149 1Department of Medical Biopathology and Biotechnology, University of Palermo, 90134 Palermo, Italy. 2Istituto Zooprofilattico Sperimentale della Sicilia ‘A. Mirri’, via G. Marinuzzi 3, 90129 Palermo, Italy. * Corresponding author at: Istituto Zooprofilattico Sperimentale della Sicilia ‘A. Mirri’, via G. Marinuzzi 3, 90129 Palermo, Italy. Tel.: +39 091 6565236, e-mail: sara.villari@izssicilia.it. Parole chiave Brucella abortus, Bovini, Cellule IFN-γ+T, Yersinia enterocolitica O:9. Riassunto Uno dei principali limiti nella diagnosi delle brucellosi animali è la cross-reattività che si verifica tra gli antigeni di superficie di Brucella e Yersinia. Con l’intento di cercare un metodo in grado di discriminare tra le infezioni causate dai due patogeni, nel presente lavoro è stata messa a confronto l’espansione di linfociti T in grado di produrre interferon gamma (IFN-γ+), ottenuti da cellule mononucleate di sangue periferico (PBMC) isolate da bovini con infezione da Brucella abortus, con quella indotta in animali sperimentalmente immunizzati con Yersinia enterocolitica O:9. I linfociti sono stati quindi analizzati mediante citofluorimetria dopo che le PBMC erano state riesposte in vitro ad antigeni di Yersinia o Brucella. I risultati ottenuti hanno evidenziato una differenza statisticamente significativa nell’espansione di linfociti T CD4+ e CD8+ / IFN-γ+ nei casi in cui le PBMC di animali immunizzati con Yersinia erano state esposte in vitro agli antigeni di Y. enterocolitica O:9, che invece non si verificava nel caso di esposizione ad antigeni di Brucella. Questo metodo, pertanto, potrebbe risultare di interesse per la conferma di casi sierologici con diagnosi dubbia e/o per l’esclusione di potenziali cross-reattività. Analisi dell’interferon gamma prodotto nelle cellule durante le infezioni da Yersinia enterocolitica O:9 e Brucella abortus nei bovini Keywords Brucella abortus, Cattle, IFN-γ+T cells, Yersinia enterocolitica O:9. Summary One of the major constraints in the diagnosis of animal brucellosis is the cross-reactivity that occurs between Brucella and Yersinia surface antigens. With the aim to find a method to distinguish Brucella from Yersinia infection, the expansion of interferon gamma producing (IFN-γ+) T cell subsets obtained from peripheral blood mononuclear cells (PBMC) isolated from cattle either infected by Brucella abortus or experimentally immunized with Yersinia enterocolitica O:9 were compared. The lymphocytes were analyzed by flow cytometry after PBMC were in vitro re-exposed to Yersinia or Brucella antigens. The results highlighted a statistically significant difference in the expansion of the CD4+ and CD8+ IFN-γ+ T cells occurring when PBMC of animals immunized with Yersinia are in vitro exposed to Y. enterocolitica O:9 antigen but not to Brucella antigen. This method could thus be suggested in those cases where results obtained by serodiagnosis need to be further clarified. Analysis of interferon-gamma producing cells during infections by Yersinia enterocolitica O:9 and Brucella abortus in cattle Veterinaria Italiana 2019, 55 (2), 149-155. doi: 10.12834/VetIt.1374.7538.2 Accepted: 31.08.2018 | Available on line: 30.06.2019 Annalisa Agnone1, Marco Pio La Manna1, Gesualdo Vesco2, Valeria Gargano2, Giusi Macaluso2, Francesco Dieli1, Guido Sireci1 and Sara Villari2* (Pappas et  al. 2006, Skendros et  al. 2011), and the Mediterranean basin is an acknowledged endemic region of human brucellosis (Seimenis et  al. 2006). The infection is transmitted to humans mostly by ruminants, through direct contact with infected materials or indirectly by ingestion of animal Introduction Brucellosis is an infectious disease mainly affecting cattle, swine, goats, sheep and dogs caused by members of the genus Brucella. Brucellosis is the most common zoonotic infection worldwide 150 IFN-γ+ cells to distinguish Brucella and Yersinia infections Agnone et al. Veterinaria Italiana 2019, 55 (2), 149-155. doi: 10.12834/VetIt.1374.7538.2 detected by Enzyme Linked Immunosorbent Assay (ELISA) (Weynants et  al. 1995). It has also been demonstrated that IFN-γ secreted by peripheral blood in response to a commercial Brucella antigen,a was not functional to distinguish between infection caused by Brucella or Yersinia enterocolitica O:9, representing the most important source of false positive reactions in the serological diagnosis of brucellosis in animals and humans (Kittelberger et al. 1997). The role of cell-mediated immunity in Brucella infection has been analyzed in murine models (Skyberg et al. 2011, Weynants et al. 1998). Protective immunity requires activated antigen-presenting cells (mainly macrophages and dendritic cells) as well as CD4+, CD8+ and γδ+T lymphocytes activation. In particular, CD4+IFN-γ+ T cell subset was described as essential for the clearance of Brucella infection in murine model activating killing mechanisms in macrophages as well as other type of cells that are reservoir of replicating bacteria (Skendros and Boura 2013). Brucella may thus survive in host macrophages and dendritic cells evading adaptive immune mechanisms (Carvalho Neta et  al. 2010, Kittelberger et al. 1997). To our knowledge, the IFN-γ- producing lymphoid subsets following initial in  vivo and then in  vitro re-exposure to Brucella and Yersinia antigens in cattle have not been identified yet. It was reported that CD4+ IFN-γ+ lymphocytes play a key role in containing Brucella infection in cattle (Skendros and Boura 2013). The novelty of our approach is represented by the effort to detect the IFN-γ+T cell subsets during natural Brucella infection and experimental Yersinia immunization after in  vitro re-exposure with specific antigens. This approach was designed to evaluate the potential diagnostic use of these parameters to discriminate between the two infections in cattle. Materials and methods Animal trial A total number of 15 female cattle were enrolled for the study. Blood samples were collected weekly by venipuncture in heparinized tubes and tubes with no anticoagulant from the groups of cattle. Sera were obtained after centrifugation and stored at - 20°C until used for diagnostic purposes. In group 1, the animals were immunized with inactivated Yersinia enterocolitica O:9 (Charolaise breed, 6-8  months old). Group 2 (Holstein breed, 2-5 years old) consisted of cattle naturally infected with B. abortus. Group 3 (Charolaise breed, 1-3 years old) was the control group, including seronegative products (milk and derivatives) or by inhalation (Olsen and Palmer 2014). Infected cows can abort, once only after the exposure to the pathogen, usually at about 7 months of pregnancy; otherwise, newborn calves are very weak and usually die shortly after birth. Brucella colonization of the udder results in a severe drop in milk production, which may also present swelling and inflamed knees (hygromas) (Olsen and Palmer 2014), while bulls with brucellosis usually become sterile (Carvalho Neta et al. 2010). Brucella is able to survive in immune cells and persist in tissues of the reticuloendothelial system (De Jong et  al. 2010), using several strategies of immune evasion. One of these includes the production of cyclic glucan molecules that impede the lipid raft-mediated vacuole maturation to interfere with the membrane transport systems (Skendros and Boura 2013). Moreover, LPS from Brucella abortus bears a non canonical lipid A active only at very high concentration, therefore even if the LPS is recognized by TLR4 receptors of phagocytes it does not induce early activation of macrophages and consequent production of pro-inflammatory cytokines (Barquero-Calvo et  al. 2007). Brucellosis diagnosis in livestock is mainly based on serological methods; the Italian national eradication program in ruminants, based on test-and-slaughter policy, prescribes to perform the official diagnosis by two serological tests, Rose Bengal Test (RBT ) and Complement Fixation Test (CFT ) (O.M. 24-06-2015). Unfortunately, they both lack of specificity due to the use of the whole lipopolysaccharide fraction of Brucella cell envelope as antigen (Corbel 1985, Nielsen et  al. 2004). In fact, the most relevant constraint of the serological diagnosis consists in the frequently observed cross-reactions after the animal exposure to other microorganisms which all share common features with Brucella polysaccharide ‘O’ chain, e.g. Yersinia enterocolitica O:9, Salmonella enterica serotype typhimurium and Escherichia coli O157 (Muñoz et  al. 2005, Nielsen et al. 2004). Despite the presence of a wide body of literature showing efforts to discover antigens that can be useful in discriminating between Brucella and other Gram negative bacteria infections (Denoel et  al. 1997, Guzman et  al. 2012, Ko et  al. 2012), the lack of Brucella specific antigen still highlights the need to develop new diagnostic methods, in order to improve the efficacy of eradication strategies and to avoid un-necessary animal sacrifices. The reaction of cattle immune system has been investigated in terms of Delayed Type Hypersensitivity, PBMC proliferative response and interferon gamma (IFN-γ) secretion by whole lymphocyte fraction in presence of Brucella antigens, 151 Agnone et al. IFN-γ+ cells to distinguish Brucella and Yersinia infections Veterinaria Italiana 2019, 55 (2), 149-155. doi: 10.12834/VetIt.1374.7538.2 commercial reagentf and then incubated with Phicoeritrina (PE) labelled  anti IFN-γ mAb (clone CC302, mouse anti cow IgG1)e for 30 minutes at 4 °C, washed three times and resuspended in 500  µl of Phosphate Buffer Solution. IFN‑γ measurement and data analysis Stained PBMC were acquired by flow cytometry analysis using a cytometerg and a commercial software.g Each acquisition was performed collecting 10,000 events of lymphocytes region gated using Forward scatter (FSC) and Side Scatter (SSC) parameters. Acquired cells were analyzed in order to detect the subset of IFN-γ producing cells; the percentage of IFN-γ+ cells detected in cultures with RPMI only (negative control) was subtracted from the same measurement observed in cells cultured with antigens. Statistical analysis of data was performed by Mann Whitney test, for any significant difference among the average percentages of a three times-repeated sample for each experimental group. In order to evaluate a parameter able to discriminate between animals immunized with Y.  enterocolitica and naturally infected with B.  abortus, the ratio between the percentages of IFN-γ+ cells in response to Brucella and Yersinia antigens was also analyzed. The confidence value was fixed at 0.05 unless otherwise specified. Results IFN‑γ+ cells expansion in response to Yersinia Results showed that in animals immunized with Y. enterocolitica an expansion of IFN-γ+ lymphocytes was detected following the re-exposure to Ye  O:9 (2.36% in immunized versus 0.36% in uninfected controls) but not to Brucella antigen (0.21%) (p < 0.05) (Figure  1A). There was a statistically significant difference in the expansion of CD4+ IFN-γ+T cells when PBMC from animals immunized with Yersinia were exposed to Ye O:9, but not to Brucella antigen (1.26% vs 0.14%) (Figure  1B). The percentage of CD8+ IFN-γ+T cells was 0.93% in Yersinia-immunized animals versus 0.08% in seronegative controls. Taking into account that γδT-lymphocytes are represented in a consistent amount in cattle in an age-dependent manner (Guzman et  al. 2012) and are relevant in murine as well as in bovine immune response to Brucella (Skyberg et  al. 2011), the role of γδ+ IFN-γ+ T cells was also analyzed (Figure  1D). Results show that these cells display a minor contribution to IFN-γ production in tested animals. The comparison of γδ+ IFN-γ+ T cells in vitro expansion was statistically different when PBMC of animals obtained from farms officially free of Brucellosis. Animal trial was accomplished following current European legislation (Directive 2010/63/UE and following Commission implementing Decisions) and the corresponding Italian law (D. Leg. 26, March 4th, 2014). The whole procedure was conducted according to the regulations of the Italian Health Ministry (Decreto Ministeriale No. 101/2006-A). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. As for Group 1, five cattle were immunized with 100 ml of heat inactivated (65  °C for 1.5 minutes) Y.  enterocolitica O:9 (1012 Colony Forming Units – CFU – per os). A 10 ml dose containing 1012  CFU with aluminium hydroxide as adjuvant was also administered subcutaneously, in the neck region, weekly for two months. Antibodies against Y.  enterocolitica O:9 were measured weekly for each animal by a specific CFT procedure, using a commercial antigen,b as recommended by the manufacturer. Sera showing at least 75% of fixation at 1:10 dilution were considered positive. Furthermore, the same serum samples from each animal were tested by RBT and CFT specific for brucellosis before the experimental procedure, resulting all negative. As for Group 2, we collected samples from naturally infected cattle resulted positive to the official serological tests. Sera showing agglutination and/or at least 50% of fixation at 1:4 (20 IU/ml) dilution were diagnosed as positive, as prescribed by the National legislation (O.M. 24-06-2015). They were also tested for Y.  enterocolitica O:9 isolation from faeces and resulted all negative. PBMC cultivation PBMC were isolated from heparinized blood samples of each experimental group as described elsewhere (La Manna et al. 2011). Then, 5 x 105 PBMC from each sample were cultured for 48 h at 37 °C-5% CO 2 with two different stimuli: YeO:9b (10 μg/ml) and B.  abortusa (10 μg/ml), in a final volume of 0.2  ml/ well of complete RPMIc in 96 well U-bottomed microplatesd. The concentration of stimuli added to the wells was chosen following a dose-response curve assay for the best antigen-specific expansion of T cell subsets (data not shown). After 48 h of culture, PBMC were collected and stained with Fluorescein Isothiocyanate (FITC) labelled monoclonal antibodies (mAb) -anti CD8 (clone CC63, mouse anti cow IgG2a),e -anti CD4 (clone 44.38, mouse anti cow IgG2a)e and -WC1 (clone 19.19,  mouse anti sheep – cross reactive with cow- IgG1a)e. After 30  minutes of incubation at 4  °C, cells were washed three times, fixed and permeabilized using a specific 152 IFN-γ+ cells to distinguish Brucella and Yersinia infections Agnone et al. Veterinaria Italiana 2019, 55 (2), 149-155. doi: 10.12834/VetIt.1374.7538.2 Yersinia treated animals were exposed to Yersinia and Brucella antigen (p < 0.05). IFN‑γ+ cells expansion in response to Brucella The results obtained in cattle naturally infected by Brucella (Figure 2) showed a statistically significant increase of IFN-γ+ lymphocytes when the cells were stimulated both by Brucella and Yersinia antigens in comparison with the control group (p  <  0.05) (Figure 2A). Data showed that in the same animals IFN-γ+ lymphocytes are similarly represented when in  vitro exposed to Yersinia (5.99%) or Brucella (5.70%). The analysis of T-cell subsets showed that CD4+ T cells produce a similar percentage of IFN-γ+ cells in response to both antigens, thus confirming cross-reactivity in cell-mediated immunity (Figure  2B). Brucella abortus-infected samples showed a higher percentage of IFN-γ+ T lymphocytes compared to controls, with statistically significant differences. IFN-γ+ PBMC and CD4+ IFN-γ+ in Brucella abortus-infected samples were also higher than in Yersinia-infected animals, maybe due to natural infections with other cross-reactive Gram-negative bacteria (Kittelberger et al. 1997). Statistical analysis In details, CD4+ IFN-γ+ T-cell subset expanded similarly in response to Yersinia (2.96% for cattle positive for Brucella abortus and in vitro stimulated with Yersinia A. IFN-γ+ T Lymphocites * * Ag Yersinia Immunized (Yersinia) Seronegative controls Ag Brucella 0 1 2 3 In vitro stimuli % o f p o si ti ve c el ls B. IFN-γ+ CD4+ T Lymphocites C. IFN-γ+ CD8+ T Lymphocites D. IFN-γ+ γδ T Lymphocites * * * * * * Ag Yersinia Immunized (Yersinia) Seronegative controls Ag Brucella 0.0 0.5 1.0 1.5 In vitro stimuli % o f p o si ti ve c el ls Ag Yersinia Immunized (Yersinia) Seronegative controls Ag Brucella 0.0 0.5 1.0 1.5 In vitro stimuli % o f p o si ti ve c el ls Ag Yersinia Immunized (Yersinia) Seronegative controls Ag Brucella 0.0 0.5 1.0 1.5 In vitro stimuli % o f p o si ti ve c el ls Figure 1. Difference of IFN-γ production between cells stimulated with the two antigens in Y. enterocolitica immunized animals. A. In Yersinia‑immunized animals the antigen‑specific stimulation resulted in a statistically significant difference of IFN‑γ+ T‑lymphocytes with respect to the seronegative controls in all the cellular subpopulation analyzed. In these animals, also a higher percentage of IFN‑γ+ T‑lymphocytes was detected, when compared with the same cells stimulated with Brucella antigen. B. Detection of CD4+ IFN‑ γ+ T‑lymphocytes. C. Detection of CD8+ IFN‑γ+ T‑lymphocytes confirmed a similar contribute of these two subsets to total IFN‑γ+ T‑lymphocytes in the two experimental groups, with a slightly higher CD4+ T cells detection. D. Detection of γδ+ IFN‑γ+ T cells amounted to 0.1% in response to YeO:9 and to 0.04% in seronegative animals. *p < 0.05. 153 Agnone et al. IFN-γ+ cells to distinguish Brucella and Yersinia infections Veterinaria Italiana 2019, 55 (2), 149-155. doi: 10.12834/VetIt.1374.7538.2 significant (p < 0.05). Following the same approach, we also compared the ratios: between CD4+ IFN-γ+ T cells and CD8+ IFN-γ+ T cells within the same groups. When the percentages of CD4+IFN-γ+ T cells are analyzed (p  <  0.05), the difference was statistically significant, whereas it was not for CD8+ IFN-γ+ T cells. Conclusions Previous data related to antigen-specific IFN-γ production measured by ELISA in Y.  enterocolitica- experimentally infected cattle re-exposed to Brucellergenea are available (Kittelberger et  al. 1997), but in the present work, for the first time, the production of antigen-specific CD4+- CD8+- and γδ- IFN-γ+ T lymphocytes were assessed by flow cytometry in animals naturally infected with Brucella antigen) and Brucella (2.77% in vitro stimulated with Brucella antigen). We did not observe any statistically significant difference when CD8+ cells were analyzed in response to the two antigens (Figure  2C). Data regarding the analysis of γδT cells revealed that the difference of IFN-γ production was not statistically significant between animals positive for Brucella and in  vitro stimulated with both antigens, while a statistically significant difference was detected when PBMC from infected and seronegative cattle were exposed to Yersinia (Figure 2D). The Mann-Whitney test has been performed to study the difference in the ratio of IFN-γ+ lymphocytes stimulated with Yersinia over those stimulated with Brucella antigen, between the animals immunized with Yersinia versus those infected with Brucella. Our results showed that this difference is statistically A. IFN-γ+ T Lymphocites * Ag Yersinia Immunized (Brucella) Seronegative controls Ag Brucella 0 2 6 8 In vitro stimuli % o f p o si ti ve c el ls B. IFN-γ+ CD4+ T Lymphocites C. IFN-γ+ CD8+ T Lymphocites D. IFN-γ+ γδ T Lymphocites * * * 4 Ag Yersinia Immunized (Brucella) Seronegative controls Ag Brucella 0 1 2 4 In vitro stimuli % o f p o si ti ve c el ls 3 Ag Yersinia Immunized (Brucella) Seronegative controls Ag Brucella 0.0 0.5 2.0 2.5 In vitro stimuli % o f p o si ti ve c el ls 1.0 1.5 Ag Yersinia Immunized (Brucella) Seronegative controls Ag Brucella 0.0 0.5 1.0 1.5 In vitro stimuli % o f p o si ti ve c el ls Figure 2. Difference of IFN-γ production between cells stimulated with the two antigens in Brucella infected animals. A. In cattle naturally infected by Brucella IFN‑γ+ T‑cells detection is similar when cells are in vitro exposed to Yersinia or Brucella antigens. B. CD4+ T cells produce IFN‑γ when exposed to both antigens. Statistical significances were assessed and percentages of IFN‑γ+ and CD4+ IFN‑γ+ T cells were compared to seronegative controls. C. No statistically significant expansion of CD8+ IFN‑γ+ T cellswas observed in response to both the antigens. D. No statistically significant expansion of γδ+ IFN‑γ+ T cellswas observed in response to both the antigens. *p < 0.05. 154 IFN-γ+ cells to distinguish Brucella and Yersinia infections Agnone et al. Veterinaria Italiana 2019, 55 (2), 149-155. doi: 10.12834/VetIt.1374.7538.2 to assess the efficacy of this method also with animals naturally infected with Yersinia. Acknowledgements We are grateful to Dr. Paolo Li Donni (Palermo University) for statistical analysis. Sources and manufacturers a. Brucellergene, OCB®Zoetis, USA. b. Yersinia enterocolitica O:9 YOP® - InstitutVirion/ Serion GmbH, Würzburg ,Germany. c. Gibco Media of Fisher Scientific UK Ltd, Loughborough, UK. d. Nunc A/S, Roskilde, Denmark. e. AbDSerotec, Kidlington, UK. f. FIX & PERM® Cell Fixation and Cell Permeabilization Kit – Thermofisher Scientific, Waltham, Massachusetts, USA. g. FACScan and Cell Quest Pro, Becton Dickinson, New Jersey, USA. Funding This study was funded by a grant of the Italian Ministry of Health “Ricerca Corrente IZS SI 10/09”. The funding body was not involved in the study design and analysis. and experimentally immunized with Yersinia after in  vitro re-exposure to the specific antigens. Our data show that in animals immunized with Yersinia there is an antigen-specific expansion of CD4+ and CD8+ T lymphocytes without any appreciable cross-reactivity, at least with Brucella. In cattle naturally infected with cross-reactivity between the in vitro Yersinia and Brucella stimulated cells has been observed and CD4+ T lymphocytes were the main producer of IFN-γ. The increase of CD4+ IFN-γ+ T cell subset was expected, since its crucial role has been already demonstrated in a murine model for the control of Brucella infection (Baldwin and Parent 2002, Vitry et al. 2012). The γδ T cells do not play a key role in response to both antigens either in vivo or in vitro, being a minor IFN-γ+ producing T cell subset when compared to CD4+ and CD8+ lymphocytes, as also reported by previous results (Vitry et al. 2012). The work carried out during this study allowed the detection, for the first time in cattle, of lymphocyte populations, i.e. CD4+ and CD8+IFN-γ+T cells, that expand differently when PBMC of animals immunized with Yersinia are in  vitro exposed to Yersinia or to Brucella. Even if this scenario was not described viceversa, the analysis of the expansion of IFN-γ+ cells in response to Brucella and Y.  enterocolitica could be useful to sort out between Yersinia and Brucella infection in cattle in case of doubtful results with the official serological methods. Anyway, further studies will be necessary 155 Agnone et al. IFN-γ+ cells to distinguish Brucella and Yersinia infections Veterinaria Italiana 2019, 55 (2), 149-155. doi: 10.12834/VetIt.1374.7538.2 Carvalho Neta A.V, Xavier M.N., Paixão T.A., Lage A.P. & Santos R.L. 2010. Pathogenesis of bovine brucellosis. Vet J, 184, 146-155. Baldwin C.L. & Parent M. 2002. 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