubated
at 30 °C ± 1 °C for 48 h. Preliminary identification was performed by
seeding pure colonies in Kligler Iron agar (Biolife) and Christensen's
Urea agar (Biolife) incubated at 25 ± 1 °C for 24 h. Lactose-negative
and urease-positive colonies were further tested for melibiose, sorbitol,
sucrose and rhamnose fermentation at 25 ± 1 °C for 24 h (Fredriksson-
Ahomaa and Simjee, 2007). Y. enterocolitica species identification was
performed using the Microgen™ GN-ID A microsubstrate system
(Microgen Bioproducts, Camberley, UK) incubated at 25 ± 1 °C for
48 h. Y. enterocolitica biotyping was carried out according to the modi-
fied Wauter's scheme (Bottone, 1999). Serotyping was performed by
slide agglutination with commercially available O-antisera for the
serogroups O:1–2, O:3, O:5, O:8 and O:9 (Yersinia enterocolitica Antisera
Set® - 293,756, Denka Seiken, Japan). O-untypeable isolates were tested
by the CNR des Yersinia, Institut Pasteur, Paris, France.
2.2. Y. pseudotuberculosis detection, enumeration and typing
Tonsils were tested for Y. pseudotuberculosis by direct plating and
cold enrichment. Tonsils were washed with sterile water before being
aseptically cut into small pieces. A 1:10 suspension was obtained dilut-
ing 10 g of tonsils in 90 ml of Phosphate Buffered Saline (Oxoid) added
with 1% mannitol (Applichem, Darmstadt, Germany) and 0.15% bile
salts (Oxoid) (PMB). The suspension was homogenized for 4 min in a
Stomacher blender.
i) Direct plating and enumeration: After resuscitation for 2 h at room
temperature, 50 μl aliquots were plated onto two plates of
Cefsulodin-Irgasan-Novobiocin Yersinia-Selective agar (CIN agar;
Oxoid) and incubated at 30 °C for 18–20 h and then at 22 °C for fur-
ther 24 h. Suspect colonies on CIN agar are small, dark red, with
“bull's eye” appearance; they are smaller than Y. enterocolitica colo-
nies, which grow faster on CIN agar medium (Niskanen et al.,
2002). Colonies resembling Y. pseudotuberculosis were counted and
five colonies per plate were selected for biochemical confirmation,
or all colonies if less than five were present.
ii) Cold enrichment: the PMB homogenates were incubated at 4 °C
for 7, 14, and 21 days. Alkali treatment (0.5 ml of the sample was
mixed with 4.5 ml of 0.25% KOH solution for 20 s) was used after
cold enrichment before cultures were streaked onto CIN agar plates.
Inoculated CIN agar plates were incubated at 30 °C for 18–20 h and
then at 22 °C for further 24 h. Five to ten suspect colonies were se-
lected for biochemical confirmation, or all colonies if less than five
were present.
Before biochemical tests, the suspect colonies were streaked onto
TSA to obtain pure cultures and incubated at 30 °C ± 1 °C for 48 h. Pre-
liminary identification was performed by seeding pure colonies in
Kligler Iron agar (Biolife) and Christensen's Urea agar (Biolife) incubat-
ed at 25 ± 1 °C for 24 h. Lactose-negative and urease-positive colonies
were tested for melibiose, sorbitol, sucrose and rhamnose fermentation
at 25 ± 1 °C for 24 h (Fredriksson-Ahomaa and Simjee, 2007). Species
identification was performed using the Microgen™ GN-ID A and B
microsubstrate systems (Microgen Bioproducts) incubated at 25 ± 1 °
C for 48 h. Y. pseudotuberculosis serotyping was carried out by the Centre
National de Référence des Yersinia, Institut Pasteur (Paris, France).
127
2.3. Detection of Y. enterocolitica and Y. pseudotuberculosis virulence genes
To identify human pathogenic Y. enterocolitica strains, PCR assays
targeting the virF and yadA genes on the plasmid pYV (plasmid for Yersinia
virulence) and the ail and ystA genes on the chromosome were performed
following Thoerner et al. (2003). To prevent plasmid loss due to
subculturing, isolates were subcultured not more than twice. The strains
used as positive controls were: i) virF, yadA genes: Y. enterocolitica
bioserotype 4/O:3, code CIP-864 (Centre National de Référence des Yersinia,
Institute Pasteur, Paris, France); ii) ail and ystA gene: Y. enterocolitica
bioserotype O:5,27, code 837 (Institute of Public Health, Oslo, Norway).
Presence of virulence genes in Y. pseudotuberculosis isolates was de-
termined targeting the virF plasmidic gene and the inv chomosomic
gene (Thoerner et al., 2003). The strain Y. pseudotuberculosis IP32953
(CNR des Yersinia, Institute Pasteur, Paris, France) was used as positive
control.
2.4. Serological analysis
Diaphragm muscle samples were frozen at −18 °C on arrival at the
laboratory, stored for 24 h and thawed at 4 °C for further 24 h. After
thawing, the meat juice was collected and stored at −18 °C until analysis.
A total of 196 meat juice samples were tested, as 5 were discharged be-
cause their quantity was too little to be analysed. Overall, 66 pig batches
were tested, as from one batch no meat juice samples could be analysed.
Samples were tested for antibodies against pathogenic Yersinia using the
commercially available indirect enzyme-linked immunosorbent assay
(ELISA) pigtype Yersinia Ab (Qiagen, Leipzig, Germany) following the
manufacturer's instructions. The presence of antibodies against Yops
(Yersinia outer proteins) of plasmid-bearing Yersinia was determined by
measuring the optical density (OD) at 450 nm. The ratio of sample OD to
mean OD of the positive control (S/P) was calculated using the mean OD
values of positive and negative controls (ODpos and ODneg, respectively):
ODsample − ODneg
S=P ¼
ODpos −ODneg
As recommended by the manufacturer, samples with S/P-ratio ≥ 0.3
were considered positive.
2.5. Resistance to antimicrobial agents
Y. enterocolitica and Y. pseudotuberculosis isolates were tested for anti-
microbial resistance by the Kirby Bauer disc-diffusion method according
to the recommendations of the Clinical and Laboratory Standards Institute
(CLSI, 2009). Mueller-Hinton agar (Oxoid) and commercial antimicrobial
susceptibility discs (Oxoid) were used. Incubation was performed at
30 ± 2 °C for 16 to 18 h. Considering their clinical use, Y. enterocolitica
strains were tested for the following 15 antimicrobial agents: ampicillin
(A, 10 μg), amoxicillin and clavulanic acid (Amc, 20/10 μg), cefotaxime
(Ctx, 30 μg), ceftazidime (Caz, 30 μg), chloramphenicol (C, 30 μg), cipro-
floxacin (Cip, 5 μg), ertapenem (Etp,10 μg), gentamicin (Cn, 10 μg), kana-
mycin (K, 30 μg), meropenem (Mem, 10 μg), nalidixic acid (Nx, 30 μg),
streptomycin (S, 10 μg), sulphonamide (Su, 300 μg), tetracycline (T,
30 μg), and trimethoprim/sulfamethoxazole (Sxt, 1.25/23.75 μg).
Y. pseudotuberculosis strains were tested for sensitivity to 12 antimi-
crobials by the CNR des Yersinia, Institut Pasteur: amoxicillin (Amx,
25 μg), amoxicillin and clavulanic acid (Amc, 20/10 μg), azithromycin
(Azm, 15 μg), cephalothin (Cf, 30 μg), cefoxitin (Fox, 30 μg), ceftriaxone
(Cro, 30 μg), ciprofloxacin (Cip, 5 μg), nalidixic acid (Nx, 30 μg),
sulphonamide (Su, 200 μg), tetracycline (T, 30 μg), ticarcillin (Tic,
75 μg) trimethoprim (Tmp, 5 μg).
Escherichia coli ATCC 25922 was used as quality control organisms.
Susceptibility results were categorized as susceptible, intermediate
or resistant according to Clinical and Laboratory Standards Institute
guidelines (CLSI, 2012).
128 S. Bonardi et al. / International Journal of Food Microbiology 235 (2016) 125–132
2.6. Statistical analyses
Differences in Y. enterocolitica prevalence between farm types (far-
row-to-finish versus finishing farms) and seasons (warm versus cold)
and the association between serological results (positive/negative)
and the presence of Y. enterocolitica in the tonsils were analysed using
logistic regressions, including farm as random effect whenever neces-
sary. All analyses were performed using STATA/MP 12.1 (StataCorp,
2011).
3. Results
3.1. Y. enterocolitica detection, enumeration and typing
Yersinia enterocolitica was detected in 55/201 pig tonsils (27.4%; 95%
CI 23.1–37.1). All isolates were typed as biotype 4 serotype O:3. The pos-
itive pigs came from 38/67 batches (56.7%) and were reared in 36/61
farms (59.0%). Y. enterocolitica-positive pigs came from 29/49 (59.2%)
finishing farms and from 7/12 (58.3%) farrow-to-finish farms. The pro-
portion of Y. enterocolitica positive pigs from finishing farms (27.8%)
was not significantly different from farrow-to-finish farms (25.6%)
(p = 0.788).
The seasonal trend in the carriage rate of Y. enterocolitica was evalu-
ated. In one year of sampling (June 2013–May 2014), Y. enterocolitica
was more frequently detected in tonsils collected during colder months
(November–April; 31.0%) compared to the warm season (June–Octo-
ber; 24.8%), though the difference was not significant (p = 0.334).
The efficiencies of Y. enterocolitica isolation procedures is shown in
Table 1. Cold enrichment in PMB was performed to detect Y. pseudotu-
berculosis, but several samples were found to be positive for Y.
enterocolitica by this method. For this reason, it is mentioned in Table
1. Overall, the ISO 10273:2003 method, direct plating and cold enrich-
ment detected 55 positive samples. None of the isolation procedures de-
tected all of the positive samples and none of the positive samples were
detected by each of the detection procedures. Thirteen samples (23.6%)
were found to be positive by the ISO 10273:2003 method, 31 (56.4%) by
direct plating, and 28 (50.9%) by cold enrichment. Among the 28 cold
enrichment-positive samples, 15 (53.6%) were found positive after
14 days of incubation at 4 °C, 9 (32.1%) after 21 days and 4 (14.3%)
after 7 days. In particular, cold enrichment in PMB detected 16 addition-
al positive samples, which resulted negative by the ISO method and di-
rect plating, and the ISO method found 6 positive samples, which
resulted negative by direct plating and cold enrichment. Overall, the
ISO 10273:2003 method recovered significantly less positive samples
compared to direct plating (p = 0.008) and cold enrichment (p =
0.027). No significant difference between cold enrichment and direct
plating was observed.
Direct plating detected 31 positive samples; 30 samples were found
to be positive by the protocol specifically used for Y. enterocolitica (sam-
ple dilution 1:10 in PSB, plating onto CIN agar) and one more was found
to be positive by the protocol specifically used for Y. pseudotuberculosis
(sample dilution 1:10 in PMB, plating onto CIN agar). The mean count
of Y. enterocolitica was 3.56 ± 0.85 log10 CFU/g (n = 31) with a mini-
mum of 2.0 log10 CFU/g and a maximum of 4.78 log10 CFU/g. The
Table 1
Number of positive samples and prevalence of positive results based on the detection
method for Y. enterocolitica and Y. pseudotuberculosis in pig tonsils.
Cultural method Y. enterocolitica Y. pseudotuberculosis
4/O:3 (%) O:1 and O:3 (%)
(N = 55) (N = 4)
Direct plating 31 (56.4%) 1 (25.0%)
ISO 10273:2003 13 (23.6%) NT
Cold enrichment in PMB 28 (50.9%) 4 (100%)
NT = not tested.
mean count was calculated without including negative samples (b-
2.0 log10 CFU/g).
3.2. Y. pseudotuberculosis detection, enumeration and typing
Y. pseudotuberculosis was isolated from 4/201 pig tonsils (2.0%; 95%
CI 0.0–4.5). Three isolates belonged to serotype O:3 and one to serotype
O:1. The positive pigs belonged to 4/67 batches (6.0%) and came from 3/
61 farms (4.9%). Y. pseudotuberculosis carriers were reared in finishing
farms only (3 out of 49 farms; 6.1%). In one (2.0%) finishing farm Y.
enterocolitica and Y. pseudotuberculosis were simultaneously present. Y.
pseudotuberculosis could be enumerated only in one sample with
4.27 log10 CFU/g.
3.3. Virulence genes detection
3.3.1. Y. enterocolitica
The distribution of virulence genes among Y. enterocolitica 4/O:3 iso-
lates was the following: 56.4% (31/55) for the yadA gene, 56.4% (31/55)
for the virF gene, 98.2% (54/55) for the ail gene, 98.2% (54/55) for the
ystA gene. One isolate (1.2%) was negative for all the genes tested. Dis-
tribution of virulence genes among isolates is shown in Table 2.
3.4. Y. pseudotuberculosis
All Y. pseudotuberculosis isolates were positive for the inv gene
(100%). The O:1 isolate and two out of three O:3 isolates were positive
for all the genes tested (inv, virF and yadA), while one O:3 isolate was
positive for the inv gene only (Table 2).
3.5. Serological test
The ELISA test demonstrated that 110 meat juice samples out of 196
(56.1%) were positive for Yersinia antibodies. Serological positivity was
found in 67.9% (36/53) of the Y. enterocolitica- and 75.0% (3/4) of the
Y. pseudotuberculosis-positive pigs (Table 3). A significant positive asso-
ciation was found between serological results and the presence of Y.
enterocolitica in the tonsils (OR = 1.97, p = 0.044). Seventeen Y.
enterocolitica- and one Y. pseudotuberculosis-positive pigs tested nega-
tive for Yersinia antibodies. In 48 out of 61 farms (78.7%), at least one se-
ropositive animal was detected. At batch level, this was in 52 out of 66
batches (78.8%). In 22 batches all of the tested animals were seroposi-
tive (Table 4).
3.6. Antimicrobial resistance
3.6.1. Y. enterocolitica
None of the isolates was susceptible to all the antimicrobials tested.
All the isolates were susceptible to amoxicillin-clavulanic acid,
Table 2
Distribution of virulence genes in Y. enterocolitica (bioserotype 4/O:3) and Y.
pseudotuberculosis (serotype O:1 and O:3) isolated from pigs. The gene inv
was not tested in Y. enterocolitica strains, and the gene ail was not tested in
Y. pseudotuberculosis strains.
Gene pattern Number of isolates (%)
Y. enterocolitica 4/O:3 (n = 55)
ail, yadA, virF, ystA 30 (54.5%)
ail, ystA 22 (40.0%)
ail, virF, ystA 1 (1.8%)
ail, yadA, ystA 1 (1.8%)
– 1 (1.8%)
Y. pseudotuberculosis (n = 4)
yadA, virF, inv 3 (75%)a
inv 1 (25%)
a
2 isolates of serotype O:3 and one isolate of serotype O:1.
 S. Bonardi et al. / International Journal of Food Microbiology 235 (2016) 125–132 129

Table 3 Table 5
Comparison between ELISA serological test and detection of Y. enterocolitica and Y. pseudo- Resistance, sensitivity or intermediate sensitivity in 55 Y. enterocolitica 4/O:3 strains isolat-
tuberculosis in pigs at slaughter. ed from pigs.

Serology Y. enterocolitica Y. pseudotuberculosis Antimicrobial Sensitive (%) Intermediate (%) Resistant (%)
(ELISA test result)
Culture Culture Culture Culture Total A 55 (100%)
negative positive negative positive AMC 55 (100%)
CTX 52 (94.5%) 3 (5.5%)
Negative 69 17 85 1 86
CAZ 55 (100%)
Positive 74 36 107 3 110
C 26 (47.3%) 29 (52.7%)
Total 143 53 192 4 196
CIP 7 (12.7%) 38 (69.1%) 10 (18.2%)
ETP 55 (100%)
CN 55 (100%)
gentamicin, ceftazidime, ertapenem and meropenem, 94.5% to cefotax- K 49 (89.1%) 6 (10.9%)
MEM 55 (100%)
ime, 89.1% to kanamycin and 78.2% to tetracycline (Table 5).
NX 23 (41.8%) 5 (9.1%) 27 (49.1%)
Twenty-one resistance patterns were observed, with one to eight re- S 3 (5.5%) 9 (16.4%) 43 (78.2%)
sistances (Table 6). The highest resistance rates were observed for am- SU 1 (1.8%) 54 (98.2%)
picillin (100%), sulphonamides (98.2%) and streptomycin (78.2%). Also T 43 (78.2%) 1 (1.8%) 11 (20.0%)
SXT 26 (47.3%) 15 (27.3%) 14 (25.4%)
resistance rates for chloramphenicol (52.7%) and nalidixic acid (49.1%)
were very common. Ampicillin resistance was associated with strepto- Legend: Ampicillin (A), amoxicillin and clavulanic acid (Amc), cefotaxime (Ctx), ceftazi-
mycin and sulfonamides resistance in 78% (43/55) of the isolates. The dime (Caz), chloramphenicol (C), ciprofloxacin (Cip), ertapenem (Etp), gentamicin (Cn),
kanamycin (K), meropenem (Mem), nalidixic acid (Nx), streptomycin (S), sulphonamide
percentage of isolates resistant to between five to eight antimicrobials (Su), tetracycline (T), trimethoprim/sulfamethoxazole (Sxt).
was 56% (31/55).
Y. pseudotuberculosis–Y. pseudotuberculosis serotypes O:3 and O:1
were susceptible to all the antimicrobials tested. England, where bioserotypes 2/O:9 and 2/O:5 are the most frequently
reported (Ortiz Martínez et al., 2010).
4. Discussion In our study, the ISO 10273:2003 method underestimated the num-
ber of positive tonsils, thus in agreement with Van Damme et al. (2013).
4.1. Y. enterocolitica The most sensitive method was the direct plating on CIN agar
confirming previous findings (Bonardi et al., 2014). Its higher recovery
The present study shows that Italian fattening pigs are frequently in- rate compared to the ISO method could be explained by the nature of
fected with human pathogenic Y. enterocolitica 4/O:3, although the iso- the samples (fresh pig tonsils), in which the microorganism is not dam-
lation rate is slightly lower than in other European countries. Overall, aged by heat/cold treatments, fermentation or preservatives and can
27.4% of nine-months old fattening pigs were positive for Y. grow without requiring enrichment. According to our results, the best
enterocolitica 4/O:3 in tonsils, thus showing higher prevalence if com- combination of cultural methods to isolate human pathogenic Y.
pared with previous data (Bonardi et al., 2003; Bonardi et al., 2013; enterocolitica 4/O:3 from pig tonsils was direct plating on CIN agar and
Bonardi et al., 2014). This could be attributed to a different analytical ap- cold enrichment in PMB. These two methods together gave a positive
proach (10 g of analysed tonsils vs 1 g; misuse vs use of the cold enrich- rate of 89.1% (49/55 positive tonsils). The tonsils positive by the ISO
ment procedure) or to some unknown changes in the pig population 10273:2003 method or cold enrichment, but negative by direct plating,
infectious status. Prevalence of pathogenic Y. enterocolitica in finishing were probably contaminated by low levels of microorganisms, i.e. below
pigs is normally higher in other EU countries, as Belgium (Van Damme the minimum countable value of 2.0 log10 CFU/g.
et al., 2014), Germany (Bucher et al., 2008), England (Ortiz Martínez
et al., 2010) and Switzerland (Fredriksson-Ahomaa et al., 2007). At
Table 6
slaughter, high numbers of infected pigs may represent a potential
Resistance pattern (R-type) of 55 Y. enterocolitica 4/O:3 isolates of porcine origin.
risk for public health due to cross-contamination of carcasses
(Nesbakken et al., 2003). R-Type N° of resistances N° strains
(%)
In our study most batches (56.7%) were infected, which is in accor-
dance with other authors (Fondrevez et al., 2014; Vanantwerpen et al., A 1 1 (1.8%)
2014). Not surprisingly, the bioserotype 4/O:3 was the only one detect- ASu 2 10 (18.2%)
ASuT 3 1 (1.8%)
ed in pig tonsils, as found by Vanantwerpen et al. (2014) in Belgium and ANxSSu 3 1 (1.8%)
Martínez et al. (2009) in Latvia, Estonia and the Leningrad region of Rus- ACSSu 4 4 (7.3%)
sia. When not the only one, the bioserotype 4/O:3 is the most prevalent ACtxSSu 4 1 (1.8%)
in porcine tonsils (Fondrevez et al., 2014; Fredriksson-Ahomaa et al., ANxSSu 4 1 (1.8%)
ASSuSxt 4 4 (7.3%)
2011; Martínez et al., 2011). The only exception is represented by
ASSuT 4 1 (1.8%)
ACNxSSu 5 8 (14.5%)
ACSSuSxt 5 3 (5.5%)
Table 4 ACSSuT 5 2 (3.6%)
Number of seropositive animals within a batch according to the culture-based Yersinia sta- ANxSSuSxt 5 2 (3.6%)
tus of the batch (n = 66). ANxSSuT 5 3 (5.5%)
ACCipNxSSu 6 6 (10.9%)
Number of seropositive Yersinia negative Yersinia positive All batches
ACCtxSSuSxt 6 1 (1.8%)
animals within the batch batchesa batchesa
ACNxSSuSxt 6 1 (1.8%)
0/3 6 8b 14 ACipNxSSuT 6 1 (1.8%)
1/3 10 6 16 ANxSSuSxtT 6 1 (1.8%)
2/3 5 9b 14 ACCipNxSSuSxt 7 2 (3.6%)
3/3 6 16 22 ACCipCtxNxSSuT 8 1 (1.8%)
Total 27 39 66 Total 55 (100%)
a
A batch was considered positive if Y. enterocolitica and/or Y. pseudotuberculosis was Legend: Ampicillin (A), cefotaxime (Ctx), chloramphenicol (C), ciprofloxacin (Cip), kana-
cultured from at least one out of three pig tonsils from that batch. mycin (K), nalidixic acid (Nx), streptomycin (S), sulphonamide (Su), tetracycline (T), tri-
b
From one batch only two meat juice samples were tested. methoprim/sulfamethoxazole (Sxt).
130 S. Bonardi et al. / International Journal of Food Microbiology 235 (2016) 125–132
The bacterial load of Y. enterocolitica 4/O:3 in tonsils of finishing pigs
was consistent with other studies (Bonardi et al., 2014; Vanantwerpen
et al., 2014; Van Damme et al., 2010) ranging from a minimum of
2.0 log10 CFU/g to a maximum of 4.78 log10 CFU/g thus confirming a
risk for carcasses, offals and fresh pig meat.
A very important finding of this study was the higher seroprevalence
(56.1%) on meat juice compared to cultural detection in tonsils (27.5%).
This discrepancy can be attributed to infections that resulted in the pro-
duction of antibodies, after which the microorganisms were cleared
from the hosts. Therefore, microbiological tests of tonsils at slaughter
cannot exclude a historical contact with enteropathogenic Yersinia
spp., as pigs may not remain life-long carriers in their tonsils (Nielsen
et al., 1996). Moreover, in 9.2% of pigs in the present study enteropatho-
genic Yersinia spp. were detected in the tonsils while the pigs were se-
ronegative, which may indicate a recent infection on farm, during
transport or lairage at the slaughterhouse. The serological results dem-
onstrated that Yersinia spp. infections are more common in the Italian
pig population than it could be evaluated by the isolation of the bacteria
at the slaughter age. The seroprevalence is similar to other countries, as
57% found in Finnish and German pigs at slaughter (Felin et al., 2014;
Meemken et al., 2014) and 65% in Belgian pigs at slaughter (Van
Damme et al., 2014), thus canceling the difference in cultural isolation
between Italy and other European countries.
In Northern Italy slaughter age of pigs is minimum 9 months, i.e.
higher if compared to pigs' age in most European countries.
Nesbakken et al. (2006) observed a decrease in the isolation rate of Y.
enterocolitica from tonsils with increasing age whereas the seropreva-
lence remained high until the pigs were approximately 5 months old.
Similarly, Vilar et al. (2013) observed the highest seroprevalence in fat-
tening pigs that were 5 months or older, whereas faecal excretion al-
ready decreased in the age group of 3-month-old pigs. Therefore, the
high seroprevalence found in our study suggests a widespread distribu-
tion of Yersinia spp. in Italian pig farms and the lower number of pigs
carrying the pathogens in the tonsils might be related to their older
slaughter age.
Y. enterocolitica 4/O:3 detected in swine showed very high levels of
antimicrobial resistance, with N50% of the isolates resistant to five to
eight antimicrobial agents and no isolates susceptible to all the antimi-
crobials tested. Comparing these data with previous ones (Bonardi et al.,
2014), we observed an increased resistance to nalidixic acid and cipro-
floxacin and high levels of intermediate sensitivity to ciprofloxacin. The
increasing resistance/intermediate sensitivity to ciprofloxacin is of con-
cern, because it has been one of the most important antimicrobials used
in Y. enterocolitica osteomyelitis and septic arthritis (Carniel et al.,
2006). High levels of resistance to β-lactam antibiotics, as ampicillin
and first generation cephalosporins, are due to β-lactamase production
by Y. enterocolitica O:3 and are well documented in literature (Bhaduri
et al., 2009; Fredriksson-Ahomaa et al., 2007). The resistant rate to
chloramphenicol was very similar to previous findings, possibly due to
coresistance to thiamphenicol (Bonardi et al., 2014). All the isolates
were sensitive to the carpabenems ertapenem and meropenem, which
have been increasingly used in humans, particularly in severe diseases
as septicemia associated with spleen and liver abscesses (Benbrika et
al., 2005; Grigull et al., 2005).
Since enteropathogenic Y. enterocolitica is widespread among pigs at
slaughter, pig meat and especially raw pork and short-cured products
could be a source of infection for Italian consumers. Furthermore, its
ability to grow at low temperature provides the microorganism an op-
portunity to multiply during storage in refrigerated food, even if vacu-
um-packed (Doherty et al., 1995). The paucity of data of human
yersiniosis cases in Italy should be probably attributed to the absence
of mandatory surveillance at national level. Microbiological investiga-
tions in case of enteric symptoms linked to the consumption of pork
and products thereof could help in discovering this “hidden zoonotic
disease”, which could be not so rare in a country where raw or
undercooked pork products are frequently consumed.
4.2. Y. pseudotuberculosis
The detection of Y. pseudotuberculosis in pig tonsils is of concern, as
tonsils may represent a contamination source for pig meat at slaughter
(Van Damme et al., 2015). Even if the prevalence of Y. pseudotuberculosis
was low (2.0%) and the enumeration was 4.27 log10 CFU/g in one sam-
ple only, being b2.0 log10 CFU/g in the remaining ones, we agree with
Laukkanen et al. (2008). that the pathogen may enter the food chain
via slaughtering of infected pigs.
Prevalence of Y. pseudotuberculosis in pig tonsils at slaughter was in-
vestigated in other countries. It ranged from 18% in Great Britain (Ortiz
Martínez et al., 2010), to 7% in Russia (Martínez et al., 2009), 5% to 3% in
Latvia (Martínez et al., 2009; Terentjeva and Bĕrzinš, 2010), 2% to 1.4%
in Belgium (Martínez et al., 2011; Van Damme et al., 2015) and 1% in Es-
tonia (Martínez et al., 2009). The variations in prevalence among coun-
tries can be due to the epidemiology of Y. pseudotuberculosis in areas
with different farm types (free-range or conventional; farrow-to-finish
or finishing; high or low production; high or low biosecurity) and pop-
ulated by different wild animals, which can act or not as natural reser-
voir of the microorganism and influence its transmission to pigs.
In this study pathogenic inv-positive Y. pseudotuberculosis belonging
to serotypes O:1 and O:3 were identified. Among EU countries, Finland
identified several outbreaks caused by Y. pseudotuberculosis O:1 and
O:3. In 2014 Y. pseudotuberculosis O:1 infection was associated with
the consumption of raw milk from a single producer (Pärn et al.,
2015), in 2006 it was linked to raw grated carrots and Y. pseudotubercu-
losis O:1 was isolated from environmental samples collected at the car-
rot processing plant, from spoiled carrots and from intestines of
common shrew (Sorex araneus) living in one of the two farms of origin
of the carrots (Kangas et al., 2008). During 2001, multiple outbreaks
caused by Y. pseudotuberculosis O:1 (68%) and O:3 (32%) were identi-
fied, but the sources of infections remained unknown (Jalava et al.,
2004). In 1998, a nationwide outbreak by Y. pseudotuberculosis O:3
was linked to iceberg lettuce probably contaminated with faeces of
roe deer (Capreolus capreolus) (Nuorti et al., 2004), which may act as
reservoir of the bacterium (Sanford, 1995).
Y. pseudotuberculosis can survive outside the host and grow at tem-
peratures as low as 4 °C, thus assuring its presence in the environment
(Bergman et al., 2010). As the microorganism can survive in surface
water (Fukushima et al., 1988), it is conceivable that wild animals
may contribute to the contamination of fresh vegetables by faecal con-
tamination of water sources or cultivated areas. Among reservoirs, an
important role could be attributable to wild boars, which are increasing
and spreading to several European areas. Both Y. enterocolitica and Y.
pseudotuberculosis were isolated from wild boars
(Fredriksson-Ahomaa et al., 2011; Sannö et al., 2014), which could
carry strains transmissible to humans.
In our study Y. pseudotuberculosis was isolated from pigs reared in
finishing farms only. This could be attributable to the periodic incoming
of piglets from farms characterized by different management and
biosecurity systems. On the contrary, management and biosecurity
can be better monitored in farrow-to-finish farms. Integrated (farrow-
to-finish) farms may therefore represent a protective factor. The farms
of origin of the pigs tested in this study were conventional farms with
high production capacity. Some authors compared production types
and capacities, finding that the prevalence was higher in organic pro-
duction than in conventional production and in conventional farms
with high rather than low production capacity (Laukkanen et al., 2008).
Antimicrobial resistance is generally less common in Y. pseudotuber-
culosis isolates than in Y. enterocolitica. A study by Bonke et al. (2011)
demonstrated that Y. pseudotuberculosis isolates bioserotypes 1/O:1, 1/
O:2 and 2/O:1 from human and animal sources, including pigs, were
susceptible to 12 antimicrobials (ampicillin, cefotaxime, chlorampheni-
col, ciprofloxacin, florfenicol, gentamicin, kanamycin, nalidixic acid,
streptomycin, sulfamethoxazole, tetracycline and trimethoprim) and
did not carry β-lactamase blaA and blaB genes. In contrast, 99% of Y.
 S. Bonardi et al. / International Journal of Food Microbiology 235 (2016) 125–132
enterocolitica isolates carried both the β-lactamase genes and 89% of the
strains expressed the β-lactamase enzymes. In Latvia, Y. pseudotubercu-
losis strains isolated from slaughter pigs exhibited resistance to erythro-
mycin and sulfamethoxazole but were sensitive to ampicillin,
amoxicillin-clavulanic acid, aztreonam, cefotaxime, ceftriaxone, cepha-
lothin, chloramphenicol, ciprofloxacin, nalidixic acid, streptomycin, tet-
racycline, trimethoprim and trimethoprim-sulfamethoxazole
(Terentjeva and Bĕrzinš, 2010). The isolates tested in our study were
sensitive to all the antimicrobials tested showing that Y. pseudotubercu-
losis is far to acquire antimicrobial resistance. This is probably due to the
wild nature of the pathogen's hosts, which did not receive antimicrobial
treatments, and to the few contacts of the microorganism with the pig
population.
Y. pseudotuberculosis infections are not routinely reported in Italy.
Only in a few countries, as Finland, Y. pseudotuberculosis infection is a
notifiable disease (Nuorti et al., 2004; Pärn et al., 2015) and therefore
small outbreaks and/or single cases of infections remain unknown in
most European states, including Italy. In our country, roe deer popula-
tion has been increasing in the last decade and consequently roe deer
can be easily found in cultivated area not protected by fences. Besides
them, the large number of wild animal reservoirs (such as rodents,
hares, wild boars) may contribute to the contamination of fresh vegeta-
bles, as suggested by the trace-back environmental studies conducted in
Finland by Nuorti et al. (2004) and Kangas et al. (2008). Furthermore,
the large number of raw RTE pork products commonly eaten after a
short curing period (like sausages or short-ripened salami) could con-
tribute to human infections. As some patients may show severe symp-
toms of bacteriemia, undergo appendectomy or suffer from chronic
sequelae, testing stool specimens for Y. pseudotuberculosis in patients
with febrile gastroenteritis and abdominal pain should be encouraged.
Acknowledgements
The study was funded by Fondazione Cariparma, Parma, Italy (Pro-
ject No 2013.0237).
The authors kindly acknowledge Dr. Gisella Pizzin, Mrs. Ida Poli and
Mrs. Giuseppina Trentadue (Department of Veterinary Science, Univer-
sity of Parma) for technical assistance.
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