Study of antibiotic resistance in freshwater ecosystems with low anthropogenic impact.

This study aimed to investigate the bacterial diversity and the background level of antibiotic resistance in two freshwater ecosystems with low anthropogenic impact in order to evaluate the presence of natural antimicrobial resistance in these areas and its potential to spread downstream. Water samples from a pre-Alpine and an Apennine river (Variola and Tiber, respectively) were collected in three different sampling campaigns and bacterial diversity was assessed by 16S sequencing, while the presence of bacteria resistant to five antibiotics was screened using a culturable approach. Overall bacterial load was higher in the Tiber River compared with the Variola River. Furthermore, the study revealed the presence of resistant bacteria, especially the Tiber River showed, for each sampling, the presence of resistance to all antibiotics tested, while for the Variola River, the detected resistance was variable, comprising two or more antibiotics. Screening of two resistance genes on a total of one hundred eighteen bacterial isolates from the two rivers showed that blaTEM, conferring resistance to ß-lactam antibiotics, was dominant and present in ~58 % of isolates compared to only ~9 % for mefA/E conferring resistance to macrolides. Moreover, ß-lactam resistance was detected in various isolates showing also resistance to additional antibiotics such as macrolides, aminoglycosides and tetracyclines. These observations would suggest the presence of co-resistant bacteria even in non-anthropogenic environments and this resistance may spread from the environment to humans and/or animals.

Holistic approach to chemical and microbiological quality of aquatic ecosystems impacted by wastewater effluent discharges.

Wastewater treatment plants (WWTPs) collect wastewater from various sources and use different treatment processes to reduce the load of pollutants in the environment. Since the removal of many chemical pollutants and bacteria by WWTPs is incomplete, they constitute a potential source of contaminants. The continuous release of contaminants through WWTP effluents can compromise the health of the aquatic ecosystems, even if they occur at very low concentrations. The main objective of this work was to characterize, over a period of four months, the treatment steps starting from income to the effluent and 5 km downstream to the receiving river. In this context, the efficiency removal of chemical pollutants (e.g. hormones and pharmaceuticals, including antibiotics) and bacteria was assessed in a WWTP case study by using a holistic approach. It embraces different chemical and biological-based methods, such as pharmaceutical analysis by HPLC-MSMS, growth rate inhibition in algae, ligand binding estrogen receptor assay, microbial community study by 16S and shotgun sequencing along with relative quantification of resistance genes by quantitative polymerase chain reaction. Although both, chemical and biological-based methods showed a significant reduction of the pollutant burden in effluent and surface waters compared to the influent of the WWTP, no complete removal of pollutants, pathogens and antibiotic resistance genes was observed.

Knowledge gaps in the assessment of antimicrobial resistance in surface waters.

The spread of antibiotic resistance in the water environment has been widely described. However, still many knowledge gaps exist regarding the selection pressure from antibiotics, heavy metals and other substances present in surface waters as a result of anthropogenic activities, as well as the extent and impact of this phenomenon on aquatic organisms and humans. In particular, the relationship between environmental concentrations of antibiotics and the acquisition of ARGs by antibiotic-sensitive bacteria as well as the impact of heavy metals and other selective agents on antimicrobial resistance (AMR) need to be defined. Currently, established safety values are based on the effects of antibiotic toxicity neglecting the question of AMR spread. In turn, risk assessment of antibiotics in waterbodies remains a complex question implicating multiple variables and unknowns reinforced by the lack of harmonized protocols and official guidelines. In the present review, we discussed current state-of-the-art and the knowledge gaps related to pressure exerted by antibiotics and heavy metals on aquatic environments and their relationship to the spread of AMR. Along with this latter, we reflected on (i) the risk assessment in surface waters, (ii) selective pressures contributing to its transfer and propagation and (iii) the advantages of metagenomics in investigating AMR. Furthermore, the role of microplastics in co-selection for metal and antibiotic resistance, together with the need for more studies in freshwater are highlighted.

SARS-CoV-2 detection in wastewater using multiplex quantitative PCR.

A multiplex reverse transcription quantitative PCR (RT-qPCR)-based method was designed for the simultaneous detection of different SARS-CoV-2 genes. In this study, we used three target genes encoding for the nucleocapsid 1 and 3 (N1, N3), and the spike (S) proteins, all commonly used in the detection of SARS-CoV-2 in human and environmental samples. The performance of the multiplex assay, compared to the single assay was assessed for the standard calibration curve, required for absolute quantification, and then, for the real environmental samples to detect SARS-CoV-2. For this latter, four environmental samples were collected at a local wastewater treatment plant (WWTP). The results showed that the cycle threshold (Ct) values of the multiplex were comparable to the values obtained by the singleplex PCR. The amplification of the three target genes indicated the presence of SARS-CoV-2 in the four water samples with an increasing trend in February and these results were confirmed in the multiplex approach, showing the robustness of this method and its applicability for the relative abundance analysis among the samples. Overall, both the laboratory and field work results demonstrated that the multiplex PCR assay developed in this study could provide a method for SARS-CoV-2 detection as robust as the single qPCR, but faster and cost-effective, reducing by three times the number of reactions, and consequently the handling time and reagents.

Estrogenicity of chemical mixtures revealed by a panel of bioassays.

Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17ß-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17ß-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75-304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays - based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions.

Antibody response to homologous epitopes of Epstein-Barr virus, Mycobacterium avium subsp. paratuberculosis and IRF5 in patients with different connective tissue diseases and in mouse model of antigen-induced arthritis.

BACKGROUND: Improved knowledge of different biomarkers is crucial for early diagnosis of rheumatic diseases and to provide important insights for clinical management. In this study, we evaluated the seroreactivity of patients with different connective tissue diseases (CTDs) (rheumatoid arthritis, RA; systemic lupus erythematosus, SLE; systemic sclerosis, SSc; and Sjogren's syndrome, SSj) to interferon regulatory factor 5 (IRF5) peptide and homologs derived from Epstein-Barr virus (EBV) and Mycobacterium avium subsp. paratuberculosis (MAP). Antigen-induced arthritis (AIA) experiments have been performed in control and IRF5 conditional knockout mice to reinforce the hypothesis that antibodies generated against the three homologous peptides are cross-reactive. METHODS: Reactivity against wild-type (wt) and citrullinated (cit) IRF5 (IRF5424-434), MAP (MAP_402718-32) and EBV (BOLF1305-320) peptides were tested by indirect ELISA in sera from 100 RA patients, 54 patients with other CTDs (14 SLE, 28 SSc and 12 SSj) and 100 healthy subjects (HCs). Antibody responses to the same wt peptides have been tested in AIA mouse sera after immunization with complete Freud's adjuvant (CFA) and methylated bovine serum albumin (mBSA) to induce arthritis in the knee joint. RESULTS: BOLF1, MAP_4027 and IRF5 peptides triggered different antibody responses in CTD diseases with a stronger reactivity in RA (p=0.0001). Similar trends were observed in AIA mice with significantly higher reactivity after 7 days from induction of arthritis. We also found statistically significant differences in antibody responses between SSc and HCs for BOLF1 (p=0.003), MAP_4027 (p=0.0076) and IRF5 (p=0.0042). Peripheral reactivity to cit peptides was lower compared to their wt counterparts, except for cit-MAP_402718-32, which induced stronger responses in RA than wt-MAP_402718-32 (46% vs. 26%, p=0.0170). Conclusion(s): Our results show differential antibody responses to BOLF1, MAP_4027 and IRF5 peptides among CTDs, highlighting their potential as diagnostic biomarkers in these diseases. Experiments performed in IRF5 conditional knockout mice support the hypothesis of cross-reactivity between the investigated homologous antigens.

IL-2 and Mycobacterial Lipoarabinomannan as Targets of Immune Responses in Multiple Sclerosis Patients.

Interleukin 2 (IL-2) is considered a key player in exacerbating multiple sclerosis (MS). Therapies targeting its receptor have been developed; however, a resolution of the disease and side effects are still an issue of concern. The involvement of other factors, such as Mycobacterium avium subspecies paratuberculosis (MAP) and envelope protein derived from human endogenous retrovirus type W (HERV-Wenv), in MS pathogenesis has been recently suggested. Here, we investigated the levels of antibodies (Abs) directed against IL-2 and HERV-Wenv in 108 MS patients, 34 patients affected by neuromyelitis optica spectrum disorder (NMOSD), and 137 healthy controls (HCs). Our results show increased levels of Abs specific to IL-2 and HERV-Wenv-su antigens in MS vs. HCs (p < 0.0001 for IL-2, p = 0.0004 for HERV-Wenv) and significantly decreased levels in NMOSD vs. MS. The assessment of different 12-month-long therapies on Abs against IL-2, HERV-Wenv, and MAP lipoarabinomannan (LAM) demonstrated the strongest effect on anti-LAM Abs (p = 0.018), a slight reduction of anti-IL-2 Abs, and small variations for anti-HERV-Wenv Abs. These results highlight the conclusion that the impact of therapy is more correlated with selected epitopes than with the therapeutic agent. Screening for anti-IL-2 and anti-HERV-Wenv Abs has a potential as additional future practice to distinguish between symptomatically similar MS and NMOSD.

Association between Lipoprotein Levels and Humoral Reactivity to Mycobacterium avium subsp. paratuberculosis in Multiple Sclerosis, Type 1 Diabetes Mellitus and Rheumatoid Arthritis.

Environmental factors such as bacterial infections may play an important role in the development of autoimmune diseases. Mycobacterium avium subsp. paratuberculosis (MAP) is an obligate pathogen of ruminants able to use the host's cholesterol for survival into macrophages and has been associated with multiple sclerosis (MS), type 1 diabetes (T1DM) and rheumatoid arthritis (RA) through a molecular mimicry mechanism. Here, we aimed at investigating the correlation between humoral reactivity against MAP and serum lipoprotein levels in subjects at T1DM risk (rT1DM) grouped by geographical background and in patients affected by MS or RA. Our results showed significant differences in HDL, LDL/VLDL and Total Cholesterol (TC) levels between patients and healthy controls (p < 0.0001). Patients positive to anti-MAP Abs (MAP+) had lower HDL levels in comparison with Abs negative (MAP-) subjects, while opposite trends were found for LDL/VLDL concentrations (p < 0.05). TC levels varied between MAP+ and MAP- patients in all three assessed diseases. These findings suggest the implication of anti-MAP Abs in fluctuations of lipoprotein levels highlighting a possible link with cardiovascular disease. Further studies will be needed to confirm these results in larger groups.

Anti-HERV-WEnv antibodies are correlated with seroreactivity against Mycobacterium avium subsp. paratuberculosis in children and youths at T1D risk.

Recent evidence points at the role that human endogenous retroviruses (HERVs) may play through the activation of genes integrated across the human genome. Although a variety of genetic/epigenetic mechanisms maintain most HERVs silenced, independent environmental stimuli including infections may transactivate endogenous elements favoring pathogenic conditions. Several studies associated exposures to Mycobacterium avium subsp. paratuberculosis (MAP) with increased anti-MAP seroreactivity in T1D patients. Here, we assessed humoral responses against HERV envelope antigens (HERV-KEnv and HERV-WEnv) and four MAP-derived peptides with human homologs in distinct populations: Sardinian children at T1D risk (rT1D) (n = 14), rT1D from mainland Italy (n = 54) and Polish youths with T1D (n = 74) or obesity unrelated to autoimmunity (OB) (n = 26). Unlike Sardinian rT1D, youths displayed increased anti-HERV-WEnv Abs prevalence compared to age-matched OB or healthy controls (24.32 vs. 11.54%, p = 0.02 for Polish T1D/OB and 31.48 vs. 11.90%, p = 0.0025 for Italian rT1D). Anti-HERV-KEnv responses showed variable trends across groups. A strong correlation between Abs levels against HERV-WEnv and homologous peptides was mirrored by time-related Abs patterns. Elevated values registered for HERV-WEnv overlaped with or preceded the detection of T1D diagnostic autoantibodies. These results support the hypothesis of MAP infection leading to HERV-W antigen expression and enhancing the production of autoantibodies in T1D.

Inflammation, Infectious Triggers, and Parkinson's Disease.

Parkinson's disease is a neurodegenerative disorder characterized by progressive loss of dopaminergic neurons of the substantia nigra pars compacta with a reduction of dopamine concentration in the striatum. The complex interaction between genetic and environmental factors seems to play a role in determining susceptibility to PD and may explain the heterogeneity observed in clinical presentations. The exact etiology is not yet clear, but different possible causes have been identified. Inflammation has been increasingly studied as part of the pathophysiology of neurodegenerative diseases, corroborating the hypothesis that the immune system may be the nexus between environmental and genetic factors, and the abnormal immune function can lead to disease. In this review we report the different aspects of inflammation and immune system in Parkinson's disease, with particular interest in the possible role played by immune dysfunctions in PD, with focus on autoimmunity and processes involving infectious agents as a trigger and alpha-synuclein protein (α-syn).

Mycobacterium avium subspecies paratuberculosis and myelin basic protein specific epitopes are highly recognized by sera from patients with Neuromyelitis optica spectrum disorder.

Epstein-Barr virus (EBV) is the main environmental agent associated to neuromyelitis optica spectrum disorder (NMOSD). Following to studies reporting an increased prevalence of antibodies against peptides derived from Mycobacterium avium subsp. paratuberculosis (MAP) homologous to EBV and human epitopes (MBP85-98, IRF5424-434) in multiple sclerosis (MS), we investigated whether seroreactivity to these antigens display a NMOSD-specific pattern. The sera of 34 NMOSD patients showed elevated levels of antibodies against MAP and MBP compared to healthy controls (44% vs. 5%, p < 0.0002 and 50% vs. 2%, p < 0.0001, respectively), while, unlike in MS, responsiveness to EBV was similar.

Interferon regulatory factor 5 is a potential target of autoimmune response triggered by Epstein-barr virus and Mycobacterium avium subsp. paratuberculosis in rheumatoid arthritis: investigating a mechanism of molecular mimicry.

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic disease characterised by a pro-inflammatory cytokines linked erosive joint damage and by humoral and cellular response against a broad range of self-peptides. Molecular mimicry between Epstein-Barr virus (EBV), Mycobacterium avium subsp. paratuberculosis (MAP) and host peptides has long been regarded as an RA pathogenetic mechanism. Using bioinformatic analysis we identified high sequence homology among interferon regulatory factor 5 (IRF5), EBV antigen BOLF1 and MAP antigen MAP_4027. Our objective was to evaluate the presence in sera of RA patients of antibodies (Abs) directed against human homologous IRF5 cross-reacting with BOLF1 and MAP_4027. METHODS: Frequency of reactivity against IRF5424-434, BOLF1305-320 and MAP_402718-32 was tested by indirect ELISA in sera from 71 RA patients and 60 healthy controls (HCs). RESULTS: RA sera show a remarkable high frequency of reactivity against IRF5424-434 in comparison to HCs (69% vs. 8%; p<0.0001). Similarly, seroreactivity against BOLF1305-320 was more frequently detected in RA sera than in HCs counterpart (58% vs. 8%; p<0.0001). Frequency of Abs against MAP_402718-32 was 17% in RA sera vs. 5% in HCs with a p-value at the threshold level (p<0.051). Prevalence of Abs against at least one of the assessed epitopes reached 72% in RA patients and 15% among HCs. Levels of Abs in RA patients were significantly related to systemic inflammation. CONCLUSIONS: IRF5 is a potential autoimmune target of RA. Our results support the hypothesis that EBV and MAP infections may be involved in the pathogenesis of RA, igniting a secondary immune response that cross-reacts against RA self-peptides.

Rheumatoid arthritis patient antibodies highly recognize IL-2 in the immune response pathway involving IRF5 and EBV antigens.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by a progressive joint damage due to largely unknown environmental factors acting in concert with risk alleles conferring genetic susceptibility. A major role has been attributed to viral infections that include past contacts with Epstein-Barr virus (EBV) and, more recently, to non-protein coding sequences of human endogenous retrovirus K (HERV-K) integrated in the human genome. Molecular mimicry between viral and self proteins is supposed to cause the loss of immune tolerance in predisposed hosts. There are evidences that anti-IL-2 antibodies (Abs) are present in subjects affected by autoimmune diseases and may be responsible for alterations in regulatory T cell responses. In this study, we evaluated the levels of Abs against IL-2, viral epitopes and interferon regulatory factor 5 (IRF5) in 140 RA patients and 137 healthy controls (HCs). Ab reactivity reached the highest levels for IRF5, EBV and IL-2 (56%, 44% and 39%, respectively) in RA with significantly lower values among HCs (7-9%, p < 0.0001), which suggests a possible cross-reaction between IRF5/EBV homologous antigens and shifts in T cell balance disrupted by anti-IL-2 Abs.

Increased seroreactivity to proinsulin and homologous mycobacterial peptides in latent autoimmune diabetes in adults.

Latent Autoimmune Diabetes in Adults (LADA) is a slowly progressing form of immune-mediated diabetes that combines phenotypical features of type 2 diabetes (T2D) with the presence of islet cell antigens detected in type 1 diabetes (T1D). Heterogeneous clinical picture have led to the classification of patients based on the levels of antibodies against glutamic acid decarboxylase 65 (GADA) that correlate with clinical phenotypes closer to T1D or T2D when GADA titers are high or low, respectively. To date, LADA etiology remains elusive despite numerous studies investigating on genetic predisposition and environmental risk factors. To our knowledge, this is the first study aimed at evaluation of a putative role played by Mycobacterium avium subsp. paratuberculosis (MAP) as an infective agent in LADA pathogenesis. MAP is known to cause chronic enteritis in ruminants and has been associated with autoimmune disorders in humans. We analyzed seroreactivity of 223 Sardinian LADA subjects and 182 healthy volunteers against MAP-derived peptides and their human homologs of proinsulin and zinc transporter 8 protein. A significantly elevated positivity for MAP/proinsulin was detected among patients, with the highest prevalence in the 32-41-year-old T1D-like LADA subgroup, supporting our hypothesis of a possible MAP contribution in the development of autoimmunity.

Mycobacterium avium subsp. paratuberculosis and associated risk factors for inflammatory bowel disease in Iranian patients.

BACKGROUND: Inflammatory bowel disease (IBD) is described as a relapsing condition with high morbidity and uncertain complex pathogenesis. The association of Mycobacterium avium ssp. paratuberculosis (MAP) with Crohn's disease (CD) in human has been debated for decades, however there is no confirmed data to verify such relations in Iran. The aim of this study was to investigate risk factors and a possible role of MAP in Iranian patients with CD. METHODS: Anti-MAP antibodies were detected in serum of IBD patients and subjects without IBD (nIBD) through ELISA; MAP DNA and viable MAP cells were identified in patients' biopsies through nested PCR and direct culture methods, respectively. Principal component analysis (PCA) was used to investigate the risk factors in relation to IBD and MAP infection. RESULTS: Positivity for IS900 PCR was detected in 64% (n = 18) of CD, 33% (n = 10) of ulcerative colitis (UC) and 9.7% (n = 6) of nIBD samples. Live MAP cells were isolated from biopsies of 2 CD patients only. Among 28 patients with CD, 46% (n = 13) and 39% (n = 11) were positive for antibodies against MAP3865c133-141 and MAP3865c125-133 peptides, respectively, whereas much lower seroreactivity was detected in UC subjects accounting for 3% (n = 1) for MAP3865c133-141 and 16.7% (n = 5) for MAP3865c125-133. A high immune reactivity to MAP epitopes among CD patients was positively correlated with consumption of fast food meals and IBD familiarity. For both CD and UC, breastfeeding period and consumption of fruit/vegetables presented negative correlation with the presence of anti-MAP antibodies. CONCLUSIONS: This study provided evidences that high prevalence of MAP DNA and anti-MAP antibodies in CD patients is significantly associated with the development of CD. Despite the role of several factors contributing to IBD, the presence of MAP DNA and anti-MAP antibodies in Iranian CD patients highlights a possible transmission of MAP from animal-derived products to humans.

Detection of Mycobacterium avium subsp. paratuberculosis in Iranian patients with type 1 diabetes mellitus by PCR and ELISA.

INTRODUCTION: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis or Johne's disease in ruminants. Its role in triggering autoimmunity, including type 1 diabetes mellitus (T1DM), has been reported in recent years. Due to the high contamination rate of MAP in Iran's livestock and the increasing outbreak of T1DM, we investigated this association in a small group of patients with T1DM in Iran. METHODOLOGY: Blood samples of 29 T1DM patients and 29 healthy control subjects were tested through enzyme-linked immunosorbent assay (ELISA) to detect antibodies against MAP3865c and ZnT8 homologous epitopes and the presence of MAP DNA. Blood samples were also cultured in mycobacterial growth indicator tubes and Herrold's egg yolk medium containing mycobactin J. RESULTS: The results of ELISA showed a significant difference between T1DM patients and healthy group. IS900 was also detected in 51.72% of T1DM patients but in none of the control group. None of the samples grew in culture media. CONCLUSIONS: Due to the presence of MAP DNA and antibodies against MAP peptides in a significant number of T1DM patients compared with healthy control subjects, we may consider MAP as a possible trigger of T1DM in Iran. This indicates that exposure to MAP occurred in the positive subjects. Identifying the sources of contamination and routes of MAP transmission to humans seems necessary to prevent and reduce the burden of MAP infection in Iran.

Correction: Seroreactivity against Specific L5P Antigen from Mycobacterium avium subsp. paratuberculosis in Children at Risk for T1D.

[This corrects the article DOI: 10.1371/journal.pone.0157962.].

Soluble BAFF Level Is Not Correlated to Mycobacterium avium Subspecies Paratuberculosis Antibodies and Increases After Interferon-ß Therapy in Multiple Sclerosis Patients.

B cells are being recognized as one of the major players in the pathogenesis of multiple sclerosis (MS). The B cell activating factor (BAFF) system plays an essential role in B cell homeostasis and function in the periphery. Mycobacterium avium subspecies paratuberculosis (MAP) has been previously associated to MS in Sardinia. Antibodies against a MAP surface protein, MAP_2694, have been found significantly associated to MS patients, and this response was modified by interferon-ß therapy. Increased BAFF levels following IFN-ß therapy have been also described in MS patients. In this study, we evaluated whether soluble BAFF levels are comparable in men and women affected by MS and performed a correlation of the reported BAFF increase in MS patients under IFN-ß therapy with changes of humoral response against MAP_2694. For these reasons, we investigated 44 MS patients before and after IFN-ß therapy. A significant difference of BAFF levels was found between men and women with MS; moreover, we confirmed that IFN-ß therapy strongly induces BAFF serum levels, but this was not related to the modification of immunological response against MAP_2694. In conclusion, our study highlights that IFN-ß therapy induces the potent B cell survival factor BAFF without alterations of the humoral immune response against MAP.

Seroreactivity against Specific L5P Antigen from Mycobacterium avium subsp. paratuberculosis in Children at Risk for T1D.

AIMS/HYPOTHESIS: Although numerous environmental agents have been investigated over the years as possible triggers of type 1 diabetes (T1D), its causes remain unclear. We have already demonstrated an increased prevalence of antibodies against peptides derived from Mycobacterium avuim subsp. paratuberculosis (MAP) homologous to human zinc transporter 8 protein (ZnT8) and proinsulin in Italian subjects at risk for or affected by T1D. In this study, we compared titers of the previously detected antibodies with seroreactivity to MAP lipopentapetide (L5P) that recently emerged as a strong immunogenic component able to specifically distinguish MAP from other mycobacteria. METHODS: Plasma of 32 children and youth at risk for T1D including follow-up samples and 42 age-matched healthy controls (HC) recruited at the Tor Vergata University Hospital in Rome was analyzed by indirect ELISA for the presence of antibodies against MAP-derived epitopes MAP3865c133-141, MAP3865c125-133, MAP2404c70-85 and MAP1,4αgbp157-173 along with their ZnT8 and proinsulin homologs. The data were analyzed through two-tailed Mann-Whitney U test and relation between variables was determined by principal component analysis. RESULTS: Responses to L5P were not detectable in subjects whose initial seroreactivity to MAP peptides and their human homologs was lost in follow-up samples, whereas anti-L5P antibodies appeared constantly in individuals with a stable immunity against MAP antigens. The overall coincidence in positivity to L5P and the four MAP epitopes both in children at risk for T1D and HC exceeded 90%. CONCLUSIONS: MAP-derived homologs may cross-react with ZnT8 and proinsulin peptides inducing immune responses at a young age in subjects predisposed for T1D. Thus, L5P may have a diagnostic value to immediately indicate the presence of anti-MAP seroreactivity when evaluation of a more complex antibody status is not required. Almost complete coincidence in responses to both types of antigens lends support to the involvement of MAP in T1D.