Selection of vaccine-candidate peptides from Mycobacterium avium subsp. paratuberculosis by in silico prediction, in vitro T-cell line proliferation, and in vivo immunogenicity.

Mycobacterium avium subspecies paratuberculosis (MAP) is a global concern in modern livestock production worldwide. The available vaccines against paratuberculosis do not offer optimal protection and interfere with the diagnosis of bovine tuberculosis. The aim of this study was to identify immunogenic MAP-specific peptides that do not interfere with the diagnosis of bovine tuberculosis. Initially, 119 peptides were selected by either (1) identifying unique MAP peptides that were predicted to bind to bovine major histocompatibility complex class II (MHC-predicted peptides) or (2) selecting hydrophobic peptides unique to MAP within proteins previously shown to be immunogenic (hydrophobic peptides). Subsequent testing of peptide-specific CD4+ T-cell lines from MAP-infected, adult goats vaccinated with peptides in cationic liposome adjuvant pointed to 23 peptides as being most immunogenic. These peptides were included in a second vaccine trial where three groups of eight healthy goat kids were vaccinated with 14 MHC-predicted peptides, nine hydrophobic peptides, or no peptides in o/w emulsion adjuvant. The majority of the MHC-predicted (93%) and hydrophobic peptides (67%) induced interferon-gamma (IFN-γ) responses in at least one animal. Similarly, 86% of the MHC-predicted and 89% of the hydrophobic peptides induced antibody responses in at least one goat. The immunization of eight healthy heifers with all 119 peptides formulated in emulsion adjuvant identified more peptides as immunogenic, as peptide specific IFN-γ and antibody responses in at least one heifer was found toward 84% and 24% of the peptides, respectively. No peptide-induced reactivity was found with commercial ELISAs for detecting antibodies against Mycobacterium bovis or MAP or when performing tuberculin skin testing for bovine tuberculosis. The vaccinated animals experienced adverse reactions at the injection site; thus, it is recommend that future studies make improvements to the vaccine formulation. In conclusion, immunogenic MAP-specific peptides that appeared promising for use in a vaccine against paratuberculosis without interfering with surveillance and trade tests for bovine tuberculosis were identified by in silico analysis and ex vivo generation of CD4+ T-cell lines and validated by the immunization of goats and cattle. Future studies should test different peptide combinations in challenge trials to determine their protective effect and identify the most MHC-promiscuous vaccine candidates.

Assessment of mental health trajectories before and after myocardial infarction, atrial fibrillation or stroke: analysis of a cohort study in Tromsø, Norway (Tromsø Study, 1994-2016).

OBJECTIVES: The increased survival rate of cardiovascular disease (CVD) implies a higher proportion of individuals who live with CVD. Using data from the Tromsø Study, we aimed to investigate mental health symptom trajectories before and after myocardial infarction, atrial fibrillation or stroke in a general population and to explore factors that contribute to the association. DESIGN: Cohort study. SETTING: Sample drawn from inhabitants of the municipality of Tromsø, Norway, who participated in the Tromsø Study (1994-2016). PARTICIPANTS: A total of 18 719 participants (52.3% women) were included, and of these 2098 (32.9% women) were diagnosed with myocardial infarction, 1896 (41.9% women) with atrial fibrillation and 1263 (42.9% women) with stroke. PRIMARY OUTCOME MEASURES: Mental health symptoms were assessed using the Hopkins Symptom Checklist-10 and the Conor Mental Health Index. RESULTS: The participants who were diagnosed with either myocardial infarction or stroke had a significant monotonous increase in mental health symptoms before myocardial infarction (p=0.029) and stroke (p=0.029) that intensified at the time of diagnosis. After the event, the study found a higher prevalence of mental health symptoms with a decline in symptom levels over time for myocardial infarction (p<0.001) and stroke (p=0.004), but not for atrial fibrillation (before: p=0.180, after: p=0.410). The risk of elevated mental health symptoms with myocardial infarction, atrial fibrillation and stroke was associated with sex (p<0.001), age (p<0.01), physical activity (p<0.001), diabetes (p<0.05) and other comorbidities (p<0.001). CONCLUSION: The study indicates that mental health problems among individuals with myocardial infarction, atrial fibrillation and stroke may have started to develop several years before the cardiovascular event and suggests that successful CVD rehabilitation may need to consider previous life factors. Future research is recommended to examine whether health promotion measures in a general population also create mental health resilience after a CVD event.

Prevalence of gestational diabetes and pregnancy outcomes in Nordland and Troms counties 2004­15. / Prevalens av diabetes blant gravide og svangerskapsutfall i Nordland og Troms 2004­15.

BACKGROUND: The aim was to investigate the prevalence of gestational diabetes and pregnancy outcomes in women with gestational diabetes in Nordland and Troms counties. MATERIAL AND METHOD: We included all 1 067 women with type 1 diabetes, type 2 diabetes and gestational diabetes among 34 915 births at four hospitals in Nordland and Troms counties from 2004 to 2015. Prevalence of diabetes was calculated based on ICD-10 codes in patient records for women with diabetes in Nordland and Troms counties, and compared with national prevalence figures from the Medical Birth Registry of Norway. Prevalence of pre-eclampsia, macrosomia (birth weight > 4 500 grams) and caesarian section was calculated for all women with diabetes and all those giving birth in the same region. RESULTS: Prevalence of type 1 diabetes and type 2 diabetes remained stable. Prevalence of gestational diabetes increased from 1.0 % to 4.0 % in Nordland and Troms counties and from 1.0 % to 3.8 % nationally. Prevalence of pre-eclampsia among all women with diabetes fell from 18.8 % in 2004-06 to 12.4 % in 2013-15. In women with diabetes, the prevalence of pre-eclampsia was 4.6 times higher, that of macrosomia was 3.5 times higher, and the proportion of caesarian sections was 2.3 times higher than in the background population. INTERPRETATION: Prevalence of gestational diabetes increased in Nordland and Troms counties, as it did nationally. Prevalence of pre-eclampsia among pregnant women with diabetes fell, but prevalence of pre-eclampsia, macrosomia and caesarean section was higher than in the background population.

(68)Ga-DOTATOC PET and gene expression profile in patients with neuroendocrine carcinomas: strong correlation between PET tracer uptake and gene expression of somatostatin receptor subtype 2.

Somatostatin receptor expression on both protein and gene expression level was compared with in vivo (68)Ga-DOTATOC PET/CT in patients with neuroendocrine carcinomas (NEC). Twenty-one patients with verified NEC who underwent a (68)Ga-DOTATOC PET/CT between November 2012 and May 2014, were retrospectively included. By real-time polymerase chain reaction, we quantitatively determined the gene expression of several genes and compared with (68)Ga-DOTATOC PET uptake. By immunohistochemistry we qualitatively studied the expression of assorted proteins in NEC. The median age at diagnosis was 68 years (range 41-84) years. All patients had WHO performance status 0-1. Median Ki67 index was 50% (range 20-100%). Gene expression of somatostatin receptor subtype (SSTR) 2 and Ki67 were both positively correlated to the (68)Ga-DOTATOC uptake (r=0.89; p<0.0001 and r=0.5; p=0.021, respectively). Furthermore, SSTR2 and SSTR5 gene expression were strongly and positively correlated (r=0.57; p=0.006). This study as the first verifies a positive and close correlation of (68)Ga-DOTATOC uptake and gene expression of SSTR2 in NEC. SSTR2 gene expression has a stronger correlation to (68)Ga-DOTATOC uptake than SSTR5. In addition, the results indicate that the gene expression levels of SSTR2 and SSTR5 at large follow one another.

Quantitative Proteomics of Gut-Derived Th1 and Th1/Th17 Clones Reveal the Presence of CD28+ NKG2D- Th1 Cytotoxic CD4+ T cells.

T-helper cells are differentiated from CD4+ T cells and are traditionally characterized by inflammatory or immunosuppressive responses in contrast to cytotoxic CD8+ T cells. Mass-spectrometry studies on T-helper cells are rare. In this study, we aimed to identify the proteomes of human Th1 and Th1/Th17 clones derived from intestinal biopsies of Crohn's disease patients and to identify differentially expressed proteins between the two phenotypes. Crohn's disease is an inflammatory bowel disease, with predominantly Th1- and Th17-mediated response where cells of the "mixed" phenotype Th1/Th17 have also been commonly found. High-resolution mass spectrometry was used for protein identification and quantitation. In total, we identified 7401 proteins from Th1 and Th1/Th17 clones, where 334 proteins were differentially expressed. Major differences were observed in cytotoxic proteins that were overrepresented in the Th1 clones. The findings were validated by flow cytometry analyses using staining with anti-granzyme B and anti-perforin and by a degranulation assay, confirming higher cytotoxic features of Th1 compared with Th1/Th17 clones. By testing a larger panel of T-helper cell clones from seven different Crohn's disease patients, we concluded that only a subgroup of the Th1 cell clones had cytotoxic features, and these expressed the surface markers T-cell-specific surface glycoprotein CD28 and were negative for expression of natural killer group 2 member D.

Gastrointestinal Surgery of Neuroendocrine Neoplasms.

Surgery is the only treatment that may cure the patient with gastroenteropancreatic (GEP) neuroendocrine neoplasms (NENs) and should always be considered as the first-line treatment if radical resection can be achieved. Even in cases where radical surgery is not possible, palliative resection may be performed to reduce local or hormone-induced symptoms and to improve quality of life. The surgical procedures for GEP-NENs are accordingly described below. In most patients life-long follow-up is required, even following radical surgery, as recurrence may occur several years later.

Diffusion blotting: a rapid and simple method for production of multiple blots from a single gel.

A very simple and fast method for diffusion blotting of proteins from precast SDS-PAGE gels on a solid plastic support was developed. Diffusion blotting for 3 min gives a quantitative transfer of 10 % compared to 1-h electroblotting. For each subsequent blot from the same gel a doubling of transfer time is necessary to obtain the same amount of protein onto each blot. High- and low-molecular-weight components are transferred equally efficiently when compared to electroblotting. However, both methods do give a higher total transfer of the low-molecular-weight proteins compared to the large proteins. The greatest advantage of diffusion blotting is that several blots can be made from each lane, thus enabling testing of multiple antisera on virtually identical blots. The gel remains on the plastic support, which prevents it from stretching or shrinking. This ensures identical blots and facilitates more reliable molecular weight determination. Furthermore the proteins remaining in the gel can be stained with Coomassie Brilliant Blue or other methods for exact and easy comparison with the developed blots. These advantages make diffusion blotting the method of choice when quantitative protein transfer is not required.

Goblet cell carcinoids: characteristics of a Danish cohort of 83 patients.

BACKGROUND: Appendiceal goblet cell carcinoids (GCCs) exhibit neuroendocrine and adenocarcinoma features. PATIENTS AND METHODS: Analysis of demography, pathology, prognostic markers, treatment and survival in 83 GCC patients (f/m: 56/27) diagnosed 1992-2013. RESULTS: Median age for f/m was 59/58 years, respectively, and similar for localized and disseminated disease. At diagnosis 54 patients had localized appendiceal disease (f/m: 29/25). According to TNM 24% had Stage I, 70% had Stage II and 6% had Stage III. Twenty-nine patients had disseminated disease (f/m: 27/2). Chromogranin A, synaptophysin and p53 were positive in >90%. Serotonin was positive in 70%. Median Ki67 index was 32% (6-75%) and higher in Tang group C (50%) compared to group A (30%; p<0.0001), and group B (30%; p<0.004). All patients had surgery. Sixty-three (76%) had radical resections including all patients with localized disease. Median OS was 83 months. The 1-, 5- and 10-year survival rates were 90%, 58%, and 38%, respectively. For localized disease OS was 164 months and 1-, 5- and 10-year survival rates were 100%, 80%, and 55%, respectively. For disseminated disease OS was 19 months and 1-, 5- and 10-year survival rates were 73%, 18% and 6%, respectively. The 1-, 5- and 10 year-survival rates for f/m were 87%/96%, 49%/76% and 31%/57%, respectively (p = 0.02). According to the Tang classification group A, B, and C OS was 118, 83 and 20 months, respectively (p = 0.0002). CONCLUSION: The Tang classification was found to be a significant prognostic factor, while the Ki67 index was not. Localized GCCs occurred equally in males and females, while disseminated GCCs were mostly seen in females. Median age of patients with localized disease and disseminated disease was identical. Cox regression analysis found Stage IV, focally positive synaptophysin and non-radical surgery as strongest negative prognostic factors.

Exploring Work-Related Causal Attributions of Common Mental Disorders.

PURPOSE: Common mental disorders (CMDs) are major causes of sickness absence and disability. Prevention requires knowledge of how individuals perceive causal mechanisms, and in this study we sought to examine work-related factors as causal attribution of CMDs. METHODS: A trial sample of n = 1,193, recruited because they struggled with work participation due to CMDs, answered an open-ended questionnaire item about what they believed were the most important causes of their CMDs. The population included participants at risk of sickness absence, and participants with reduced work participation due to sickness absence, disability or unemployment. We used thematic content analysis and categorized responses from 487 participants who reported work-related factors as causal attributions of their CMDs. Gender differences in work-related causal attributions were also examined. RESULTS: The participants attributed their CMDs to the following work-related factors; work stress, leadership, reduced work participation, job dissatisfaction, work conflict, social work environment, job insecurity and change, workplace bullying, and physical strain. Women tended to attribute CMDs to social factors at work. CONCLUSION: Findings from this study suggest several work-related risk factors for CMDs. Both factors at the workplace, and reduced work participation, were perceived by study participants as contributing causes of CMDs. Thus, there is a need to promote work participation whilst at the same time targeting aversive workplace factors. Further, our findings indicate that work-related factors may affect women and men differently. This illustrates that the association between work participation and CMDs is complex, and needs to be explored further.

Neuroendocrine Carcinomas of the Gastroenteropancreatic System: A Comprehensive Review.

To date, empirical literature has generally been considered lacking in relation to neuroendocrine carcinomas (NECs), the highly malignant subgroup of neuroendocrine neoplasms. NECs are often found in the lungs or the gastroenteropancreatic (GEP) system and can be of small or large cell type. Concentrating on GEP-NECs, we can conclude that survival times are poor, with a median of only 4-16 months depending on disease stage and primary site. Further, this aggressive disease appears to be on the rise, with incidence numbers increasing while survival times are stagnant. Treatment strategies concerning surgery are often undecided and second-line chemotherapy is not yet established. After an analysis of over 2600 articles, we can conclude that there is indeed more empirical literature concerning GEP-NECs available than previously assumed. This unique review is based on 333 selected articles and contains detailed information concerning all aspects of GEP-NECs. Namely, the classification, histology, genetic abnormalities, epidemiology, origin, biochemistry, imaging, treatment and survival of GEP-NECs are described. Also, organ-specific summaries with more detail in relation to disease presentation, diagnosis, treatment and survival are presented. Finally, key points are discussed with directions for future research priorities.

Topotecan monotherapy in heavily pretreated patients with progressive advanced stage neuroendocrine carcinomas.

BACKGROUND: Neuroendocrine carcinomas (WHO grade 3) are highly aggressive tumors with an immense tendency to metastasize and with a poor prognosis. In advanced disease, there is no standard treatment beyond first-line platin/etoposide-based chemotherapy. Topotecan is widely used as second-line treatment in small cell lung cancer, which also responds markedly on first-line platin/etoposide. Hence, we investigated the feasibility of topotecan in previously treated patients with neuroendocrine carcinomas. MATERIAL AND METHODS: Retrospective analysis of 22 patients with disseminated and progressive neuroendocrine carcinomas (Ki67>20%, G3) successively treated with oral topotecan 2.3 mg/m(2) d1-5 every 3 weeks. All patients had previously received treatment with carboplatin/etoposide. Demographic, clinical and pathological features were recorded. CT-evaluations according to RECIST 1.1 were performed after every three courses. Hematological toxicity was assessed by CTC-criteria. RESULTS: Twenty-two eligible patients received a median of 2 courses [range1-6]. Median age: 65 years [35-77]. Male/female: 11/11. Median Ki-67 index: 95% [25-100%]. Median number previous chemotherapy regimens: 2 [1-3]. All patients were evaluable for response: Five achieved stable disease (SD) and 17 progressed (PD). The median overall survival for the 22 patients was 3.2 months and the median progression-free survival was 2.1 months. The one-year survival was 18%. There were no treatment related deaths. The treatment was well tolerated: Haematological toxicity comprised leukopenia CTC grade 3 (14%), grade 4 (9%) and thrombocytopenia grade 3 (14%). CONCLUSION: Topotecan monotherapy shows modest anti-tumor activity in heavily treated patients with progressive disseminated G3 neuroendocrine carcinomas.

Novel insights into transmission routes of Mycobacterium avium in pigs and possible implications for human health.

Mycobacterium avium infection is a severe condition in humans, whereas pigs are often subclinically infected. Pig carcasses represent a possible source of human infection. Faecal excretion of M. avium was recently demonstrated in experimentally infected pigs, along with detection of M. avium in apparently normal lymph nodes. The present study investigates faecal excretion in naturally infected herds and the presence of live mycobacteria in lymph nodes. Two pig herds (A and B), with a history of sporadically suspected M. avium infection were sampled. Herd B used peat, as opposed to Herd A. Samples from peat, sawdust, drinking water, faeces and lymph nodes were collected. Identification of mycobacteria was performed by 16S rDNA sequencing and PCR. Mycobacterium avium isolates were analysed by Multi-Locus Variable Number of Tandem repeat Analysis (MLVA). Mycobacterium avium subsp. hominissuis was detected in samples of faeces, peat and lymph nodes from Herd B, often with identical MLVA profiles. Additionally, other non-tuberculous mycobacteria (NTM) were found in the same material. The absence of macroscopic lymph node lesions in the presence of M. avium subsp. hominissuis was frequently demonstrated. In Herd A, only one NTM isolate, which proved not to be M. avium, was found. Faeces might facilitate transmission of M. avium subsp. hominissuis between pigs and maintain the infection pressure in herds. The low incidence of macroscopic lesions together with the massive presence of M. avium subsp. hominissuis in lymph nodes from pigs kept on peat raises questions related to animal husbandry, food safety and human health.

Intracellular growth of Mycobacterium avium subspecies and global transcriptional responses in human macrophages after infection.

BACKGROUND: Mycobacterium avium subsp. avium (Maa) and M. avium subsp. hominissuis (Mah) are environmental mycobacteria and significant opportunistic pathogens. Mycobacterium avium infections in humans and pigs are mainly due to Mah. It is not known whether this is caused by a difference in virulence or difference in exposure to the two subspecies. The aim of the present study was to investigate the ability of the M. avium subspecies to replicate intracellularly and to characterise the gene expression program triggered by infection of human primary macrophages. RESULTS: All isolates were able to invade and persist within human macrophages. However, intracellular replication was only evident in cells infected with the two Maa isolates. Transcriptional responses to the isolates were characterized by upregulation of genes involved in apoptosis, immune- and inflammatory response, signal transduction and NF-kB signaling, cell proliferation and T-cell activation. Although similar pathways and networks were perturbed by the different isolates, the response to the Maa subspecies was exaggerated, and there was evidence of increased activation of type I and II interferon signaling pathways. CONCLUSION: Mycobacterium avium isolates of different genetic characteristics invaded monocytes and induced different degree of macrophage activation. Isolates of Maa were able to replicate intracellularly suggesting that differences in exposure, uptake or induction of adaptive immunity are more likely explanations for the difference in prevalence between M. avium subspecies.

Increased frequency of intestinal CD4+ T cells reactive with mycobacteria in patients with Crohn's disease.

OBJECTIVE: The aim of this study was to assess the frequency of mycobacteria and Escherichia coli reactive T cells in intestinal biopsies from patients with Crohn's disease (CD) and ulcerative colitis (UC). MATERIALS AND METHODS: The biopsies were obtained by colonoscopy from adult patients with active CD (n = 5) and active UC (n = 4). The number of CD4+ T cell clones expanded and screened from each patient varied from 383 to 3972 giving a total of 16639 individual clones. The T cell clones were tested for responses to Mycobacterium avium subspecies paratuberculosis (MAP) and E. coli. The cytokine profile of 42 individual T cell clones from four CD patients was assessed. RESULTS: The frequency of mycobacteria reactive T cell clones in CD patients ranged from 0.17 to 1.63% and was higher (p = 0.038) than the frequency of E. coli reactive T cells ranging from 0 to 0.18%. No or very low numbers of mycobacteria reactive clones were detected in three UC patients while the fourth UC patient had a frequency similar to what was observed in CD patients. The frequencies of E. coli reactive T cell clones in UC patients ranged from 0 to 0.52%. T cell clones (n = 42) from CD patients all produced IL-17 and/or IFN-γ. Several clones were also able to produce IL-10. CONCLUSIONS: The high frequency of intestinal tissue resident T cells reactive to mycobacteria suggests that an adaptive immune response have taken place and argues that these bacteria may contribute to the chronic inflammation in CD.

Pitfalls in determining the cytokine profile of human T cells.

Secretion of cytokines by T cells in vitro can be influenced by the methods chosen for T cell activation. However, the awareness of this fact appears insufficient. Two of the most widely applied methods for activation of T cells are phorbol 12-myristate 13-acetate (PMA) together with Ionomycin or anti-CD3/anti-CD28 stimulation. We analyzed production of IL-4, IFN-γ, IL-17 and IL-10 by a panel of human CD4 T-cell clones isolated from intestinal biopsies using the Bio-Plex™ assay and also flow-cytometry for the latter three cytokines. Higher levels of IL-17 and IFN-γ were produced by stimulation with PMA/Ionomycin compared to anti-CD3/anti-CD28. Some T-cell clones which were assigned to produce both cytokines by stimulation with PMA/Ionomycin, were only assigned to produce IFN-γ by anti-CD3/anti-CD28 stimulation. IL-10 production was higher after anti-CD3/anti-CD28 stimulation. Furthermore the dose response curve for PMA/Ionomycin differed for IL-10 compared to IL-17 and IFN-γ as it was biphasic with no IL-10 production at higher PMA/Ionomycin concentrations. These results demonstrated that the cytokine profile may be differently determined depending on the assay and conditions used and illustrate that care should be taken when designing and interpreting studies of cytokine production by T cells.

Temozolomide as second or third line treatment of patients with neuroendocrine carcinomas.

BACKGROUND: Knowledge of the clinical efficacy in recurrent neuroendocrine carcinomas is sparse. Treatment with temozolomide alone or in combination with capecitabine and bevacizumab has recently shown promising results. PATIENTS AND METHODS: Analysis of consecutive patients with neuroendocrine carcinomas (Ki-67 proliferation index >20%) and performance status 0-2 treated with temozolomide 200 mg/sqm orally days 1-5 every 28 days after at least one previous platin-containing chemotherapy regimen. RESULTS: Twenty-eight eligible patients received a median of 3 courses. Sixteen patients were evaluable for response: Six achieved stable disease and ten progressed. The median survival for the 28 patients was 3.5 months. Survival in patients with tumors of pancreatic origin (n = 7) was 7.0 months versus 2.9 months in non-pancreatic origin (n = 21). Patients in PS 0-1 (n = 22) had a median survival of 4.5 months versus 1.1 months in patients in PS 2 (n = 6). Ki-67 index ≥ 50% was associated with a significantly shorter median survival than Ki-67 index <50% (2.7 months versus 10.9 months). The treatment was well tolerated. CONCLUSION: Temozolomide monotherapy has limited effect in treatment of recurrent neuroendocrine carcinomas. Second line treatment with temozolomide in combination with other compounds should be further investigated in patients in good performance with Ki-67 index <50%.

A comparative study of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis in experimentally infected pigs.

BACKGROUND: Mycobacterium avium subsp. avium (Maa) and Mycobacterium avium subsp. hominissuis (Mah) are opportunistic pathogens that may infect several species, including humans and pigs. Mah is however more frequently isolated from pigs than Maa, and it is unclear if this is due to difference in virulence or in exposure to the two organisms. Clinical isolates of each subspecies were administered perorally to ten domestic pigs, respectively. The animals were sacrificed at six and 12 weeks after inoculation. At necropsy, macroscopic lesions were recorded, and tissue samples were collected for mycobacterial culture, IS1245 real time PCR and histopathological examination. Culturing was also performed on faecal samples collected at necropsy. RESULTS: Macroscopic and histopathological lesions were detected in pigs infected with each subspecies, and bacterial growth and histopathological changes were demonstrated, also in samples from organs without gross pathological lesions. Six weeks after inoculation, live Mah was detected in faeces, as opposed to Maa. The presence of live mycobacteria was also more pronounced in Mah infected tonsils. In comparison, the Maa isolate appeared to have a higher ability of intracellular replication in porcine macrophages compared to the Mah isolate. CONCLUSIONS: The study shows that both subspecies were able to infect pigs. Additionally, the more extensive shedding of Mah might cause pig-to-pig transmission and contribute to the higher incidence of infection caused by this subspecies.