Veterinary Immunology and Immunopathology 163 (2015) 77–85

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Research Paper

Clinical and histological features of brucellin skin test responses in Brucella suis biovar 2 infected pigs ˜ a , I. Moriyón c , J.M. Blasco a,∗ L. Dieste-Pérez a , M. Barberán b , P.M. Munoz a Unidad de Sanidad Animal, Centro de Investigación y Tecnología Agroalimentaria (CITA) del Gobierno de Aragón, Av. Monta˜ nana 930, 50059 Zaragoza, Spain b Departamento de Patología Animal, Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain c Instituto de Salud Tropical y Departamento Microbiología y Parasitología, Universidad de Navarra, 31008 Pamplona, Spain

a r t i c l e

i n f o

Article history: Received 23 June 2014 Received in revised form 28 August 2014 Accepted 13 November 2014 Keywords: Porcine brucellosis Brucellin Hypersensitivity Histopathology

a b s t r a c t Current serological tests for swine brucellosis detect antibodies to the Brucella Opolysaccharide (O/PS). However, when infections by bacteria carrying cross-reacting O/PS occur, these tests suffer from false positive serological reactions (FPSR), and the skin test with Brucella soluble protein extracts is the best diagnostic alternative to differentiate true Brucella suis infections from FPSR in pigs. Since this test has been seldom used in B. suis infected swine, the clinical and histological features involved have not been described properly. Here, we describe the clinical and histological events in B. suis biovar 2 infected pigs skin tested with a cytosoluble O/PS free protein extract from rough Brucella abortus Tn5::per mutant. A similar extract from rough Ochrobactrum intermedium was also used for comparative purposes. No relevant differences were evidenced between the homologous and heterologous allergens, and the main clinical feature was an elevated area of the skin showing different induration degrees. Moreover, an important vascular reaction with hyperemia and haemorrhage was produced in most infected sows 24–48 h after inoculation, thus facilitating the clinical interpretation of positive reactions. Histologically, combined immediate (type III) and delayed (type IV) hypersensitivity reactions were identified as the most relevant feature of the inflammatory responses produced. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Brucellosis produced by Brucella suis biovar 2 is emerging in Europe, causing reproductive failure in pigs and economic losses for the swine industry (Garin Bastuji et al., 2000; Godfroid and Kasbohrer, 2002; EFSA, 2009). Both the wild boar (Sus scrofa) and European hare (Lepus europaeus) are the wild reservoirs of this biovar (Garin Bastuji et al., 2000; Abdoel et al., 2008; Cvetnic et al., ˜ et al., 2010), and the infection can be readily 2009; Munoz

∗ Corresponding author. Tel.: +34 976 716460. E-mail address: [email protected] (J.M. Blasco). http://dx.doi.org/10.1016/j.vetimm.2014.11.009 0165-2427/© 2014 Elsevier B.V. All rights reserved.

transmitted to domestic pigs through contacts with those wild animals (Garin Bastuji et al., 2000; Abdoel et al., ˜ 2008; Cvetnic et al., 2009; Munoz et al., 2010). Because of this, sporadic outbreaks occur in both outdoor and backyard farms in several European countries (Garin Bastuji et al., 2000; Godfroid and Kasbohrer, 2002; Hubálek et al., 2002; Cvetnic et al., 2003; Al Dahouk et al., 2005; Gennero et al., 2006; Leuenberger et al., 2007; EFSA, 2009), and active surveillance programmes using suitable diagnostic tools have to be implemented in domestic swine. The tests currently recommended for the serological diagnosis of swine brucellosis (i.e. rose bengal –RBT –, complement fixation, indirect and competition/blocking enzyme-linked immunosorbent assays – ELISA –, and the fluorescence

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polarisation assay) detect antibodies to the Brucella Opolysaccharide (O/PS) of the smooth lipopolysaccharide (S/LPS) (Nielsen et al., 1999; Paulo et al., 2000; OIE, 2012). However, many pig farms in swine brucellosis-free countries are affected by false positive reactions (FPSR) in all these O/PS based tests (Corbel, 1985; Perry and Bundle, 1990; EFSA, 2009; Weiner et al., 2013). This generates cumbersome and costly retesting schedules that represent one of the most frequent causes of trade restrictions in swine in the EU (Jungersen et al., 2006). Obviously, the important derived sanitary and economic consequences make necessary to unequivocally confirm a positive serological result, and this has been attempted using several serological test combinations (Weynants et al., 1996; Nielsen et al., 2006; McGiven et al., 2012). However, such combinations of tests are not only cumbersome and expensive but also lack 100% specificity to differentiate brucellosis from FPSR. In contrast to O/PS tests, immunoassays using Brucella cytosoluble proteins (brucellin) are not affected ˜ by FPSR (Baldi et al., 1996; Munoz et al., 2005; Riber and Jungersen, 2007). Unfortunately, these tests lack sensitivity at individual level in infected pigs (McGiven, 2013). On the other hand, as B. suis infection induces also cellular mediated immune-responses to these protein antigens, the skin test performed with brucellin-type or, alternatively, with cytosoluble protein extracts is considered of choice in FPSR contexts (EFSA, 2009; OIE, 2012; Olsen et al., 2012; Dieste-Pérez et al., 2014). However, these studies are scanty in swine (EFSA, 2009), and the clinical and histological features of the responses involved have not been described properly. As an alternative to the classical brucellin obtained from Brucella melitensis B115, which contains O/PS and may thus cause specificity problems (Dieste-Pérez et al., 2014), we demonstrated recently that cytosoluble extracts from rough Brucella abortus mutants devoid of O/PS are suitable for skin testing B. suis biovar 2 naturally infected pigs (Dieste-Pérez et al., 2014). Similar cytosoluble extracts from Ochrobactrum intermedium, a phylogenetic Brucella relative, induce positive skin test reactions in Brucella ovis infected sheep (Velasco et al., 1997), but this cross-reactivity has been never proven in pigs. The objective of this work is to describe the main clinical and histological features of skin test responses produced in B. suis biovar 2 naturally infected pigs after intradermal inoculation with either homologous B. abortus or heterologous O. intermedium O/PS free cytosolic extracts.

a very close phylogenetic Brucella relative (Velasco et al., 1998). The growth conditions of both strains and the extraction and characterisation of the corresponding allergenic cytosoluble extracts were made as described previously (Blasco et al., 1994; Velasco et al., 1997; DiestePérez et al., 2014). Briefly, both strains were grown in 1.7% casitone (Pronadisa, Madrid, Spain), 0.3% soy tone (E. Merck, Darmstadt, Germany), 0.5% yeast extract (Merck, Darmstadt, Germany), 0.25% K2 HPO4 , 2% glucose, 0.5% NaCl, and 0.01% A-butyl acetate (1:3) (Sigma Chemical CO., St. Louis, MO, USA), in a 15 l Biostat fermentor (B. Braun Melsungen AG, Leinfelden, Germany) for 48–72 h at 36 ◦ C. Bacteria were then inactivated with phenol (0.5%, 37 ◦ C, 48 h), washed twice with sterile saline (0.85% NaCl), and harvested by centrifugation (9000 rpm, 12 min). Cytosolic extracts were obtained from bacterial suspensions in 0.1 M phosphate-buffered saline (pH 7.2) containing DNA-ase and RNA-ase (50 ␮g per ml of suspension) by disintegration in a 40 K French Pressure cell (SLM Instruments Inc., Urbana, IL, USA), operating at 40,000 psi. The total protein contents of the cytosoluble fractions were calculated colorimetrically (Markwell et al., 1978) using bovine serum albumin as standard. The total protein content in both cytosolic extracts was around 45%. Moreover, the B. abortus Tn5::per and O. intermedium fractions were further characterised using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by periodate silver staining (Tsai and Frasch, 1982) and immunoelectrophoresis (Grabar et al., 1955) with sera from B. suis biovar 2 infected and brucellosis free pigs. SDS-PAGE profiles of both cytosols showed only minor qualitative and/or quantitative differences, and all consisting of 20–25 major polypeptides ranging from apparent molecular masses of over 100 kDa to less than 10 kDa (not shown). Immunoelectrophoretic analyses of B. abortus Tn5::per cytosolic extracts revealed 3–8 protein bands when tested against the sera of ten sows infected by B. suis biovar 2 (reference serum collection of CITA, Gobierno de Aragón, Zaragoza, Spain). When O. intermedium cytosol was tested against these same sera, 2–4 precipitin bands were observed. Western blots with the same sera evidenced major reactivity bands in the ranges of 10–20 kDa and 35–60 kDa in both cases (not shown). Similar assays carried out with the sera from ten Brucella-free pigs (belonging to the same CITA reference serum collection), resulted always in absence of reactivity.

2. Materials and methods

2.2. Animals and skin test experiments

2.1. Bacterial strains, growth conditions and allergen extraction and characterisation

All procedures were performed in accordance with the current European (Directive 2010/63/UE, DOUE L 276/33), National (Real Decreto 1201/2005 and 53/2013, in BOE number 252 and 34, respectively) and Regional (Ley 11/2003, in BOA number 35) rules, and approved by the CITA Ethics Committee for Animal Experimentation (report No. 2011-2). The animals belonged to two different herds (A and B), both clinically affected by abortions and reduced fertility after a brucellosis outbreak caused by the same ˜ et al., 2010) confirmed B. suis biovar 2 genotype (Munoz by culture of aborted sows (Dieste-Pérez et al., 2014). The negative controls were obtained from an officially

For obtaining the homologous allergen we used B. abortus Tn5::per, a rough mutant from the smooth B. abortus 2308 reference strain carrying a mini-Tn5 inserted in per (ORF BAB1 0544), the putative perosamine synthase gene, and thus devoid of O/PS (Monreal et al., 2003). For obtaining the heterologous allergen, we used O. intermedium LMG 3301 (Velasco et al., 1998), which also lacks S-LPS. O. intermedium is an opportunistic human pathogen belonging to the alpha 2 subgroup of Proteobacteria, and

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brucellosis-free accredited herd not affected by the FPSR problem. 2.2.1. Experiment 1: assessment of the optimal allergen doses and reaction reading times In the first experiment, the optimal conditions for using the B. abortus Tn5::per and O. intermedium cytosolic extracts were determined. Positive controls were 10 B. suis biovar 2 infected sows (all culture-positive) belonging to herd A, and negative controls were 10 sows belonging to the accredited brucellosis-free herd. All animals were inoculated intradermally – ID – by the same researcher (for each group, 5 with the homologous and 5 with the heterologous allergens) in four close sites (two on each side of the perineal region close to the tail) with 0.1 ml of four allergen dilutions (0.06 mg/ml, 0.12 mg/ml, 0.5 mg/ml and 2 mg/ml) in sterile saline (i.e. each animal received 6 ␮g, 12 ␮g, 50 ␮g and 200 ␮g of each allergen). Whenever possible, non-pigmented areas of the skin were selected for the inoculations to facilitate an assessment (both visual inspection and palpation) of the clinical responses which was done independently by three trained researchers in order to avoid personal biases at 20 min, 24, 48 and 72 h after inoculation. This experiment showed that the conditions resulting in 100% agreement among these researchers, and in the best clinical assessment of the positive skin test responses were inoculation of 50 ␮g of allergen/animal (no matter the allergen), and a post-inoculation time of 48 h. 2.2.2. Experiment 2: comparative study of B. abortus Tn5::per and Ochrobactrum intermedium skin test responses Using the optimal conditions obtained in the Experiment 1, we conducted a second trial to characterise the main clinical and histological features of the hypersensitivity responses and to assess the agreement between the two allergens. For this purpose, 7 B. suis infected sows of herd B (all culture positive for B. suis 2) were ID inoculated by the same researcher involved in the first experiment with 50 ␮g of each allergen on both sides of the perineal region. The same procedure was applied to 7 sows from the officially brucellosis free accredited herd (negative controls). The clinical features of the hypersensitivity reactions were assessed 48 h after inoculation by the same three trained researchers involved in Experiment 1 by visual inspection and palpation of the inoculated area, and by measuring also the skin thickness with the same vernier-scaled calliper. Moreover, skin biopsy samples from both inoculation sites were taken by the same researcher for histological examination. 2.2.3. Experiment 3: kinetics of the clinical and histological responses after skin test with B. abortus Tn5::per allergen To further determine the kinetics of the main clinical and histological features of skin responses to the B. abortus Tn5::per allergen, we used 64 herd A sows of unknown bacteriological status, all positive in RBT and ˜ indirect ELISA, performed as described previously (Munoz et al., 2012). These sows were allotted at random in 4 groups of 16 animals each, and then skin tested by the

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same researcher involved in the above experiments with a single ID inoculation of 50 ␮g of B. abortus Tn5::per allergen. The development of skin reactions was assessed clinically 20 min after inoculating the first group of sows, and then 24, 48 and 72 h thereafter in second, third and fourth groups, respectively. Again, the clinical reactions were assessed macroscopically by visual inspection and palpation in a blind manner by the same three researchers involved in Experiments 1 and 2. Moreover, biopsy samples for histological study were taken as above from all animals at the corresponding reading intervals. 2.2.4. Experiment 4: effects of repeated inoculation with B. abortus Tn5::per allergen A final trial was conducted to assess if the clinical and histological features of skin responses could be affected by repeated inoculation. For this we used a total of 75 herd B sows of unknown bacteriological status, all positive in both ˜ et al., 2012) and also in a first skin RBT and iELISA (Munoz testing performed as in Experiment 2 with the B. abortus Tn5::per allergen. Immediately after this skin testing, the sows were allotted at random in 5 groups of 15 animals each. The animals from group 1 were then re-inoculated similarly one week after the first inoculation, while the groups 2, 3, 4 and 5 were re-inoculated again 2, 3, 4 and 6 weeks thereafter, respectively. The clinical features of the hypersensitivity responses were assessed 48 h after inoculation in a blind manner as above, and biopsy samples for histological study were taken simultaneously. 2.3. Clinical and histological procedures The macroscopic appearance of the skin reaction was evaluated by visual inspection and palpation of the inoculated area (Riber and Jungersen, 2007; OIE, 2012; Dieste-Pérez et al., 2014) and lesions classified as erythema (skin redness), papule (swelling smaller than 1 cm diameter) or nodule (evident local swelling bigger than 1 cm diameter), accompanied or not with erythema. When appropriate, the diameter of reaction was also measured with a vernier-scaled type calliper. For histological studies, biopsy samples of representative tissue (containing the epidermis, and superficial and deep dermis) were taken by the same trained researcher using 5 mm diameter biopsy punchers (Henry Schein, Madrid, Spain). Biopsy samples were kept in a 10% formaldehyde neutral solution for 24 h, then included in paraffin and processed and stained with haematoxylin–eosin (HE) by standard procedures. All preparations were analysed by the same pathologist using an Olympus Vanox AHBS3 microscope and the images recorded with an Olympus DP12 digital camera. To quantify the intensity of histological lesions for comparative purposes, a semi-quantitative system was used converting into figures the most relevant histological features identifying either a type III immediate (T3) or type IV delayed (T4) hypersensitivity responses. For the former, each of the main characteristics (i.e. hyperemia, oedema, haemorrhage and presence of polymorphonuclear – PMN – cells in the epidermis, blood vessels or infiltrated in the connective tissue) was scored as 0 (absence), 1 (mild), 2 (moderate)

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Fig. 1. Macroscopical features of the skin test reaction produced after intradermal inoculation with 50 ␮g of either the homologous B. abortus Tn5::per or heterologous O. intermedium allergens in the perineal region of a B. suis biovar 2 infected sow. Notice the important erythematous nodules at the injection sites produced at 48 h after inoculating simultaneously both the homologous (left arrow) and heterologous (right arrow) allergens.

or 3 (severe) using the same preparation representative of each intensity grade as a reference. For T4 reactions, the abundance of macrophages or lymphocytes in connective tissue was scored similarly from 0 to 3. Mean of the scores observed in individual lesions were calculated when appropriate to obtain a value for the overall reaction intensity.

did not show a clinical response 24 h after inoculation, and all displayed very clear clinical signs 48 h after inoculation. No matter the allergen, the response was always more evident at 48 h than at 24 h, with diffuse to nodular skin elevated areas varying in induration degrees and colour from reddish to an almost black (Fig. 1). Although macroscopic reactions at 72 h did not differ significantly from those observed at 48 h, only nodules or papules were evident and the erythema decreased or even disappeared in some infected sows. Altogether, the optimal interval to detect the most evident clinical inflammatory responses was 48 h, no matter the allergen. No significant discrepancies were obtained in the clinical assessments made in a blind manner by the three different researchers involved. In general, the intensity of the clinical responses increased with the allergen dose, but no significant differences were evidenced among 50 ␮g and 200 ␮g doses, no matter the allergen. Accordingly, 50 ␮g was selected as the optimal dose of both allergens for further experiments. Neither clinical signs nor histological responses were evidenced in the ten brucellosis-free control sows used in this experiment at any time after inoculation, and no matter the allergen inoculated. 3.2. Experiment 2: comparative study of B. abortus Tn5::per and Ochrobactrum intermedium skin test responses

3. Results

As expected (Velasco et al., 1997), the clinical results obtained with B. abortus Tn5::per and O. intermedium cytosols in the seven B. suis biovar 2 infected sows showed a high concordance, with a similar appearance of nodules and erythema and no differences that could be appreciated by palpation/visual inspection (Fig. 1). However, when the skin thickness was compared, the B. abortus Tn5::per cytosol induced stronger reactions than its O. intermedium counterpart (13.57 ± 4.46 mm versus 12.79 ± 2.91 mm; p < 0.05). In agreement with this, a somewhat higher lesion scores (not shown) were obtained when examining biopsy samples with the homologous allergen. No matter the allergen, most histological features in B. suis biovar 2 infected sows were characteristic of a combined immediate (T3) and delayed-type IV (T4) hypersensitivity response (10 out of the 14 skin samples analysed showed this combined response and the remaining four presented a typical T4 response). Since the above-described results indicate a clearer interpretation for routine diagnosis when using the B. abortus Tn5::per allergen, the experiments described below were performed using only this allergen.

3.1. Experiment 1: assessment of the optimal allergen doses and reaction reading times

3.3. Experiment 3: kinetics of skin test responses with B. abortus Tn5::per allergen

The 10 B. suis biovar 2 infected sows tested showed evident clinical skin responses upon inoculation with either the homologous or heterologous allergen (Fig. 1). Although not evidenced in all cases, a mild focal erythema was the most frequent clinical sign in the early 20 min after allergen inoculation, and the intensity of the skin reaction increased thereafter. Only one of these infected sows

The 64 seropositive sows selected from herd A showed a positive skin test response after inoculation with the B. abortus Tn5::per allergen. The main histological features representative of T3 and T4 hypersensitivity responses at different time intervals are summarised in Fig. 2. Although some samples taken 20 min after inoculation showed hyperemia and oedema in the superficial papillary

2.4. Statistical analyses When applicable, the statistical analysis of both clinical and histological results was made using the R Commander interface to R (R Foundation for Statistical Computing, Vienna, Austria). Differences in both the diameter of the reaction and the scores of the clinical lesions were analysed by the paired Student’s t-test, after verifying the normality assumption. In both cases, differences were tested at a p < 0.05 level. Since the normality assumption was rejected when comparing the scores of lesions after repeated allergen inoculation (Section 2.2.4), statistical analysis of data was made with the Kruskal–Wallis one-way analysis of variance, using also a p < 0.05 level. Finally, the chi-square test was used for analysing categorical variables to determine the association between the reading intervals and the intensity of histological lesions.

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Fig. 2. Microscopic features representative of the three lesion degrees evidenced after inoculation of pigs with the B. abortus Tn5::per allergen, and scored as Grades 1 (mild; panels A and B), 2 (moderate; panels D–F) and 3 (severe; panels G–I). Panel C: negative control showing the absence of lesions after allergen inoculation in a Brucella-free sow. Panels E and H: upper and deep dermis, respectively; panels F and I: deep dermis and hypodermis, respectively; Panels A, D and G: examples of immediate type III (T3) hypersensitivity responses; panels B, E, F, H and I: examples of combined T3 immediate and type IV (T4) delayed type hypersensitivity responses. Notice the hyperemia, hemorrage and cellular infiltration by neutrophils in the dermis ( ), and the cellular infiltration by neutrophils in the deep dermis ( ). Notice also the infiltrates of lymphocytes and macrophages around blood vessels in the dermis ( ) and hypodermis ( ). These vascular and cellular reactions are shown with detail in the inserts of panels D, G, F and I. H&E stain. Panel E size bar (200 ␮m) is representative for all panels.

dermis (not shown), these were only of low intensity and not indicative of a typical hypersensitivity reaction. In contrast, all samples taken 24 h after inoculation showed reactions characterised by vasodilation, hyperemia and recruitment of PMNs in vascular lumens of arterioles and capillaries, haemorrhage, and deposition of oedematous fluid with fibrin in the perivascular connective tissue. Cellular infiltration adjacent to the blood vessels in the deep dermis, was composed essentially by PMNs and some macrophages (panels D and G). These microscopical features are considered as characteristic of an immediate T3 hypersensitivity reaction. Forty-eight hours after inoculation, the reaction was predominantly of cellular type, localised around blood vessels in the dermis (panel E) and hypodermis (panels F, H and I), and composed of mononuclear cells, essentially lymphocytes (panel I insert) and macrophages (panel F insert). Hyperemia and oedema were not evident but fibrin and haemorrhages were still present (panels E, F, H and I). Altogether, these microscopical features evidenced 48 h after inoculation were representative of a combined immediate (T3) and delayed-type (T4) hypersensitivity response. However, 72 h after allergen inoculation (not shown), a delayed T4 hypersensitivity reaction was dominant, as

evidenced by the large infiltrates of mononuclear inflammatory cells, with a special increase of lymphocytes. The kinetics and mean score values of the main histological features observed 24, 48 and 72 h after inoculating the B. abortus Tn5::per allergen in these 64 seropositive herd A sows are shown in Fig. 3. The oedema was relatively constant throughout with low to moderate average scores the first two time intervals, that did not increase significantly at 72 h. Hyperemia and haemorrhage reached moderate to high scores at early times (24 h), then decreasing significantly (p < 0.01 and p < 0.001, respectively). The PMN infiltrates in the connective tissue remained relatively constant at moderate to high scores, but a significant decrease (p < 0.05) was evidenced after 24–48 h. As expected, infiltrates of both lymphocytes and macrophages increased directly and significantly (p < 0.001) according the time interval, being both cells very abundant at 48 h and 72 h. 3.4. Experiment 4: effect of repeated inoculations with B. abortus Tn5::per allergen The 75 herd B seropositive sows used, all of which were positive in the first skin testing with the B. abortus Tn5::per allergen, resulted also positive after allergen

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Fig. 3. Kinetics and intensity (mean scores) of the main histological features evidenced at different time intervals after skin testing with the B. abortus Tn5::per allergen. Statistically significant associations among reading intervals and the intensity of the histological features are indicated (*p < 0.05; **p < 0.01; ***p < 0.001).

re-inoculation, no matter the time elapsed between inoculations. No significant difference in the intensity of the reactions was observed between the inoculation and reinoculation reactions either by simple visual inspection and palpation of inoculation site or by measuring the diameter of skin reaction. Moreover, most (61 out of 75) of the clinically positive skin samples examined histologically after re-inoculation also showed features of a combined immediate (T3) and delayed (T4) hypersensitivity response. 4. Discussion The precise causes of the FPSR problem have not been investigated extensively but Y. enterocolitica O:9 is considered the major source in swine (Thibodeau et al., 2001; EFSA, 2009). Because of the high prevalence of FPSR and the important consequences that swine brucellosis has for international trade, any positive serological result has to be confirmed using suitable differential tests based in specific antigens not shared between Brucella and the bacteria causing FPSR. Thus far, the best differential strategy is based in a simple “in vivo” skin test using Brucella soluble protein extracts (brucellin or cytosoluble proteins) that detect skin hypersensitivity reactions in brucellosis infected animals but not in those affected by FPSR (Pouillot et al., 1997; Saegerman et al., 1999; EFSA, 2009; OIE, 2012; Olsen et al., 2012; Dieste-Pérez et al., 2014). With the exception of O. intermedium and some plant-associated relatives (Velasco et al., 1997), all of which are considered of no relevance in Brucella natural hosts, no Brucella soluble proteins have been proven to cross-react with similar proteins of other bacteria, including those causing FPSR (Díaz, 1974; Riber and Jungersen, 2007). As it could have some practical interest to obtain antigens for diagnosis, we were interested in investigating if some clinical and histological differences could be evidenced after skin testing brucellosis infected swine with the homologous and heterologous allergens. As it has been previously proven in

comparative experiments using B115 brucellin in sheep (Velasco et al., 1997), our results demonstrate that O. intermedium cytosolic proteins induce skin reactions in B. suis biovar 2 infected pigs, similar to that evoked by the homologous B. abortus Tn5::per allergen. However, although this shows their efficacy for diagnosing swine brucellosis, the homologous allergen allowed a clearer interpretation of the positive skin test responses in B. suis biovar 2 infected pigs. It is accepted that two main skin hypersensitivity reactions are produced in brucellosis infected animals after intradermal inoculation of Brucella antigens: (i) an immediate T3 hypersensitivity response due to antigen–antibody complexes activating complement, which attracts neutrophils and cause inflammation and tissue damage; and (ii) a delayed T4 hypersensitivity response mediated by cytokine expression at the inoculation sites, attracting monocytes from bloodstream. Both T3 and T4 hypersensitivity reactions lead to clinical and histological signs that are of potential diagnostic value. The skin test has been seldom used in pigs and, therefore, there is very limited information on the nature, kinetics and characteristics of the clinical and histological features of the hypersensitivity responses in swine brucellosis (Delez et al., 1947; Stuart et al., 1987). This paucity of histopathological information also extends to cattle brucellosis (Cheville et al., 1994), and in both animal species is negatively affected by the inappropriateness of the antigens used. Some authors (Delez et al., 1947) reported that B. suis biovar 3 infected pigs skin tested with crude soluble extracts of smooth Brucella developed reactions characterised by a predominant PMN infiltration during the first 48 h followed by a mononuclear cell infiltration at later times. However, as expected and demonstrated by the increase in agglutination titres in the inoculated pigs, these crude extracts contained S-LPS, a molecule known to induce unspecific skin responses in healthy animals (Jones et al., 1973). Therefore, the reactions described were probably unspecific, an intepretation consistent with the fact that PMN infiltrates were also

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observed in healthy pigs 48 h after inoculation with the crude extracts but not with saline (Delez et al., 1947). In studies in B. abortus infected cattle, the histological features of skin lesions (determined only 48 h after inoculation) evidenced a cellular reaction involving mainly monocytes and macrophages (Cheville et al., 1994). However, the antigen used was also obtained from a smooth strain (B. abortus 2308) and the probable presence of S-LPS was not ruled out, which also precludes a clear-cut interpretation of those results. Delayed-type hypersensitivity reactions have also been described in pigs experimentally infected with B. abortus 544 inoculated ID with brucellin obtained from rough B. melitensis B115. These reactions were characterised by infiltrations of lymphocytes and plasma cells and some PMNs (Stuart et al., 1987), the histological signs characteristic of a combined T3/T4 hypersensitivity. However, although unable to synthesise a complete S-LPS, B. melitensis contains O/PS linked to the inner leaflet of the cytosolic membrane (Cloeckaert et al., 1992) and, in fact, a rise in agglutination titres was noticed after B115 brucellin inoculation in several of the B. abortus 544 experimentally infected pigs (Stuart et al., 1987). Thus, the reactions observed in this study are likely to be due in part to the reaction of anti-S-LPS antibodies with the O/PS in the extracts. We have recently proven that cytosoluble protein extracts obtained from rough B. abortus mutants (including per mutants) are free of O/PS and suitable alternatives to the classical B. melitensis B115 brucellin for diagnosing swine brucellosis in FPSR contexts (Dieste-Pérez et al., 2014). Accordingly, the B. abortus Tn5::per (and the O. intermedium LMG 3301, since this strain lacks S-LPS) allergens used in our study were free from O/PS and composed essentially by cytosoluble proteins that do not cross react with bacteria causing FPSR. No matter the allergen inoculated, most clinical and histological features identified in B. suis biovar 2 infected sows corresponded to a combined immediate (T3) and delayed (T4) hypersensitivity response (Fig. 2) causing clinical lesions relatively easy to assess (Fig. 1), and of practical diagnostic value. In agreement with other studies (Riber and Jungersen, 2007; OIE, 2012; Dieste-Pérez et al., 2014), the most practical method for interpreting the skin test responses in B. suis infected swine is assessing the clinical inflammatory response by simple visual inspection and palpation of the inoculated zone (under many current pig management conditions, the tail base is preferred to the ear) 48 h after inoculation. Measuring the diameter of the skin lesion with a vernier-type calliper is also feasible, but this procedure is cumbersome and does not offer clear advantages over the visual inspection and palpation. In fact, no sows showing positive reactions by calliper measurement resulted skin test negative by latter method in any of experiments conducted. Because antibodies against cytosolic proteins are common during the natural course of brucel˜ et losis (Kittelberger et al., 1995; Baldi et al., 1996; Munoz al., 2005), the T3 reactions are an expected event after skin testing with cytosoluble proteins. Indeed, a high proportion of the infected sows used in our experiments reacted also in gel precipitation tests using cytosolic proteins as antigen (not shown). Thus, antibody-mediated T3 reactions

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were produced in most cases, causing erythema, oedema and vascular lesions after inoculation. This T3 reaction began to decrease at 48 h, and in most cases the predominant histological features from 48 h (Fig. 2 panels B and E–I) to 72 h (not shown) were those typical of a delayed T4 hypersensitivity response, that resulted in clearly visible/palpable clinical alterations. This is consistent with the total agreement obtained in the blinded assessment of clinical features made by the three independent researches involved in the experiments. Repeated skin testing is frequently required for routine swine brucellosis surveillance and confirmation (DiestePérez et al., 2014), and this could be a cause of anergy. This phenomenon has been described both in tuberculosis infected cattle (Radunz and Lepper, 1985) and brucellosis infected sheep (Blasco et al., 1994), but did not occur in B. suis biovar 2 infected pigs. However, the persistence of cellular reactivity after allergen re-inoculations was not absolute, and biopsy samples taken at 48 h from some animals after repeated allergen inoculation showed only T3 reactions (not shown). Moreover, this T3 antibody mediated response was present in all cases after re-inoculation, even in those sows in which T4 predominated (not shown).

5. Conclusion No significant differences in the clinical and histological features after skin testing B. suis biovar 2 infected sows have been evidenced using homologous (B. abortus Tn5::per) and heterologous (O. intermedium) allergens, but the overall interpretation was clearer using the homologous allergen. The clinical features of skin testing were characterised by elevated areas of the skin with different induration degrees. A clearly evident vascular reaction characterised by hyperemia and haemorrhage was present in most infected sows 24–48 h after allergen inoculation. These evident clinical features facilitated the macroscopical assessment of positive skin reactions, eliminating the need for measuring skin thickness or lesion diameters with a calliper. Histologically, a combined immediate (T3) and delayed (T4) hypersensitivity reaction was identified as the most relevant feature of the inflammatory responses produced after skin testing B. suis biovar 2 infected pigs.

Conflict of interest None declared.

Acknowledgements This research was supported in part by a PhD grant (L. Dieste-Pérez) from the Aragón Government (2011–2015) and by the following projects: INIA project RTA201100103-00-00, MICIN project AGL2011-30453-C04, and consolidated group from Aragón Government A14. We wish to acknowledge A. Delgado and P. Eseverri for the excellent technical assistance.

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