Study of the helminth fauna in eagle owl (Bubo bubo) in the south of Spain.

In this study we show the results of the eagle owls' (Bubo bubo) helminthfauna found in Andalusia. A total number of 50 specimens have been analysed in a period of 10 years (from 2011 to 2020). Prevalence ( P % ), mean intensity (IM) and mean abundance (AM) of parasitation have been obtained. The percentage of parasitation in the total sample was 80% (40 out of 50 eagle owls): 78% nematodes, 8% trematodes, 6% cestodes and 4% acantocephalans. 7 species of helminths were identified: 6 nematodes, and 1 trematode. In the case of cestodes and acantocephalans it was not possible to determine species and only the genus was identified. The intestinal nematode Capillaria tenuissima ( P %  = 58% (44-71.2); IM = 11,52 (5.83-28.9)) was the core species whereas Synhimantus laticeps (P% = 16 (7.5-28.8); IM = 4 (1.75-7.25)) and Hartertia hispanica (P% = 16 (7.5-28.8); IM = 1,5 (1-2)) were the secondary species. The remainder species were considered satellite species, with low prevalence and average abundance. Likewise, descriptive parameters of the helminth community were determined: species richness, 1.56 (1.29-1.94), total abundance, 12 (7.24-26.40), Brillouin's diversity index, 0.18 (0.10-0.29) and Berger-Parker dominance index, 0.88 (0.81-0.93). The data from this study show a non-diverse helminthic community, without species dominance with C. tenuissima as the central species, followed by S. laticeps and H. hispanica as secondary species. Worth mentioning is the presence of H. hispanica, which is considered an endemic species in Spain and specifically in Andalusia. To the authors' knowledge, this is the largest population sample taken in parasitological studies about helminths of this raptor in Europe and the first one carried out in the south of Spain (Andalusia).

Evaluation of a Cooperia oncophora double-domain ASP-based vaccine against Cooperia spp. infections in cattle and sheep.

A double-domain activation-associated secreted protein (dd-Co-ASP) isolated from the bovine small intestinal parasite Cooperia oncophora was previously shown to be an effective vaccine candidate to protect calves against a homologous challenge infection. The aim of this study was to investigate whether the dd-Co-ASP protein, purified from a Belgian C. oncophora isolate, would offer protection against a C. oncophora isolate from the southern hemisphere as well as other Cooperia species such as C. punctata in cattle and C. curticei in sheep. Two vaccination studies were performed, i.e. one in cattle and one in sheep, in which the protective effects of dd-Co-ASP, supplemented with Quil A as an adjuvant, were compared with an adjuvant control. Whereas our results showed a 75 % reduction in Cooperia spp. cumulative faecal egg counts, the results obtained in sheep demonstrated that dd-Co-ASP was ineffective in raising a protective immune response against a C. curticei challenge infection. Even though sequence analysis of the dd-Co-ASP gene revealed restricted sequence heterogeneity in the double domain ASP within and between bovine Cooperia species, the results of the vaccine study suggest that there is sufficient conservation at the protein level to yield cross-protection, holding promise for the development of a general Cooperia vaccine for use in cattle.

Immunization with the recombinant myosin regulatory light chain (FhrMRLC) in Adjuplex® adjuvant elicits a Th1-biased immune response and a reduction of parasite burden in Fasciola hepatica infected rats.

The aim of this study was to assess the immune response and the protective efficacy elicited by the vaccination with the recombinant Fasciola hepatica myosin regulatory light chain (FhrMRLC) in Adjuplex® adjuvant against the infection with F. hepatica in rats. Four groups of 15 animals each were used for the study, one group was immunized with the recombinant F. hepatica MRLC in Adjuplex® adjuvant and the other groups remained as adjuvant, positive and negative control groups. The parasitological study showed that a statistically significant reduction of 65.1% and 82.1% in fluke burden and fecal egg count, respectively, was detected in vaccinated animals. In addition, vaccination with FhrMRLC induced a well-defined humoral and cellular immune response characterized by a significant production of specific IgG and IL-2, IL-12, TNF-α and IFN-γ; which confirms the immunogenic capacity of the FhrMRLC.

Pathological, immunological and parasitological study of sheep vaccinated with the recombinant protein 14-3-3z and experimentally infected with Fasciola hepatica.

In this study, the immunogenicity and protective capacity of a new recombinant vaccine candidate, the rFh14-3-3z protein was analysed in sheep experimentally challenged with Fasciola hepatica, in terms of fluke burden, faecal egg counts, hepatic damage and humoral immune response. Three groups of 8 animals each were used for study, group 1 was immunised with the rFh14-3-3z in Montanide adjuvant, whereas group 2 and 3 remained as adjuvant control and infection control groups, respectively. The parasitological analysis showed that no significant reduction in fluke burden, fluke size and faecal egg counts was detected. The extent of hepatic damage was very similar between groups. Nonetheless, animals immunised with the rFh14-3-3z protein induced the development of specific IgG1 and IgG2, being the IgG1 the predominant antibody; which confirms the immunogenicity of this protein in sheep. This is the first report of the 14-3-3z proteins as vaccine against the infection with F. hepatica.

Distribution of Foxp3+ T cells in the liver and hepatic lymph nodes of goats and sheep experimentally infected with Fasciola hepatica.

Foxp3 regulatory T cells (Tregs) are now considered to play a key role in modulation of immune responses during parasitic helminth infections. Immunomodulation is a key factor in Fasciola hepatica infection; however, the distribution and role of Foxp3+ Tregs cells have not been investigated in F. hepatica infected ruminants. The aim of this study was to evaluate the presence of Foxp3+ Tregs in the liver and hepatic lymph nodes from experimentally infected sheep and goats during acute and chronic stages of infection. Three groups of goats (n=6) and three groups of sheep (n=6) were used in this study. Goats in groups 1-2 and sheep in groups 4-5 were orally infected with metacercarie of ovine origin. Groups 1 and 4 were killed during the acute stage of the infection, at nine days post infection (dpi); groups 2 and 5 were killed during the chronic stage, at 15 and19 weeks post infection respectively (wpi). Groups 3 (goats) and 6 (sheep) were left as uninfected controls. Fluke burdens and liver damage were assessed and the avidin-biotin-complex method was used for the immunohistochemical study. At nine dpi in acute hepatic lesions, the number of both Foxp3+ and CD3+ T lymphocytes increased significantly in goats and sheep. In the chronic stages of infection (15-19wpi), the number of Foxp3+ and CD3+ T lymphocytes were also significantly increased with respect to control livers, particularly in portal spaces with severely enlarged bile ducts (response to adult flukes) while the increase was lower in granulomas, chronic tracts and smaller portal spaces (response to tissue damage). Foxp3+ Tregs were increased in the cortex of hepatic lymph nodes of sheep (chronic infection) and goats (acute and chronic infection). The estimated proportion of T cells which were Foxp3+ was significantly increased in the large bile ducts and hepatic lymph node cortex of chronically infected goats but not sheep. This first report of the expansion of Foxp3+ Tregs in acute and chronic hepatic lesions in ruminants suggests that these cells may be involved in both parasite survival and modulation of hepatic damage. Future studies should be focused on the investigation of parasite molecules and cytokines involved in this process.

Peripheral blood lymphocyte subsets in Fasciola hepatica infected and immunised goats.

The proportions of CD4(+), CD8(+) and WC1+ T lymphocytes from peripheral blood using flow cytometry were investigated in goats infected with Fasciola hepatica and previously immunised with recombinant Cathepsin-L1 (rCL1) and Glutathione-S-transferase sigma class (GST). The immunisation trial did not induce protective responses, and no significant differences were recorded between immunised and non-immunised groups. However, there was a significant decrease in the proportion of CD4(+) T lymphocytes in the infected groups both at 5 weeks post-infection (wpi), coinciding with the migratory stage of the infection, and at 12 wpi in the biliary stage of the infection. The proportional decrease in this circulating population may be related to the recruitment of CD4(+) T cells in liver and hepatic lymph nodes and also to the immunomodulatory effect of the parasite through the interaction of F. hepatica excretory-secretory products (FhESP) with this cell population. To date, this is the first report about the effect of F. hepatica infection in peripheral lymphocyte subsets in goats.

Early hepatic and peritoneal changes and immune response in goats vaccinated with a recombinant glutathione transferase sigma class and challenged with Fasciola hepatica.

Changes and local immune response were evaluated in the peritoneal cell populations, duodenal lamina propria and liver from goats immunized with recombinant glutathione transferase sigma class (rFhGST-S1) during early stages of infection with Fasciola hepatica. Group 1 (n=7) was unimmunized and uninfected; group 2 (n=10) was immunized with adjuvant Quil A and infected; group 3 (n=10) was immunised with rFhGST-S1 and infected. Three goats from each group were killed at 7-9 days post-infection (dpi) to evaluate early changes and immune response. The remaining goats were killed at 15 weeks post-infection (wpi). rFhGST-S1 vaccination induced variable response: three goats showed low fluke burden at 15 wpi and two goats showed low hepatic damage at early infection stages. This response was associated to a severe infiltrate of eosinophils in peritoneal fluid and hepatic necrotic foci, high iNOS expression in peritoneal cells and abundant infiltrate of eosinophils surrounding hepatic migrating flukes. T lymphocyte subsets were found in the vicinity of necrotic areas but they were absent in the vicinity of migrating larvae. No significant variation for T cell subsets, except for CD4 and γδ T lymphocytes, that were higher in the Quil A group compared to the rFhGST-S1 group. Expression of IL4 and IFN-γ in the hepatic inflammatory infiltrates was very occasional.

Early and late peritoneal and hepatic changes in goats immunized with recombinant cathepsin L1 and infected with Fasciola hepatica.

The aim of the present study was to study peritoneal and hepatic changes during early [7-9 days postinfection (dpi)] and late [15 weeks postinfection (wpi)] infection of goats immunized with recombinant F. hepatica pro cathepsin L1 (rCL1) in Quil A and challenged with Fasciola hepatica. Despite finding no significant reduction in fluke burdens between the control and immunized group, at 15 dpi the rCL1-vaccinated group showed significantly higher weight gain and reduced severity of hepatic lesions compared with the control group that received only Quil A. In the rCL1-vaccinated group, two of three goats sacrificed at 7-9 dpi had little hepatic damage and had a higher percentage of peritoneal eosinophils and elevated induced nitric oxide synthase (iNOS) expression in peritoneal cells than the goats from the control group. Moreover, while these two goats showed a heavy infiltration of eosinophils surrounding migrating flukes, the remaining animals examined at 7-9 dpi had no inflammatory infiltration surrounding migrating flukes. Two out of seven goats in the rCL1-vaccinated group had low fluke burdens and little hepatic damage at 15 wpi, suggesting an effective protective response in some of the vaccinated goats. This protective response did not correlate with peripheral eosinophilia or with serum titres of anti-rCL1 immunoglobulin (Ig) G. The results of the present work suggest that an eosinophil-mediated immune response plays a crucial role in the early effective host response against F. hepatica in goats. Adjuvants designed to increase cell-mediated immunity should be tested in future vaccine trials against F. hepatica.

Humoral immune response in goats immunised with cathepsin L1, peroxiredoxin and Sm14 antigen and experimentally challenged with Fasciola hepatica.

The humoral immune response was analysed in goats immunised with FhCL1, FhPrx, Sm14, and experimentally challenged with Fasciola hepatica. All immunised animals developed significant levels of anti-fluke specific antibodies and those immunised with FhCL1 showed the highest antibody titre. After experimental infection, an increase in the antibody level was detected only in goats immunised with FhCL1. In the adjuvant-control animals, the experimental challenge induced significant production of specific antibodies against FhCL1, FhPrx and Sm14. While liver fluke specific humoral responses were seen in all groups, no significant protection in any of the vaccinated groups was found.

Evaluation of hepatic damage and local immune response in goats immunized with native glutathione S-transferase of Fasciola hepatica.

Worm burden, hepatic damage and local cellular and humoral immune responses were assessed in goats immunized with glutathione-S-transferase and challenged with Fasciola hepatica. Infected but unimmunized and uninfected control groups were also studied. Hepatic damage was evaluated grossly and microscopically. Local immune response was evaluated by (1) microscopical examination of hepatic lymph nodes (HLNs); (2) analysis of the distribution of CD2(+), CD4(+), CD8(+), T-cell receptor gammadelta(+) lymphocytes and immunoglobulin (Ig) G(+) plasma cells; and (3) investigation of the distribution of cells expressing interleukin (IL)-4 and interferon (IFN)-gamma in the hepatic inflammatory infiltrates and HLNs. Immunized animals did not have significant reduction in fluke number, but there was significant (P<0.05) reduction of fluke size relative to the control groups. The lesions in the two infected groups were similar and consisted of fibrous perihepatitis and white tortuous tracts, mainly involving the left hepatic lobe. Microscopical lesions were similar in both infected groups and were typical of chronic fascioliosis. These included portal fibrosis, inflammatory infiltration with plasma cells, formation of lymphoid follicles, accumulation of haemosiderin-laden macrophages and granulomatous foci. Both infected groups had a marked local immune response characterized by infiltration of CD2(+), CD4(+) and CD8(+) T lymphocytes, and IgG(+) plasma cells in hepatic lesions and in HLNs. There was no expression of IL-4 or INF-gamma by cells in the hepatic inflammatory infiltrate, but expression of INF-gamma in HLNs was much lower than that of IL-4, suggesting an immune response dominated by T helper 2 cells.

Immune response of goats immunised with glutathione S-transferase and experimentally challenged with Fasciola hepatica.

Glutathione S-transferase (FhGST) purified from Fasciola hepatica adult worms was used to immunise goats against F. hepatica in an experimental infection; the level of protection, in terms of fluke burden, faecal egg counts and hepatic damage was determined, as well as the humoral and cellular immune response elicited. Animals were allocated into three groups of six animals each: group 1 (immunised with FhGST and infected), group 2 (unimmunised and infected), and group 3 (unimmunised and uninfected). There was no significant reduction of fluke burden (9.3%) or faecal egg counts; hepatic damage was also similar in both infected groups. However, immunisation with FhGST induced the development of a well-defined immune response, characterized by the production of specific-FhGST antibodies as well as the appearance of circulating IL-4.

Study of the local immune response to Fasciola hepatica in the liver and hepatic lymph nodes of goats immunised with a peptide of the Sm14 antigen.

The nature of the local immune response was assessed studying the distribution of CD2(+), CD4(+), CD8(+), gammadelta(+) T lymphocytes, IgM(+) B cells, IL-4(+) and IFN-gamma(+) cells in the liver and hepatic lymph nodes (HLN) of goats immunised with a synthetic peptide of the Sm14 antigen from Schistosoma mansoni and challenged with Fasciola hepatica. A morphometric study of HLN was also carried out in order to evaluate the hyperplasia of lymphoid follicles. Despite the decrease in fluke burdens found in the immunised group (45.9%) respect to the infected control group, this difference was not statistically significant due to the high individual variability. In liver, a significant increase of CD2(+), CD4(+), CD8(+), gammadelta(+) T lymphocytes was found in the infected groups respect to the uninfected control and in the infected control respect to the immunised group. HLN showed a significant enlargement due to the hyperplasia of lymphoid follicles and infiltration of CD2(+), CD4(+), CD8(+), gammadelta(+) T lymphocytes in both infected groups respect to the uninfected control, with no significant differences between the infected control and immunised group. IFN-gamma(+) lymphoid cells was absent or very occasional in HLN where the number of IL-4(+) cells was higher than that of IFN-gamma, suggesting a polarized Th2 response in immunised and in infected control group.

A study of the liver of goats immunized with a synthetic peptide of the Sm14 antigen and challenged with Fasciola hepatica.

A synthetic peptide of the Sm14 antigen of Schistosoma mansoni was used to immunize goats against experimental challenge with Fasciola hepatica. The goats used consisted of: group 1 (unimmunized and uninfected [controls]); group 2 (unimmunized and infected); group 3 (immunized and infected). Compared with group 2, the animals of group 3 showed at necropsy a reduction in hepatic worm burden; however, because of variability in the two groups this reduction was not statistically significant. A gross morphometric study of the hepatic changes revealed a correlation between lesions and worm burdens in group 3. No significant differences in damaged areas were found between groups 2 and 3, except that group 2 had more severe bile duct hyperplasia. A striking hepatic inflammatory infiltration of CD3+ T lymphocytes and IgG+ plasma cells was found in both groups 2 and 3, especially the latter.

Induction chemotherapy followed by concurrent chemoradiotherapy in advanced head and neck squamous cell carcinoma.

BACKGROUND: A phase II study was carried out to investigate an induction regimen with cisplatin, paclitaxel followed by radiotherapy concurrent with weekly cisplatin for locally advanced squamous cell carcinoma of the head and neck. PATIENTS AND METHODS: Stage III-IV disease patients were eligible. Two cisplatin (100 mg/m2) and paclitaxel (175 mg/m2) courses were administered every 21 days followed by standard fractionated external beam radiotherapy (approximately 70 Gy), concomitant to weekly cisplatin (30 mg/m2). RESULTS: Thirty-five patients were enrolled: over 70% had unresectable disease with bulky lesions. Grade 3-4 neutropenia developed in 14% and G3 mucositis in 23%. Locoregional control was achieved in 51%. Median time to progression and overall survival were 10,7 and 17 months respectively; 2- and 3-year survival rates were 30% and 25% respectively. CONCLUSION: Our induction two-drug regimen followed by chemoradiotherapy with concurrent weekly cisplatin was well tolerated with low acute toxicity and good locoregional control and survival rate.

Cellular phenotypes in the abomasal mucosa and abomasal lymph nodes of goats infected with Haemonchus contortus.

The distribution of T-cell subsets (CD2, CD4, CD8, and gammadelta) and B cells (IgM) was examined at 3, 6, 10 and 13 days post-infection (dpi) in the abomasal mucosa and abomasal lymph nodes of goats primarily infected with Haemonchus contortus. In the abomasal mucosa a mild (3 and 6 dpi) or marked (10 and 13 dpi) increase of T cells, particularly CD4+ and gammadelta+ lymphocytes, was observed, whereas the increase in CD8+ cells was less pronounced. B cells and IgG+ plasma cells also showed a marked increase in the abomasal mucosa at 10 and 13 dpi. The abomasal lymph nodes showed an increase in size, particularly at 10 and 13 dpi, and a decrease in the proportion of T cells, particularly CD8+ lymphocytes, due to the increased proportion of B cells. The proportion of CD4+ and gammadelta+ lymphocytes did not change significantly during the infection in the abomasal lymph nodes, but their absolute numbers were augmented as a result of the enlargement of the nodes. The results revealed a strong cellular and humoral immune response during the early post-infection stages. However, as indicated by the worm burdens, this rapid host response was unable to induce larval expulsion.

Spontaneous osteoclastogenesis is a predictive factor for bone metastases from non-small cell lung cancer.

Lung cancer is a widespread disease and its incidence is growing. Since therapies have increased the life expectancy of lung cancer patients, the development of bone osteolytic metastases is becoming a common cause of morbidity. Osteolysis is caused by an increased osteoclast activity and may be reduced by inhibiting their formation and activity. We studied 60 male patients affected by NSCLC, divided in early and advanced stage disease. Patients' blood and urinary samples were collected at tumor diagnosis and at follow-up. PBMCs were cultured to investigate the spontaneous osteoclastogenesis. IL-7 was dosed in serum and its quantitative gene expression was evaluated on tumor and healthy tissues by RQ-PCR. Both at diagnosis and follow-up, osteolytic bone patients showed high spontaneous osteoclastogenesis level compared to non-bone metastatic and healthy controls. The presence of spontaneous osteoclastogenesis correlated with urinary crosslinks increase. Serum IL-7 levels were higher in bone metastatic patients than in patients without bone lesions and healthy controls. The serum IL-7 increase correlated with the osteoclastogenesis and, at least in part, depended on an increased IL-7 production by tumor cells. At follow-up, patients with increased osteoclastogenesis and serum IL-7 levels, were subjected to standard clinical analysis, which showed early secondary bone lesions. The in vitro assay for spontaneous osteoclastogenesis and serum IL-7 dosage could be useful for diagnostic purposes and it might be able to monitor cancer patients with a high risk to develop osteolytic metastases at follow-up, especially after a curative treatment.

[The evaluation of workplace visual characteristics and ophthalmologic requirements to define fitness to work]. / Valutazione delle esigenze visive del posto di lavoro e requisiti oftalmologici per l'idoneità alla mansione specifica.

It's necessary to proceed to an evaluation of visual function to define principal ophthalmologic requirements and fitness to work in relation of workplace characteristics. We also have to consider the effects of age on visual function, the specific work risks on eyes and sight care especially for near sight. The evaluation of these parameters permits to verify the compatibility between the psychophysics characteristics of workers and the requests of tasks and then to define fitness to its work. Moreover to the visual functions we have to consider organic visual conditions like conjunctivitis, blepharitis, stenosis of lacrimal gland, dacriocystitis, retinic diseases, muscular motility alteration of the eyes, glaucoma. Our study shows the relation between ophthalmologic requirements of workers and some job groups like professional drivers, technical clerks, precision work, height level workers and proposes a standard method to define fitness to work. Our standard method permits a specific and homogeneous evaluation of fitness to work.

Cancer mortality in a cohort of asbestos textile workers.

A cohort of 889 men and 1077 women employed for at least 1 month between 1946 and 1984 by a former Italian leading asbestos (mainly textile) company, characterised by extremely heavy exposures often for short durations, was followed up to 1996, for a total of 53,024 person-years of observation. Employment data were obtained from factory personnel records, while vital status and causes of death were ascertained through municipality registers and local health units. We observed 222 cancer deaths compared with 116.4 expected (standardized mortality ratio, SMR=191). The highest ratios were found for pleural (SMR=4105), peritoneal (SMR=1817) and lung (SMR=282) cancers. We observed direct relationships with duration of employment for lung and peritoneal cancer, and with time since first employment for lung cancer and mesothelioma. Pleural cancer risk was independent from duration (SMR=3428 for employment <1 year, 7659 for 1-4 years, 2979 for 5-9 years and 2130 for > or =10 years). Corresponding SMRs for lung cancer were 139, 251, 233 and 531. Nonsignificantly increased ratios were found for ovarian (SMR=261), laryngeal (SMR=238) and oro-pharyngeal (SMR=226) cancers. This study confirms and further quantifies the central role of latency in pleural mesothelioma and of cumulative exposure in lung cancer.

Fluorescence polarization assay for the diagnosis of bovine brucellosis: adaptation to field use.

A fluorescence polarization assay (FPA) was used to test whole blood samples prepared by mixing blood cells from cattle without exposure to Brucella abortus (B. abortus) with sera from animals with confirmed (bacteriologically) infection. A cut-off value between negative and positive values was initially established to be 87.2mP. This value was changed to 95mP to increase assay specificity without loss of sensitivity when testing blood samples from negative animals. The FPA technology was applied to whole blood samples in the field and to stored whole blood samples using two diluent buffers. Relative sensitivity and specificity values for the FPA performed in the field, based on buffered antigen plate agglutination test and competitive enzyme immunoassay results were 95.3 and 97.3%, respectively. However, to obtain maximum sensitivity and specificity, a cut-off value of 105mP was determined for fresh whole blood samples. The relative sensitivity and specificity values of the FPA when testing stored whole blood samples were 100% each using a 95mP cut-off.The usefulness of the FPA for testing whole blood samples in the field was demonstrated.