Food Microbiology 73 (2018) 85e92

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Food Microbiology
journal homepage: www.elsevier.com/locate/fm

Development of a rapid method for the detection of Yersinia

enterocolitica serotype O:8 from food
Mirella Luciani a, Maria Schirone b, *, Ottavio Portanti a, Pierina Visciano b,
Gisella Armillotta a, Rosanna Tofalo b, Giovanna Suzzi b, Luigia Sonsini a, Tiziana Di Febo a
a
Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, 64100, Teramo, Italy
b
Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, 64100, Teramo, Italy

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

Article history: In this study, a new and alternative method based on monoclonal antibodies (MAbs) for the rapid
Available online 16 January 2018 detection of Yersinia enterocolitica O:8 was developed. This microorganism is an emerging foodborne
pathogen causing gastrointestinal disease in humans. The transmission can occur through contaminated
Keywords: food such as raw or undercooked meat, milk and dairy products, water and fresh vegetables. Nine MAbs
Yersinia enterocolitica (46F7, 54B11, 54C11, 62D10, 64C7, 64C10, 72E8, 72E10, 72G6) were characterized and selected versus Y.
Monoclonal antibodies
enterocolitica O:8, and only 2 of them showed also a weak cross-reaction with Campylobacter jejuni. The
Capture-ELISA
MAb 54B11 was used for the development of Y. enterocolitica capture-ELISA in food matrices, i.e. meat
Food
and dairy products (n ¼ 132). The method was validated by ISO 16140:2003 and compared with the
official method for the detection of presumptive pathogenic Y. enterocolitica (ISO 10273:2003). Relative
accuracy, sensitivity and specificity corresponded to 100%. The selectivity was evaluated on other food
samples (n ¼ 126) showing a lower confidence limit of 90.3% and an upper confidence limit of 100%. The
results from this study demonstrated that the developed method was rapid and cheap, specific and
sensitive for the screening of the pathogen in food.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction second most common serotype isolated from clinical material in

Poland (Zadernowska and Chaje˛ cka-Wierzchowska, 2017).
The genus Yersinia, belonging to the family Enterobacteriaceae, Yersinia enterocolitica causes a human infection known as yer-
includes three zoonotic pathogens, Yersinia pestis, Yersinia pseudo- siniosis that is the third most commonly reported zoonosis in
tuberculosis and some strains of Yersinia enterocolitica (Bursov
a Europe with 7,202 confirmed cases in 2015, after campylobacter-
et al., 2017). The last species is divided into six biotypes (1A, non- iosis and salmonellosis. The highest specific total cases reported in
pathogenic, 1B, and 2e5 causing human and/or animal in- the Member States were in the following decreasing order: Ger-
fections), and more than 70 serotypes based on their O lipopoly- many (2,752), Czech Republic (678), France (624) and Finland (582)
saccharide determinants O:3, O:8, O:9 and O:5,27 (Peruzy et al., (EFSA and ECDC, 2016; Rohde et al., 2017). Moreover, outbreaks
2017; Platt-Samoraj et al., 2017; Saraka et al., 2017). According to were described in other countries in the world, such as Finland,
report of the EFSA (2015) 4/O:3 and 2/O:9 are the primary bio/se- Japan, India and United States (Gnanasekaran et al., 2017). The
rotypes revealed in humans in Europe, while biotype 1B and symptoms range from mild and self-limiting gastroenteritis to
serotype O:8 have been called for many years the American sero- acute enteritis with diarrhea and abdominal pain, mesenteric
types due to the occurrence area, and both food import and export lymphadenitis and pseudo-appendicular syndromes indistin-
and transfer of people spread it also to the European countries. guishable from an acute appendicitis particularly in children older
However, Bottone (2015) reported that Y. enterocolitica O:8 has than 5 years, or septicemia in elderly and immuno-compromised
been isolated also in Japan and Europe, and it was considered the individuals (Petsios et al., 2016; Saraka et al., 2017). Besides post-
infection events, Y. enterocolitica can cause also primary cutaneous
infections, endocarditis, pneumonia and other nosocomial in-
fections, such as meningitis, septicemia, osteomyelitis, pharyngitis
* Corresponding author.
and conjunctivitis (Bonardi et al., 2016; Bottone, 2015). Some
E-mail address: [email protected] (M. Schirone).

https://doi.org/10.1016/j.fm.2018.01.009
0740-0020/© 2018 Elsevier Ltd. All rights reserved.
86 M. Luciani et al. / Food Microbiology 73 (2018) 85e92

sequelae such as reactive arthritis or erythema nodosum have also detection methods (Petsios et al., 2016). So, the aim of this study
been described (Bozcal et al., 2017). was the development of a rapid and easy method for its detection in
The principal source of yersiniosis in humans is the contami- food matrices of animal origin, based on the use of monoclonal
nated food and pigs can be considered a key reservoir, due to the antibodies (MAbs) specific for the lipopolysaccharide of Y. enter-
high prevalence of strains with high virulence (Ye et al., 2016). More ocolitica serotype O:8. Moreover, the developed method was
specifically, pigs can asymptomatically carry the microorganism in compared with the official qualitative microbiological assay of ISO
lymph nodes, tonsils and/or intestinal tract and during the 10273:2003.
slaughtering process it can spread to the carcass, near head and
sternum (Van Damme et al., 2017). Carcass refrigeration can facil-
itate its proliferation because Y. enterocolitica is a typically psy- 2. Materials and methods
chrophile microorganism (Bancerz-Kisiel et al., 2016). With regards
to temperature, it can grow in a wide range from 2 to 42  C and 2.1. Bacterial strains
therefore it can be found in food stored at refrigeration temperature
(Peruzy et al., 2017). In addition, the pathogen can hardly survive A total of 20 strains, distinguished in 7 Y. enterocolitica strains
cooking treatment due to its heat sensitivity (Wang et al., 2016). and 13 belonging to other microorganisms were obtained from the
Yersinia enterocolitica can be isolated also from livestock (horses, American Type Culture Collection (ATCC). Escherichia coli O14 was
sheep, cattle, rabbits), pets (dogs and cats) and rodents (Zhang provided from the Bundesinstitut für Gesundheitlichen Ver-
et al., 2015). Besides raw pork meat, milk and dairy products, braucherschutz und Veterina €rmedizin (BGVV), one strain of E. coli
fruits, vegetables and water have been often involved in human O157:H7 was obtained from the Istituto Superiore di Sanit a (ISS);
outbreaks (Gensberger and Kosti
c, 2017). Yersiniosis has been re- 14, 5 and 1 strains of Brucella genus were provided from the Na-
ported in Norway and United States after consumption of ready-to- tional Collection of Type Cultures (NCTC), the Central Veterinary
eat salad mix and pasteurised milk, respectively (Longerberger Laboratory (CVL, Weybridge) and from the Agence Française de
et al., 2014; MacDonald et al., 2016). The presence of Y. enter- Se
curite

Sanitaire des Aliments (AFSSA), respectively. Ochrobactrum
ocolitica in the latter product can be due to different conditions anthropi was obtained from the Deutsche Sammlung von Mik-
such as pasteurization failure, post-pasteurization contamination roorganismen und Zellkulturen (DSMZ) and Vibrio cholerae El Tor
and/or post-pasteurization addition of raw milk or other in- from the Istituto Sieroterapico of Milano. Other 37 bacterial strains
gredients (Bursova
et al., 2017). and 6 Y. enterocolitica field strains belonging to the collection of the
Both conventional and molecular methods have been described Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G.
for the detection of Y. enterocolitica in food samples. The first are Caporale” (IZSAM) were also used (Table 1).
culture-based methods through cold and/or selective enrichment
followed by phenotypic identification. They are laborious, time
consuming and sometimes they do not provide the possibility to 2.2. Preparation of bacterial antigens
precisely determine the pathogenicity of the isolates. Moreover,
they are characterized by a low sensitivity when the pathogen is The bacterial strains were grown in Brain Heart Infusion (BHI)
present at low concentrations and therefore it is difficult to detect Broth (Oxoid Ltd., London, UK) at 37  C for 14e16 h to obtain a final
amongst high background microbiota (Petsios et al., 2016). On the concentration of 2  108 cfu/ml and then inactivated at 60  C for
contrary the molecular methods, such as hybridization or poly- 1 h. Then they were centrifugated at 5500 g for 30 min, washed 3
merase chain reaction (PCR) including multiplex PCR and real-time times with 0.01 M phosphate buffered saline (PBS) pH 7.2 and
PCR, that are capable to detect also a variety of specific genes (i.e. resuspended in PBS. The cell suspensions were stored at 20  C
ail, inv, yadA, yops, yst or virF) are highly sensitive, rapid and specific until use.
(Bancerz-Kisiel and Szweda, 2015; Petsios et al., 2016). However, These antigens were used for the MAbs characterization and for
most of molecular methods as well as immunological assays cannot the determination of cross-reaction by capture-ELISA. Yersinia
distinguish between living and dead bacteria, unlike other tests for enterocolitica O:8 ATCC 23715, used for mice and rabbit immuni-
live/dead differentiation, such as the viable count method (Rohde zation and hybridoma screening, was sonicated on ice 2 times for
et al., 2017). 2.5 s and between the 2 rounds of sonication, the sample was put
Some authors reported other methods for the enumeration of Y. on ice for 5 min. The total protein concentration of the sonicated
enterocolitica such as an impedance method also reliable to antigen was determined using the Bicinchoninic Acid (BCA) Protein
enumerate the cells growing in biofilms in a more convenient way Assay Kit (Thermo Scientific, Rockford, IL, USA).
with regards to time and effort if compared with a standard plate
count (Wang et al., 2016). Zhang et al. (2015) described a new
isothermal and cross-priming amplification assay combined with 2.3. Preparation of Y. enterocolitica lipopolysaccharide
immunoblotting analysis for the rapid detection of the pathogen in
milk powders, that could be achieved in 90 min after pre- Yersinia enterocolitica lipopolysaccharide (LPS) was prepared
enrichment. This method is highly specific and sensitive and con- using the phenolic extraction method with some modifications
stitutes an alternative to PCR assay. (Portanti et al., 2006). Yersinia enterocolitica O:8 ATCC 23715 was
Other immunological methods for the detection of Yersinia spp. grown in BHI broth and centrifugated at 4000 g for 20 min; the
using monoclonal and polyclonal antibodies were developed pellet was resuspended in sterile deionized water and added with

(Hochel and Skvor, 2007; Balakrishna et al., 2012; Rütera et al., 85% phenol. Then the mixture was heated at 66e70  C for 20 min
2014). These methods are rapid and require only few hours to and centrifugated at 20000 g for 20 min at 4  C. The phenol extract
detect the pathogen. Generally antibody based tests are based on was diluted 1:10 with deionized water and dialyzed against NaCl
simple, quick and cheap detection system (Wangman et al., 2017). physiological saline solution. The LPS was precipitated overnight
However, the prevalence of this pathogen in food is often under- at 20  C with three volumes of methanol/sodium acetate; the
estimated due to different factors, such as the low concentration, pellet was then re-suspended in sterile deionized water and stored
the similarities with other Y. enterocolitica-like species as well as at 80  C. The quantity of LPS was determined by 2-keto-3-
the heterogeneity of isolates, but not least some hurdles in the deoxyoctonate (KDO) assay (Karkharis et al., 1978).
 M. Luciani et al. / Food Microbiology 73 (2018) 85e92 87

Table 1
List and number (N) of bacterial strains.

Bacterial strains N Bacterial strains N

Bacillus cereus ATCC 11778 1 2 1 E. coli O6 ATCC 25922 1 2 3 1

B. cereus (IZSAM) 2 1 E. coli O157:H7 (ISS) 1 2 3 1
Bacillus subtilis (IZSAM) 2 1 Escherichia fergusonii (IZSAM) 1 2 1
Bordetella bronchiseptica (IZSAM) 2 2 Klebsiella oxytoca ATCC 49131 1 2 3 1
Brucella abortus 99W (IZSAM) 1 3 1 Klebsiella pneumoniae (IZSAM) 1 2 3 1
Br. abortus S19 (IZSAM) 1 2 1 Listeria innocua ATCC 33090 1 2 1
Br. abortus S99 (CVL Weybridge) 1 2 3 1 Listeria ivanovii ATCC 19119 1 2 1
Br. abortus RB51 (IZSAM) 1 2 1 Listeria monocytogenes ATCC 7644 1 2 1
Br. abortus biovar 1 NCTC 624 1 2 3 1 L. monocytogenes 1 2 2
Br. abortus biovar 2 NCTC 10501 1 2 3 1 Ochrobactrum anthropi DSMZ 6882 1 1
Br. abortus biovar 3 NCTC 10502 1 2 3 1 Proteus vulgaris ATCC 49132 1 2 1
Br. abortus biovar 4 NCTC 10503 1 2 1 Pseudomonas aeruginosa (IZSAM) 2 1
Br. abortus biovar 5 NCTC 10504 1 2 1 Salmonella enterica subsp. enterica serovar Bredeney (IZSAM) 1 2 3 1
Br. abortus biovar 6 NCTC 10505 1 2 1 S. enterica subsp. enterica serovar Derby (IZSAM) 1 2 3 2
Br. abortus biovar 7 NCTC 10506 1 2 1 S. enterica subsp. enterica serovar Enteritidis ATCC 13076 1 2 3 1
Br. abortus biovar 9 NCTC 10506 1 2 1 S. enterica subsp. enterica serovar Enteritidis (IZSAM) 2 7
Brucella melitensis Rev 1 (AFSSA)1 2 1 S. enterica subsp. enterica serovar Hadar (IZSAM) 1 2 3 1
1 2 3
Br. melitensis biovar 1 (CVL Weybridge) 1 S. enterica subsp. enterica serovar Muenchen (IZSAM) 1 2 3 1
1 2 3
Br. melitensis biovar 2 (CVL Weybridge) 1 S. enterica subsp. enterica serovar Panama (IZSAM) 1 2 3 1
1 2 3
Br. melitensis biovar 3 (CVL Weybridge) 1 S. enterica subsp. enterica serovar Saintpaul (IZSAM) 1 2 3 1
Br. melitensis biovar 3 NCTC 8334 1 2 1 S. enterica subsp. enterica serovar Typhimurium ATCC 14028 1 2 3 1
Brucella ovis REO 198 (IZSAM) 1 2 1 Shigella flexneri ATCC 12022 1 2 3 1
Brucella suis biovar 1 NCTC 10316 1 2 1 Staphylococcus aureus ATCC 25923 1 2 1
Br. suis biovar 1 NCTC 5061 1 2 1 Staphylococcus epidermidis (IZSAM) 2 2
Br. suis biovar 1 (CVL Weybridge) 1 2 1 Staphylococcus faecalis ATCC 29212 2 1
Br. suis biovar 2 NCTC 10095 1 2 1 Vibrio cholerae El Tor (Sieroterapico Milano)1 2 1
Br. suis biovar 3 NCTC 10511 1 2 1 Yersinia enterocolitica O:8 ATCC 23715 1 2 1
Br. suis biovar 4 NCTC 11364 1 2 1 Y. enterocolitica O:8 ATCC 27739 1 2 1
Campylobacter jejuni ATCC 33291 1 2 3 1 Y. enterocolitica O:8 ATCC 49397 1 2 1
Citrobacter freundii (IZSAM) 1 2 3 1 Y. enterocolitica O:8 ATCC 51871 1 2 1
Enterobacter agglomerans (IZSAM) 1 2 3 1 Y. enterocolitica O:8 ATCC 9610 1 2 1
Enterobacter amnigenus (IZSAM) 1 2 3 1 Y. enterocolitica O:9 ATCC 55075 1 2 3 1
Enterobacter cloacae (IZSAM) 1 2 3 3 Y. enterocolitica O:9 ATCC 700823 1 2 3 1
Enterococcus faecium (IZSAM) 2 3 2 Y. enterocolitica O:9 (IZSAM) 1 2 3 3
Escherichia coli O14 (BGVV) 1 2 3 1 Y. enterocolitica O:3 (IZSAM) 1 2 3 3
1
Used for MAbs characterization.
2
Used for cross-reactivity determination of capture-ELISA-Y. enterocolitica.
3
Non-target strains used for selectivity tests with food matrices.

2.4. Immunization of mice 2.5. Characterization of monoclonal antibodies

Female BALB/c mice, 6/8 weeks of age, were inoculated with The MAbs were characterized by indirect ELISA (i-ELISA) versus
heat inactivated and sonicated Y. enterocolitica O:8 ATCC 23715. The Y. enterocolitica O:8 ATCC 23715 (whole antigen and LPS) and all the
antigen, diluted with sterile PBS to a protein concentration of 50 mg/ other bacterial strains used in this study. The 96-well microplates
ml, was emulsified with complete Freund adjuvant (CFA) (Sigma, St. (PolySorp, Nunc, Roskilde, DK) were coated with 5 mg/ml of the
Louis, MO, USA) and administered intraperitoneally; 14 days later a different bacterial antigens and blocked with 1% yeast extract to
second immunization was performed using the same concentration test hybridoma supernatants. As secondary antibody, ECL anti-
of antigen emulsified with incomplete Freund adjuvant (IFA) mouse IgG conjugated with horseradish peroxidase (GE Health-
(Sigma). After 27 and 56 days, 50 mg/ml of antigen in sterile PBS was care, Little Chalfont, Buckinghamshire, UK) was used; the 3,30 ,5,50 -
obtained. After the mice sacrification and spleen recovery, the Tetramethylbenzidine (TMB) (Sigma) was adopted as chromogenic
collected splenocytes were fused with murine myeloma cells Sp2/ substrate. The reading was performed with a microplate reader
O-Ag-14 (ATCC CRL-1581™) (Schulman et al., 1978). The (Bio-Rad, Hercules, CA, USA) at an optical density of 450 nm
antibody-secreting hybridomas were grown for 2 weeks in Dul- (OD450). The MAbs showing OD450 greater than 0.300 with a bac-
becco's Modified Eagle's Medium (DMEM) containing 20% fetal terial antigen, were considered positive for that specific antigen.
bovine serum, 2 mM glutamine, 100x Penicillin-Streptomycin- Then the MAbs were isotyped using the Mouse-Typer Isotyping
Amphotericin B, 50 mg/ml gentamicin, 10000 UI/ml nistatin and Panel (Bio-Rad) and those showing IgG isotype were further char-
50x HAT. Hybridomas were then cloned by the limiting dilution acterized by immunoblotting.
method (Luciani et al., 2006). The different bacterial antigens and Y. enterocolitica O:8 LPS (5
The animal experimentation was carried out in compliance with mg/well) were subjected to SDS-PAGE electrophoretic separation
the Italian national law in force until 2014, i.e. Legislative Decree 27 using NuPAGE 12% Bis-Tris Gels Mini (Life Technologies, Carlsbad,
January 1992 No 116 implementing Directive 86/609/EEC of the CA, USA) at 200 V; gels were then stained with Simply Blue Safe
Council of the European Communities on the protection of animals Stain (Life Technologies) and SilverQuest Silver Staining kit (Life
used for experimental and other scientific purposes (EC, 1986). The Technologies) to visualize, respectively, protein bands and LPS
protocol was approved by the Italian Ministry of Health with bands. The electrophoretic separation was repeated as indicated
number 5146 of 26/04/2012. above, and separated proteins were transferred onto nitrocellulose
membrane with iBlot Dry Blotting System (Life Technologies).
Membranes were blocked with 5% skim milk in PBS containing
88 M. Luciani et al. / Food Microbiology 73 (2018) 85e92

0.05% Tween 20 (PBST) for 2 h at room temperature (RT), washed bacterial concentration of the serially diluted Y. enterocolitica O:8
with PBST and incubated overnight at 4  C with MAb supernatants ATCC 23715 versus OD450nm and S/N values.
diluted 1:2 with PBST containing 2.5% skim milk. The detection of
immune-complexes was performed using the ECL anti-mouse IgG 2.8. Validation of Yersinia enterocolitica capture-ELISA
HRP-conjugated (GE Healthcare) and the chemiluminescent sub-
strate ECL Select Western Blotting Detection Reagent (GE Health- The Y. enterocolitica capture-ELISA method was validated as
care). The analysis of the results was performed using the indicated in ISO 16140:2003 for qualitative methods in food
ChemiDoc MP (Bio-Rad) and Quantity One Quantitation Software matrices. A total of 132 food samples, 68 meat (chicken flesh) and
version 4.3 (Bio-Rad). 64 dairy products (caciotta cheese) were analyzed both with the
The MAbs with IgG isotype, specific for Y. enterocolitica, were official method (ISO 10273:2003) and Y. enterocolitica capture-
produced in vitro on a large scale and purified by affinity chroma- ELISA. Among samples, 38 and 30 of meat and dairy products
tography using a column HiTrap rProtein A FF, 5 ml (GE Healthcare) respectively, were artificially contaminated with strains of
according to the manufacturer's instructions. The purified MAbs Y. enterocolitica O:8 ATCC 23715. In particular, 25 g of each sample
were labeled with horseradish peroxidase (HRP) (Sigma) as indi- were inoculated with suspensions at a final concentration of
cated in the literature (Nakane and Kawaoi, 1974). 102 cfu/g Y. enterocolitica O:8 ATCC 23715. The inoculated food
matrices were diluted with ITC Broth, homogenized in the stom-
2.6. Polyclonal antibodies productions acher and then incubated at 25  C for 48h.
Intra-laboratory tests were used to evaluate relative accuracy,
Polyclonal antibodies against Y. enterocolitica O:8 ATCC 23715 relative sensitivity, relative specificity and relative limits of detec-
were produced in New Zealand female rabbits. The heat inactivated tion and selectivity. Relative accuracy, sensitivity and specificity
and sonicated bacteria were diluted in FCA to a protein concen- were determined by analyzing the above reported 132 samples, of
tration of 200 mg/ml and injected subcutaneously 6 times over 50 which 68 artificially contaminated and 64 uncontaminated
days. Antiserum with adequate titer, affinity, and specificity was samples.
obtained 2 months after the first immunization. The IgG-rich The relative limit of detection was determined using both the
fraction was purified using the same procedure for the MAbs pu- official method and Y. enterocolitica capture-ELISA to analyze the
rification. Rabbit IgG protein concentration was measured by above cited 132 samples from each of the two food matrices
spectrofotometer at OD280. inoculated at 4 concentration levels (5, 10, 100 and 1000 cfu/g) of
The protocol of the animal experimentation was approved by Y. enterocolitica O:8 ATCC 23715, and one uncontaminated sample
the Italian Ministry of Health with number 11520 of 24/10/2013. of both matrices as negative control. The concentration of
Y. enterocolitica strain used to prepare the inoculated samples was
2.7. Yersinia enterocolitica capture-ELISA established by plate titration and measurements of OD450.
The selectivity was determined by analyzing samples inoculated
Twenty-five g of each food matrices were diluted with Irgasan with target strains of Y. enterocolitica (inclusivity) and non-target
Ticarcillin Chlorate (ITC) Broth (Oxoid), homogenized in a stom- bacterial strains (exclusivity) as reported in Table 1. More in
acher and then incubated at 25  C for 48 h. All the food samples detail, a total of 126 samples from the two food matrices (half meat
were tested in parallel using the official method ISO 10273:2003 and half dairy products) were analyzed, of which 62 were
and the Y. enterocolitica capture-ELISA after heat treatment. contaminated with a strain of Y. enterocolitica O:8 ATCC 23715 and
The 96-wells ELISA microplates (High Binding Costar, Corning, other non-target strains, and the remaining 64 samples were
New York, NY, USA) were coated with 100 ml/well of rabbit IgG inoculated with only non-target strains.
versus Y. enterocolitica O:8 ATCC 23715 at a concentration of 20 mg/
ml in 50 mM carbonate/bicarbonate buffer, pH 9.6, and incubated 2.9. Statistical analysis
overnight at RT. The microplates were then washed three times
with PBST. One hundred ml/well of culture positive and negative The comparison between the Y. enterocolitica capture-ELISA and
controls and of previously enriched and heat-treated food samples the official method ISO 10273:2003 was assessed using Cohen's
were added into the microplates and incubated under gentle Kappa statistical test (Microsoft Office Excel 2010).
shaking for 1 h at 37  C. After further washings, 100 ml/well of the
MAbs conjugated with peroxidase were dispensed into all wells 3. Results and discussion
and incubated for 1 h at 37  C. The microplates were washed as
described above and incubated with 100 ml/well of TMB for In the present study, a new and rapid method for the detection
30 min at RT; the enzyme reaction was stopped by adding 50 ml/ of Y. enterocolitica in food has been developed and compared to ISO
well 0.5 N sulphuric acid. The OD was measured at 450 nm with a 10273:2003. A total of 423 hybridomas, showing by i-ELISA an
microplate reader. The examined samples were diagnosed positive OD450  0.300 versus Y. enterocolitica O:8 ATCC 23715, was ob-
or negative for the presence of Y. enterocolitica based on the ratio S/ tained. The MAbs were first screened versus Y. enterocolitica O:8
N between the OD450nm of tested samples (S) and the OD450nm ATCC 23715, E. coli O157:H7 (ISS), Salmonella enterica subsp.
produced by the negative controls (N). Yersinia enterocolitica O:8 enterica serovar Typhimurium ATCC 14028 and Listeria mono-
ATCC 23715 in PBS was used as positive control and ITC Broth was cytogenes ATCC 7644. Only 37 MAbs reacted with Y. enterocolitica
used as negative control. O:8 ATCC 23715. A second screening was performed by testing the
The cut-off was determined by titration of a serially diluted 37 MAbs with a greater number of bacterial strains. In particular, 7
Y. enterocolitica O:8 ATCC 23715 culture in ITC broth with a known MAbs (46F7, 54B11, 54C11, 62D10, 72E8, 72E10, 72G6) reacted with
bacterial concentration (7.1  105 cfu/ml). The number of cfu/ml in Y. enterocolitica O:8 ATCC 23715. Other 2 MAbs (64C7 and 64C10)
the Y. enterocolitica O:8 ATCC 23715 culture was determined by responded not only with Y. enterocolitica O:8 ATCC 23715 but
plate count on Cefsulodin-Irgasan-Novobiocin-agar (Oxoid). The showed also a weak cross-reaction (values above 0.400) with
cut-off value, expressed as S/N, was calculated by interpolating the Campylobacter jejuni ATCC 33291.
mean absorbance of negative ITC broth plus three times the stan- The OD450 values of the 9 reported MAbs versus Y. enterocolitica
dard deviation on the calibration curve obtained by plotting the were shown in Table 2. The highest OD450 values were expressed
 M. Luciani et al. / Food Microbiology 73 (2018) 85e92 89

Table 2
MAbs characterization of the 9 selected MAbs tested versus Yersinia enterocolitica and Campylobacter jejuni by i-ELISA.

MAb (OD450 nm)

Bacterial strains 46F7 54B11 54C11 62D10 64C7 64C10 72E8 72E10 72G6
Yersinia enterocolitica O:8 ATCC 23715 2.192 2.519 2.404 2.514 1.305 1.390 2.486 2.521 2.451
Y. enterocolitica O:8 ATCC 27739 2.112 2.605 2.293 2.584 1.689 1.795 2.641 2.602 2.321
Y. enterocolitica O:8 ATCC 49397 1.836 2.346 2.078 2.451 1.213 1.097 2.418 2.428 2.084
Y. enterocolitica O:8 ATCC 51871 0.653 2.621 2.608 2.683 1.282 1.411 2.687 2.738 2.690
Y. enterocolitica O:8 ATCC 9610 1.675 2.463 2.337 2.523 0.972 1.086 2.521 2.526 2.291
Y. enterocolitica O:9 ATCC 55075 0.052 0.077 0.043 0.045 0.046 0.069 0.044 0.043 0.053
Y. enterocolitica O:9 ATCC 700823 0.049 0.074 0.052 0.045 0.047 0.061 0.044 0.043 0.051
Y. enterocolitica O:9 0.050 0.076 0.044 0.043 0.047 0.060 0.045 0.045 0.047
Y. enterocolitica O:9 0.058 0.090 0.050 0.052 0.055 0.072 0.054 0.050 0.059
Y. enterocolitica O:9 0.054 0.078 0.050 0.049 0.049 0.068 0.051 0.050 0.051
Y. enterocolitica O:3 0.056 0.095 0.053 0.057 0.053 0.063 0.051 0.054 0.056
Y. enterocolitica O:3 0.051 0.077 0.045 0.046 0.047 0.063 0.045 0.044 0.051
Y. enterocolitica O:3 0.048 0.071 0.043 0.044 0.043 0.061 0.044 0.043 0.050
Campylobacter jejuni ATCC 33291 0.077 0.067 0.062 0.064 0.473 0.431 0.063 0.057 0.072

versus serotype O:8 of Y. enterocolitica ranging from a minimum of

0.653 to a maximum of 2.738. All the other bacterial strains
considered in the present study resulted not cross-reactive and
therefore they were not reported in Table 2.
With regards to the results of i-ELISA of the 9 selected MAbs
tested versus Y. enterocolitica O:8 ATCC 23715 both whole antigen
and LPS, only MAbs 64C7 and 64C10 did not react with LPS. The
OD450 of all the tested MAbs were reported in Table 3. In particular,
OD450 of MAbs 64C7 and 64C10 were below 0.300 corresponding to
0.050 and 0.020, respectively.
The immunoblotting results also confirmed that the tested
MAbs showed the typical LPS-ladder pattern with the exception of
64C7 e 64C10 that reacted with protein bands of molecular weights
comprised between 80 and 25 kDa (Fig. 1).
The MAb 46F7 had IgM isotype; the others eight MAbs had IgG2b
anti k isotype. Among these MAbs with IgG isotype, the MAbs
54B11, 54C11, 62D10, 72E8, 72E10 and 72G6, specific for LPS, were
tested in capture-ELISA and showed similar results. For the stan-
dardization and validation of Y. enterocolitica capture-ELISA, the
MAb 54B11 was selected as secondary antibody because clone
54B11 showed the best IgG productivity, in comparison with the
other 5 selected clones. MAb 54B11 was used at the working
dilution of 1:56000 to obtain an OD450 value of the positive control Fig. 1. MAbs characterization by immunoblotting. Lane 1: 46F7; lane 2: 54B11; lane 3:
between 1.800 and 2.200. 54C11; lane 4: 62D10; lane 5: 72E8; lane 6: 72E10; lane 7: 72G6; lane 8: 64C7; lane 9:
In the ELISA standardization step, food samples enriched in ITC 64C10.

broth at 25  C for 48 h and in Peptone Sorbitol Bile (PSB) broth at

25  C for 48e72 h were tested. Best results in terms of lower
found to be 1.75. When the food samples produced a S/N ratio
background at higher sensitivity were obtained with ITC broth
greater than or equal to the cut-off value they were considered
enrichment, so the validation step was done using only food sam-
positive for Y. enterocolitica O:8 ATCC 23715, on the contrary they
ples enriched in ITC broth.
were negative with a lower cut-off. The bacterial strains not
The cut-off value of the capture-ELISA, expressed as S/N, was
belonging to Y. enterocolitica showed no cross-reaction. The results
were reported in Table 4. The highest values of S/N ratio were
observed for the serotype O:8 up to a maximum of 5.3.
Table 3 For the repeatability and reproducibility of capture-ELISA, one
MAbs characterization of the 9 selected MAbs tested versus Y. enterocolitica O:8 positive and one negative sample were analyzed with 80 and 120
whole antigen and lipopolysaccharide by i-ELISA. repetitions for the first and second parameter, respectively
MAbs Yersinia enterocolitica O:8 ATCC 23715 (Table 5).
whole antigen LPS
The results for relative accuracy, sensitivity and specificity for
132 meat and dairy product samples analyzed in this study were
46F7 3.000 3.000
shown in Table 6. The 68 meat product samples analyzed by ISO
54B11 3.600 2.000
54C11 3.600 2.500 10273:2003 and capture-ELISA were distinguished in 38 positive
62D10 3.100 3.000 and 30 negative samples with both the two methods, so the
64C7 1.300 0.050 mentioned parameters corresponded to 100%. The same results
64C10 1.500 0.020 were observed with the dairy product samples showing a total of
72E8 2.500 3.000
72E10 3.200 2.900
30 positive and 34 negative samples, also in that case relative ac-
72G6 2.600 3.200 curacy, sensitivity and specificity were 100%.
90 M. Luciani et al. / Food Microbiology 73 (2018) 85e92

Table 4
Cross-reactions of Yersinia enterocolitica by capture-ELISA.

Bacterial strains S/Na Bacterial strains S/N

Bacillus cereus ATCC 11778 1.0 E. coli ATCC 25922 0.8

B. cereus 0.9 E. coli O157:H7 0.9
Bacillus subtilis 0.9 Escherichia fergussoni 0.8
Bordetella bronchiseptica 0.9 Klebsiella oxytoca ATCC 49131 0.9
Brucella abortus S19 0.9 Klebsiella pneumoniae 0.8
Br. abortus S99 0.8 Listeria innocua ATCC 33090 0.9
Br. abortus RB51 0.7 Listeria ivanovii ATCC 19119 1.0
Br. abortus biovar 1 (624) 0.7 Listeria monocytogenes ATCC 7644 0.8
Br. abortus biovar 2 (10501) 0.8 L. monocytogenes 0.8
Br. abortus biovar 3 (10502) 0.8 Proteus vulgaris ATCC 49132 1.0
Br. abortus biovar 4 (10503) 0.7 Pseudomonas aeruginosa 0.9
Br. abortus biovar 5 (10504) 0.9 Salmonella enterica subsp. enterica serovar Bredeney 0.9
Br. abortus biovar 6 (10505) 0.8 S. enterica subsp. enterica serovar Derby 0.9
Br. abortus biovar 7 (10 506) 0.8 S. enterica subsp. enterica serovar Enteritidis ATCC 13076 0.9
Br. abortus biovar 9 (10 506) 0.8 S. enterica subsp. enterica serovar Enteritidis 0.9
Brucella melitensis Rev 1 0.8 S. enterica subsp. enterica serovar Hadar 0.9
Br. melitensis biovar 1 0.7 S. enterica subsp. enterica serovar Muenchen 0.8
Br. melitensis biovar 2 0.7 S. enterica subsp. enterica serovar Panama 1.1
Br. melitensis biovar 3 0.8 S. enterica subsp. enterica serovar Saintpaul 0.9
Br. melitensis biovar 3 (8334) 0.7 S. enterica subsp. enterica serovar Typhimurium ATCC 14028 0.9
Brucella ovis REO 198 0.7 Shigella flexneri ATCC 12022 0.9
Brucella suis biovar 1 (10316) 0.7 Staphylococcus aureus ATCC 25923 1.59
Br. suis biovar 1 (5061) 0.7 Staphylococcus epidermidis 0.8
Br. suis biovar 1 0.8 Staphylococcus faecalis ATCC 29212 1.0
Br. suis biovar 2 (10095) 0.7 Vibrio cholerae El Tor 0.7
Br. suis biovar 3 (10511) 0.8 Yersinia enterocolitica O:8 ATCC 23715 5.3
Br. suis biovar 4 (11364) 0.7 Y. enterocolitica O:8 ATCC 27739 4.8
Campylobacter jejuni ATCC 33291 0.8 Y. enterocolitica O:8 ATCC 49397 4.7
Citrobacter freundii 0.9 Y. enterocolitica O:8 ATCC 51871 4.9
Enterobacter agglomerans 0.9 Y. enterocolitica O:8 ATCC 9610 4.6
Enterobacter amnigenus 1.0 Y. enterocolitica O:9 ATCC 55075 0.7
Enterobacter cloacae 0.9 Y. enterocolitica O:9 ATCC 700823 0.7
Enterococcus faecium 1.0 Y. enterocolitica O:9 0.8
Escherichia coli O14 (BGVV) 0.9 Y. enterocolitica O:3 0.7
a
S/N  1.75: positive; S/N < 1.75: negative.

Table 5 with a selectivity parameter of 100% and with a lower confidence

Repeatability (80 repetitions) and reproducibility (120 repetitions) of capture-ELISA. limit of 90.3 and an upper confidence limit of 100%.
Samples Repeatability Reproducibility In literature, several culture methods for isolation of Y. enter-
ocolitica are reported, but they require more steps (pre-enrichment,
S/N SDa CV%b S/N SD CV%
selective/secondary enrichment and post-enrichment) and subse-
Positive 32.3 3.186 9.9 33.2 2.948 8.9
quent selective plating protocols, with different periods of time to
Negative 0.9 0.083 9.3 0.9 0.080 8.9
obtain the results (Petsios et al., 2016). While the direct plate
a
SD ¼ standard deviation. isolation from food samples by conventional methods could be
b
CV ¼ coefficient of variation.
difficult due to the low concentration of the pathogen as well as the
large number of background microbiota, the method developed in
Table 6 the present study could be considered a valid and useful alternative
Relative accuracy, sensitivity and specificity expressed as percentage (%) of capture- assay for a rapid screening. More in detail, after the 48 h enrich-
ELISA compared to the official method (ISO 10273:2003). ment step, it needed only about 3 h to detect the presence of Y.
Parameters Meat samples Dairy product samples enterocolitica against 3e4 days required by ISO 10273:2003 and
allowed the simultaneous analysis of a great number of samples.
Percentage LCLa UCLb Percentage LCL UCL
Moreover, the comparison of the two methods showed that the
Relative accuracy 100.0 95.8 100.0 100.0 95.5 100.0 upper confidence limit for relative accuracy, sensitivity and speci-
Relative sensitivity 100.0 92.6 100.0 100.0 90.8 100.0
ficity was 100% in both cases. A total of 9 MAbs with a specific re-
Relative specificity 100.0 90.8 100.0 100.0 91.8 100.0
Kappa index ¼ 1.0 p < .05 p < .05 action versus Y. enterocolitica O:8 were obtained and only 2 of them
a
showed a weak cross-reaction with C. jejuni. The latter result could
LCL ¼ lower confidence limit.
b
UCL ¼ upper confidence limit. be affected by the presence of some common epitopes between the
2 bacteria.
Moreover the obtained 9 MAbs showed no reactions with the
With regards to the relative limit of detection of the two tested Brucella species. Yersinia enterocolitica O:9 is known to have a
methods, it corresponded to 5 and 10 cfu/g for both meat and dairy similar O-antigen with Brucella spp. and therefore cross-reacting
products with ISO 10273:2003 and capture-ELISA, respectively antibodies can be detected (O'Grady et al., 2016). Nielsen et al.
(Table 7). (2004) reported that this serotype shares the major O-poly-
The results of selectivity obtained by analyzing the other 126 saccharide, 1,2-linked 4,6-dideoxy-4-formamido-a-D-mannopyr-
samples were the same for the 2 food matrices. A total of positive anosyl almost completely with B. abortus. Cross-reactions of MAbs
(62) and negative (64) samples were obtained by the two methods used in the present study with other species or serotypes of Y.
 M. Luciani et al. / Food Microbiology 73 (2018) 85e92 91

Table 7
Relative limit of detection of capture-ELISA compared to the official method (ISO 10273:2003).

Samples Contamination level (cfu/g) Percentage

ISO 10273:2003 Capture-ELISA

Meat/Dairy products 5 40 0
10 100 100
100 100 100
1000 100 100

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