Brucella abortus Strain RB51 Administered to Prepubescent Water Buffaloes, from Vaccination to Lactation: Kinetics of Antibody Response and Vaccine Safety.

Brucella RB51 is a live modified vaccine. Its use in water buffalo has been proposed using a vaccination protocol different to that used for cattle, but knowledge of the long-term effects of RB51 vaccination in this species remains incomplete. The aim of the study was to evaluate the safety and kinetics of antibody responses in water buffaloes vaccinated according to the protocol described for the bovine species in the WOAH Manual, modified with the use of a triple dose. Water buffaloes were vaccinated with the vaccine RB51. A booster vaccination was administered at 12 months of age. When turning 23-25 months old, female animals were induced to pregnancy. RB51-specific antibodies were detected and quantified using a CFT based on the RB51 antigen. Vaccinated animals showed a positive serological reaction following each vaccine injection, but titers and the duration of the antibody differed among animals. For 36 weeks after booster vaccination, the comparison of CFT values between vaccinated and control groups remained constantly significant. Afterwards, antibody titers decreased. No relevant changes in antibody response were recorded during pregnancy or lactation. In conclusion, results indicated that the vaccination schedule applied is safe and allows for vaccinated and unvaccinated controls to be discriminated between for up to 8 months after booster vaccination.

Lentilactobacillus kefiri SGL 13 and Andrographis paniculata alleviate dextran sulfate sodium induced colitis in mice.

Introduction: Inflammatory bowel diseases (IBD) are chronic inflammatory conditions that typically involve diarrhea, abdominal pain, fatigue, and weight loss, with a dramatic impact on patients' quality of life. Standard medications are often associated with adverse side effects. Thus, alternative treatments such as probiotics are of great interest. The purpose of the present study was to evaluate the effects of oral administration of Lentilactobacillus kefiri (basonym: Lactobacillus kefiri) SGL 13 and Andrographis paniculata, namely, Paniculin 13™, on dextran sodium sulfate (DSS)- treated C57BL/6J mice. Methods: Colitis was induced by administering 1.5% DSS in drinking water for 9 days. Forty male mice were divided into four groups, receiving PBS (control), 1.5% DSS, Paniculin 13™ and 1.5% DSS + Paniculin 13™. Results: The results showed that body weight loss and Disease Activity Index (DAI) score were improved by Paniculin 13™. Moreover, Paniculin 13™ ameliorated DSS-induced dysbiosis, by modulating the gut microbiota composition. The gene expression of MPO, TNFα and iNOS in colon tissue was reduced and these data matched with the histological results, supporting the efficacy of Paniculin 13™ in reducing the inflammatory response. No adverse effects were associated to Paniculin 13™ administration. Discussion: In conclusion, Paniculin 13™ could be an effective add-on approach to conventional therapies for IBD.

Assessment of Welfare in Groups of Horses with Different Management, Environments and Activities by Measuring Cortisol in Horsehair, Using Liquid Chromatography Coupled to Hybrid Orbitrap High-Resolution Mass Spectrometry.

Horses have always been animals used for companionship, work, transportation, and performance purposes over the history of humanity; there are different ways of managing horses, but studies on how horse welfare is influenced by different activities and managements are scanty. Understanding how the management, the environment, and the different uses of horses can affect the level of stress and well-being is important not only for people associated with horses. Three groups of horses with different management, environments, and activities were selected: (1) stabled horses ridden frequently, (2) horses that perform public order service under the Italian state police, and (3) free-ranging horses. Cortisol analysis was carried out on horsehair samples using liquid chromatography coupled to hybrid orbitrap high-resolution mass spectrometry (LC-HRMS/MS), a laboratory technique used for the first time to quantify horsehair cortisol. The selection of horses to be included in the three groups was carried out by including only subjects with positive welfare assessment in accordance with the horse welfare assessment protocol (AWIN). These analyses demonstrated that the cortisol levels detected in the horsehair of free-ranging animals were significantly higher compared to those detected in stabled and working horses. These results may have been a consequence of complex environmental, managerial, and behavioral factors, which should be worth further investigation.

Animal Welfare Assessment in Antibiotic-Free and Conventional Broiler Chicken.

The poultry sector is moving towards antibiotic-free production, both to challenge the increasing spread of the antibiotic resistance phenomenon and to meet market demands. This could negatively impact the health and welfare of the animals. In this study, the welfare of 14 batches of 41-47-day-old broilers raised by the same integrated company with and without antibiotics was assessed using the Welfare Quality® protocol. The total welfare score did not significantly differ between the two systems: the good-feeding principle was, on average, higher in the conventional batches, with statistical significance (t = -2.45; p = 0.024), while the other welfare principles (good housing, good health and appropriate behaviour) were slightly better in the antibiotic-free batches. Despite stocking densities averagely higher in the antibiotic-free batches, the absence of antibiotics did not seem to impact the good-health principle; in particular, hock burns, foot pad dermatitis and lameness were significantly less severe in the antibiotic-free batches (p < 0.0001, p = 0.018, p < 0.0001, respectively), which showed also a lower death rate (2.34% vs. 2.50%). Better management of antibiotic-free batches was reported, particularly concerning litter conditions. Further studies would be required to identify and standardise a set of managerial methodologies in order to improve the health of broilers raised without antibiotics.

Humoral immune response and delayed-type hypersensitivity in rabbits infected with Trypanosoma equiperdum.

Trypanosoma equiperdum is the causative agent of dourine, a parasitic venereal disease of equids. In this work, rabbits were infected with T. equiperdum strain OVI; serological tests (complement fixation test, ELISA and immunoblotting), used for the diagnosis of dourine in horses, were applied to study rabbit humoral immune response and to characterise T. equiperdum antigen pattern recognised by antibodies from infected rabbits. Moreover a protein extract of T. equiperdum strain OVI was produced and tested in skin tests on infected rabbits to detect the cell-mediated response induced by T. equiperdum, in order to evaluate its use in the field diagnosis of dourine. Sera of infected rabbits recognized in immunoblotting Trypanosoma protein bands with molecular weight below 37 kDa, providing a serological response comparable with that already observed in dourine infected horses. Moreover the trypanosome protein extract was capable to produce in vivo delayed-type hypersensitivity (DHT Type IV) in rabbits and proved itself to be non-toxic and non-sensitizing.

Study on the Public Perception of "Community-Owned Dogs" in the Abruzzo Region, Central Italy.

The Abruzzo Regional Law Nr. 47/2013, following a circular from the Italian Ministry of Health and OIE recommendations, allows the local municipalities to release free-roaming dogs (FRDs) caught on the territory once the local veterinary services (LVSs) have rated the dogs as unowned and not aggressive, have neutered them, as well as identified them through a microchip and a visible collar. The responsibility of these "community-owned dogs" (CODs) falls under the mayor of the local municipality that can entrust their custody to qualified people. The present study was conducted in the Abruzzo region, located in central Italy, to investigate public perception towards CODs, and in particular whether their presence is perceived as a problem or a benefit by the local communities. The data were collected by both direct interviews and an online survey, based on a questionnaire developed by a multidisciplinary team. The questionnaire was distributed in 31 municipalities sampled on the basis of the urbanization rate, and 497 people were interviewed over a 9-month period. More than half of the respondents (54%) stated that CODs can contribute to the control of stray dogs. The majority (83%) believed that a greater commitment is needed to involve the local communities on issues regarding CODs. The findings of this study highlighted the general difficulty for people to distinguish stray dogs from CODs that are not fully known, as evidenced by the fact that 59% of the respondents did not know the aforementioned Regional Law that defines and regulates the presence of the CODs.

Alternative methods to reduce the animal use in quality controls of inactivated BTV8 Bluetongue vaccines.

The acceptance of serology data instead of challenge for market release of new batches of commercial vaccine is under evaluation by regulatory agencies in order to reduce the use of animals and costs for manufacturers. In this study two vaccines for Bluetongue virus serotype 8 were submitted to quality controls required by the European Pharmacopoeia and tested on sheep in comparison with a commercial inactivated vaccine. Body temperature, antibody titres and viraemia of vaccinated and controls sheep were recorded. In addition IL4 and IFNγ in sera and supernatant derived from in vitro stimulation of blood cells were also quantified using two commercial ELISA kit. The outer-capsid protein VP2 contained in vaccine formulations was quantified using a home-made capture-ELISA. Results obtained indicates that in-lab evaluation of cell-mediated and humoral immune response are useful parameters to predict the efficacy of BTV inactivated vaccines avoiding the challenge phase required to release new batches of vaccines with proven clinical efficacy and safety. The correlation observed between serology data and VP2 protein concentration of final product could be useful in-process control to predict if a new vaccine batch of BTV must be discarded or released to the market.

Production of monoclonal antibodies binding to the VP7 protein of African horse sickness virus.

Monoclonal antibodies (MAbs) against AHSV were produced by immunising BALB/c mice with AHSV serotype 9 and six clones able to recognize specifically the VP7-AHSV with a strong reactivity were selected. The specificity of the MAbs was assessed in i-ELISA against a commercial VP7-AHSV and in immunoblot against a home-made VP7-AHSV, expressed by a Baculovirus expression system; potential cross-reactions with related orbiviruses (Bluetongue virus and Epizootic Haemorrhagic Disease virus) were investigated as well. One of the six MAbs selected, MAb 7F11E14, was tested in direct immunofluorescence and reacted with all nine AHSV serotypes, but didn't cross-react with BTV and EHDV. MAb 7F11E14 was also used to develop a competitive ELISA and was able to detect AHSV antibodies in the sera of AHS infected animals.

Development of an indirect ELISA for the serological diagnosis of dourine.

Dourine is a parasitic venereal disease of equines caused by T. equiperdum. Humoral antibodies are found in infected animals, but diagnosis of dourine must include history, clinical, and pathological findings in addition to serology. Complement Fixation Test (CFT) is the Office International des Epizooties (OIE) recommended test for international trade; however, some uninfected equines may give inconsistent or nonspecific reactions in CFT due to the anticomplementary effects of their sera. In this study an Indirect Enzyme Linked Immunosorbent Assay (iELISA) was developed. This test could be used to confirm positive serological cases of dourine or to solve inconclusive results obtained by CFT, in addition to Indirect Fluorescent Antibody Test (IFAT) and a Chemiluminescent Immunoblotting Assay (cIB). Six-hundred-and-six CFT negative sera and 140 sera positive to CFT and IFAT were tested by iELISA using OVI T. equiperdum as antigen. Results were expressed as percentage of positivity and the optimum cut-off value determined sensitivity and specificity of 100%. All positive sera, tested by cIB, were confirmed as positive. Additionally, twenty seven sera, low-positive at CFT and negative by IFAT, were tested with iELISA and cIB. All samples resulted negative by cIB and one of them was positive in ELISA. Our results suggest that iELISA and cIB may be used as alternative or supplementary confirmatory tests whenever other recommended serological methods are inconclusive or doubtful.

Trypanosoma equiperdum Low Molecular Weight Proteins As Candidates for Specific Serological Diagnosis of Dourine.

The diagnosis of dourine can be difficult because the clinical signs of this disease in horses are similar to those of surra, caused by Trypanosoma evansi. Moreover, T. equiperdum and T. evansi are closely related and, so far, they cannot be distinguished using serological tests. In a previous work, the T. equiperdum protein pattern recognized by antibodies from dourine-infected horses and the humoral immune response kinetics were investigated by immunoblotting assay; a total of 20 sera from naturally and experimentally infected horses and from healthy animals were tested. Immunoblotting analysis showed that antibodies from infected horses specifically bind T. equiperdum low molecular weight proteins (from 16 to 35 kDa), which are not recognized by antibodies from uninfected horses. In this work, we tested other 615 sera (7 from naturally infected horses and 608 sera from healthy horses and donkeys): results confirmed the data obtained previously. In addition, six SDS-PAGE bands with molecular weight ranging from 10 to 37 kDa were analyzed by mass spectrometry, in order to identify immunogenic proteins that could be used as biomarkers for the diagnosis of dourine. A total of 167 proteins were identified. Among them, 37 were found unique for T. equiperdum. Twenty-four of them could represent possible candidate diagnostic antigens for the development of serological tests specific for T. equiperdum.

Bone Regeneration Induced by Bone Porcine Block with Bone Marrow Stromal Stem Cells in a Minipig Model of Mandibular "Critical Size" Defect.

Introduction. Adding stem cells to biodegradable scaffolds to enhance bone regeneration is a valuable option. Different kinds of stem cells with osteoblastic activity were tested, such as bone marrow stromal stem cells (BMSSCs). Aim. To assess a correct protocol for osteogenic stem cell differentiation, so BMSSCs were seeded on a bone porcine block (BPB). Materials and Methods. Bone marrow from six minipigs was extracted from tibiae and humeri and treated to isolate BMSSCs. After seeding on BPB, critical-size defects were created on each mandible of the minipigs and implanted with BPB and BPB/BMSSCs. After three months, histomorphometric analysis was performed. Results. Histomorphometric analysis provided percentages of the three groups. Tissues present in control defects were 23 ± 2% lamellar bone, 28 ± 1% woven bone, and 56 ± 4% marrow spaces; in BPB defects were 20 ± 5% BPB, 32 ± 2% lamellar bone, 24 ± 1% woven bone, and 28 ± 2% marrow spaces; in BPB/BMSSCs defects were 17 ± 4% BPB/BMSSCs, 42 ± 2% lamellar bone, 12 ± 1% woven bone, and 22 ± 3% marrow spaces. Conclusion. BPB used as a scaffold to induce bone regeneration may benefit from the addition of BDPSCs in the tissue-engineered constructs.

Validation of the Socially Acceptable Behaviour (SAB) test in a Central­Italy pet dog population.

Aggressiveness is reported to be the most important public health issue related to both owned and free­roaming dogs. Common approaches to assess canine aggressiveness are temperament tests. The main aim of this study was to evaluate whether one such test, the Socially Acceptable Behaviour (SAB) test, created to evaluate aggressive and fearful behaviour in dogs in the Netherlands, could be used reliably to assess dog aggression and fear in a population of owned dogs in Central Italy. Reactions to the test were recorded and compared to the owners' perception of their dog's aggressiveness using a validated questionnaire (C­BARQ). Dogs showing aggressive reactions during the test obtained significantly higher (more aggressive) scores on the C­BARQ subscales 'stranger­directed aggression' (SDA p<0.001), 'owner­directed aggression' (ODA p = 0.03), and 'familiar dog aggression' (FDA p = 0.006), than dogs who did not react aggressively. Logistic regression analysis revealed that 7 of the SAB­subtests were predictive of the SDA score. The findings indicated that aggression directed toward unfamiliar people can be reliably assessed using the SAB test for a population of Italian pet dogs.

Quick, Accurate, Smart: 3D Computer Vision Technology Helps Assessing Confined Animals' Behaviour.

Mankind directly controls the environment and lifestyles of several domestic species for purposes ranging from production and research to conservation and companionship. These environments and lifestyles may not offer these animals the best quality of life. Behaviour is a direct reflection of how the animal is coping with its environment. Behavioural indicators are thus among the preferred parameters to assess welfare. However, behavioural recording (usually from video) can be very time consuming and the accuracy and reliability of the output rely on the experience and background of the observers. The outburst of new video technology and computer image processing gives the basis for promising solutions. In this pilot study, we present a new prototype software able to automatically infer the behaviour of dogs housed in kennels from 3D visual data and through structured machine learning frameworks. Depth information acquired through 3D features, body part detection and training are the key elements that allow the machine to recognise postures, trajectories inside the kennel and patterns of movement that can be later labelled at convenience. The main innovation of the software is its ability to automatically cluster frequently observed temporal patterns of movement without any pre-set ethogram. Conversely, when common patterns are defined through training, a deviation from normal behaviour in time or between individuals could be assessed. The software accuracy in correctly detecting the dogs' behaviour was checked through a validation process. An automatic behaviour recognition system, independent from human subjectivity, could add scientific knowledge on animals' quality of life in confinement as well as saving time and resources. This 3D framework was designed to be invariant to the dog's shape and size and could be extended to farm, laboratory and zoo quadrupeds in artificial housing. The computer vision technique applied to this software is innovative in non-human animal behaviour science. Further improvements and validation are needed, and future applications and limitations are discussed.

Validation of value of actual micromotion as a direct measure of implant micromobility after healing (secondary implant stability). An in vivo histologic and biomechanical study.

OBJECTIVES: The osseointegration process replaces the surgically damaged bone with newly formed bone in contact to the implant surface. This involves some loss of primary stability, which will continue until new bone is formed providing a new stability, known as "secondary stability." A direct measurement of secondary implant stability appears fundamental to determine the period and modalities for implant loading. The aim of this study was to validate the measurement of the implant micromotion to test secondary implant stability. MATERIALS AND METHODS: Twenty-four 3.8 × 11.5 mm implants (Dynamix, Cortex, Shlomi, Israel) were inserted in sheep iliac crests. The animals were sacrificed after 2 months, and the freshly retrieved bone blocks were immediately fixed on a customized device to calculate the value of actual micromotion (VAM) according to a previously described technique. Implant stability quotient (ISQ) values, reverse torque value (RTV), %bone-to-implant contact (%BIC), bone volume percentage (%BV) and crestal bone loss (CBL) were also calculated for each implant. Statistical correlations between VAM and the other parameters were calculated. RESULTS: Data correlation analysis between the examined parameters showed that VAM significantly correlates (P < 0.05) to RTV, %BIC, ISQ and CBL. CONCLUSIONS: As VAM showed to be statistical correlated to the other parameters of osseointegration, it may be used to clinically check the amount of implant osseointegration, secondary stability and CBL. Future studies are needed to confirm these results moreover. An instrument to measure VAM in the oral cavity still needs to be developed.

New Osseodensification Implant Site Preparation Method to Increase Bone Density in Low-Density Bone: In Vivo Evaluation in Sheep.

PURPOSE: The aim of this study was to evaluate a new surgical technique for implant site preparation that could allow to enhance bone density, ridge width, and implant secondary stability. MATERIALS AND METHODS: The edges of the iliac crests of 2 sheep were exposed and ten 3.8 × 10-mm Dynamix implants (Cortex) were inserted in the left sides using the conventional drilling method (control group). Ten 5 × 10-mm Dynamix implants (Cortex) were inserted in the right sides (test group) using the osseodensification procedure (Versah). After 2 months of healing, the sheep were killed, and biomechanical and histological examinations were performed. RESULTS: No implant failures were observed after 2 months of healing. A significant increase of ridge width and bone volume percentage (%BV) (approximately 30% higher) was detected in the test group. Significantly better removal torque values and micromotion under lateral forces (value of actual micromotion) were recorded for the test group in respect with the control group. CONCLUSION: Osseodensification technique used in the present in vivo study was demonstrated to be able to increase the %BV around dental implants inserted in low-density bone in respect to conventional implant drilling techniques, which may play a role in enhancing implant stability and reduce micromotion.

Laterality as an indicator of emotional stress in ewes and lambs during a separation test.

We assessed motor laterality in sheep to explore species-specific brain hemi-field dominance and how this could be affected by genetic or developmental factors. Further, we investigated whether directionality and strength of laterality could be linked to emotional stress in ewes and their lambs during partial separation. Forty-three ewes and their singleton lambs were scored on the (left/right) direction of turn in a y-maze to rejoin a conspecific (laterality test). Further, their behavioural response (i.e. time spent near the fence, vocalisations, and activity level) during forced separation by an open-mesh fence was assessed (separation test). Individual laterality was recorded for 44.2% ewes (significant right bias) and 81.4% lambs (equally biased to the left and the right). There was no significant association in side bias between dams and offspring. The Chi-squared test revealed a significant population bias for both groups (p < 0.05). Evolutionary adaptive strategies or stimuli-related visual laterality may provide explanation for this decision-making process. Absolute strength of laterality (irrespective of side) was high (Kolmogorov-Smirnov test, dams: D = 0.2; p < 0.001; lambs: D = 0.36, p < 0.0001). The Wilcoxon test showed that lateralised lambs and dams spent significantly more time near each other during separation than non-lateralised animals (p < 0.05), and that lateralised dams were also more active than non-lateralised ones. Arguably, the lateralised animals showed a greater attraction to their pair because they were more disturbed and thus required greater reassurance. The data show that measures of laterality offer a potential novel non-invasive indicator of separation stress.

Effect of Implant Thread Geometry on Secondary Stability, Bone Density, and Bone-to-Implant Contact: A Biomechanical and Histological Analysis.

PURPOSE: This study evaluated the effect of 2 different thread designs on secondary stability (micromotion) and osseointegration rate in dense and cancellous bones. MATERIALS AND METHODS: Forty large threaded and 40 small threaded implants (Cortex) were placed in low- (iliac crest) and high-density (mandible) bone of sheep. Two months later, micromobility tests and histological analysis were performed to measure secondary stability, osseointegration (bone-to-implant contact percentage [%BIC]), and bone density (bone volume percentage [%BV]). The value of actual micromotion of implant is introduced as a new parameter to evaluate secondary stability. RESULTS: Large threaded implants showed significantly higher %BIC and %BV than small threaded implants in low-density bone and statistically higher secondary stability in cancellous and cortical bones. CONCLUSIONS: Implants in dense bone reach higher secondary stability than those in cancellous bone, despite the lower %BIC. Implant geometry and bone density play a key role in secondary stability. Large thread design improves bone anchorage mechanically and histologically as compared with small threaded implants.

Insufficient irrigation induces peri-implant bone resorption: an in vivo histologic analysis in sheep.

OBJECTIVES: To measure in vivo impact of dense bone overheating on implant osseointegration and peri-implant bone resorption comparing different bur irrigation methods vs. no irrigation. MATERIALS AND METHODS: Twenty TI-bone implants were inserted in the inferior edge of mandibles of sheep. Different cooling procedures were used in each group: no irrigation (group A), only internal bur irrigation (group B), both internal and external irrigation (group C), and external irrigation (group D). The histomorphometric parameters calculated for each implant were as follows: %cortical bone-implant contact (%CBIC) and %cortical bone volume (%CBV). Friedman's test was applied to test the statistical differences. RESULTS: In group A, we found a huge resorption of cortical bone with %CBIC and %CBV values extremely low. Groups B and C showed mean %CBIC and %BV values higher than other groups The mean %CBV value was significantly different when comparing group B and group C vs. group A (P < 0.05). Significant differences in %CBIC were found also between group C and group A (P < 0.05). CONCLUSIONS: Thermal injury, due to insufficient irrigation, of hard bone caused massive resorption of the cortical bone and implant failure. Drilling procedures on hard bone need an adequate cooling supply because the bone matrix overheating may induce complete resorption of dense bone around implants. Internal-external irrigation and only internal irrigation showed to be more efficient than other types of cooling methods in preventing bone resorption around implants.

Methods of Control of the Leishmania infantum Dog Reservoir: State of the Art.

Leishmania infantum is a protozoan parasite causing severe vector-borne visceral diseases both in humans and dogs. The latter are the most important natural reservoir and therefore should be the main target of control measures. The real efficacy of seropositive dogs culling as a direct control method is still debated, and the new sensitivity of large part of population considers ethically unacceptable this kind of approach. Treatment of infectious dogs with one of the available therapeutic protocols is recommendable as it allows to reduce parasite burdens and therefore the possibility of transmission of Leishmania infantum to vectors. Vaccination has been proven to be a very effective control tool, but the absence of a commonly recognized diagnostic method able to distinguish vaccinate from seropositive individuals is still an important limit. Concerning indirect control methods, a number of studies have demonstrated the efficacy of topical insecticides treatment (collars, spot-on, and sprays) in reducing incidence and prevalence of L. infantum. Also, the reduction of the odds of seroconversion in humans in endemic areas has been reported after the application of indirect control measures on dogs. The contemporary use of direct and indirect methods is even more effective in reducing seroprevalence in dogs.

Leishmania infantum in a Central Italy dog shelter: retrospective study of serologic reactivity during a 4-year period in a confined dog population subjected to preventive and therapeutic treatment.

Leishmaniasis from Leishmania infantum is a parasitary zoonotic disease and a serious problem to public health. Guidelines from Italian Health Authority (Istituto Superiore di Sanità) suggest to control the zoonotic visceral leishmaniasis canine reservoir in endemic areas using an association of preventive and therapeutic tools. Moreover, in literature there are no studies about the long term effects on the disease seroprevalence and incidence in relation to this "holistic" approach. Past research has considered the effects of the alternative employment of preventive or therapeutic treatment, usually for limited periods. In this retrospective study the patterns of seroprevalence and incidence of leishmaniasis in a dog shelter sited in an endemic area of Central Italy are described throughout a 4-year period. Both preventive spot-on tools (imidacloprid/permetrin) and therapeutic protocols based on antimonials and allopurinol were administered. The results showed a progressive reduction of prevalence and incidence of serological reactivity to L. infantum, corroborating the effectiveness of the treatment administered to the animals. Significant improvements from the beginning to the end of the 4-year period were reported, considering both prevalence and incidence. A very low rate of relapses (8% in a pool of 67 subjects positive since 2004; 10.2% among all subjects enrolled in the study) was achieved.