Antimicrobial, antioxidant and anti-tyrosinase properties of extracts of the Mediterranean parasitic plant Cytinus hypocistis

Background: Cytinus is an endophytic parasitic plant occurring in South Africa, Madagascar, and in the Mediterranean region. We have extracted the inflorescences (the only visible part of the plant, emerging from the host roots at the time of blossom) of Cytinus hypocistis collected in Sardinia, Italy, and explored the antimicrobial, antioxidant, anti-tyrosinase, and cytotoxic activities of the extracts. Methods: Extracts from C. hypocistis were prepared using increasing polarity solvents: cyclohexane, ethanol, and water. Phenolic composition were determined through spectrophotometric assays, and antioxidant activity with both electron-transfer and hydrogen-atom assays. Nine different bacterial strains, including clinical isolate methicillin-resistant Staphylococcus aureus, were used in agar diffusion method. Cytotoxicity was tested using against the B16F10 melanoma cell line. Results: While cyclohexane extracts where biologically inactive, ethanolic and aqueous extracts displayed an intriguing activity against several Gram-positive bacterial strains, including methicillin-resistant S. aureus, and against the Gram-negative Acinetobacter baumanii. Compared to the conventional antibiotics like cloxacillin, ampicillin, and oxytetracycline, C. hypocistis extracts were less active in absolute terms, but displayed a wider spectrum (notably, cloxacillin and ampicillin were inactive against methicillin-resistant S. aureus). The ethanolic extract of C. hypocistis was found to be particularly rich in polyphenols, in most part hydrolysable tannins. The antioxidant activity of extracts, tested with several methodologies, resulted to be particularly high in the case of ethanolic extracts, in accordance with the composition in phenolics. In detail, ethanol extracts presented about a twofold higher activity than the water sample when tested through the oxygen radical absorbance capacity-pyrogallol red (ORAC-PYR) assay. Cytotoxicity analysis against the B16F10 melanoma cell line showed that both extracts have not significant cytotoxic effect, even at the highest dose (1000 μg/mL). Tests showed that ethanolic extracts also had the greatest tyrosinase inhibition activity, indicating that C. hypocistis-derived substances could find application in food formulations as anti-browning agents. Conclusions: Overall, these results point to the need of further studies on C. hypocistis extracts, aimed at isolating and fully characterizing its biologically active compounds. © 2015 Zucca et al

Analysis of cell hyperplasia and parietal cell dysfunction induced by Ostertagia ostertagi infection

Infections in cattle with the gastric nematode Ostertagia ostertagi are associated with decreased acid secretion and profound physio-morphological changes of the gastric mucosa. The purpose of the current study was to investigate the mechanisms triggering these pathophysiological changes. O. ostertagi infection resulted in a marked cellular hyperplasia, which can be explained by increased transcriptional levels of signaling molecules related to the homeostasis of gastric epithelial cells such as HES1, WNT5A, FGF10, HB-EGF, AREG, ADAM10 and ADAM17. Intriguingly, histological analysis indicated that the rapid rise in the gastric pH, observed following the emergence of adult worms, cannot be explained by a loss of parietal cells, as a decrease in the number of parietal cells was only observed following a long term infection of several weeks, but is likely to be caused by an inhibition of parietal cell activity. To investigate whether this inhibition is caused by a direct effect of the parasites, parietal cells were co-cultured with parasite Excretory/Secretory products (ESP) and subsequently analyzed for acid production. The results indicate that adult ESP inhibited acid secretion, whereas ESP from the L4 larval stages did not alter parietal cell function. In addition, our data show that the inhibition of parietal cell activity could be mediated by a marked upregulation of inflammatory factors, which are partly induced by adult ESP in abomasal epithelial cells. In conclusion, this study shows that the emergence of adult O. ostertagi worms is associated with marked cellular changes that can be partly triggered by the worm's Excretory/secretory antigens

Comparison of faecal techniques including FLOTAC for copromicroscopic detection of first stage larvae of Angiostrongylus vasorum

Angiostrongylus vasorum is a metastrongylid nematode that resides in the pulmonary arteries and the right heart chambers. In dogs, infection results in respiratory, bleeding and neurological disorders and further clinical signs. In the present study, FLOTAC was evaluated for the detection of first-stage larvae (L1) of A. vasorum in canine faecal samples. This technique is based on the counting of parasitic stages (eggs, larvae, oocysts and cysts) in chambers after spinning of faecal samples onto a surface. In a first step, nine flotation solutions were evaluated using faeces of two experimentally infected dogs. Zinc sulphate (specific gravity (s.g.) 1.2) and zinc sulphate plus potassium iodomercurate (s.g. 1.45) gave good results. However, with the latter technique, the larvae were slightly deformed. Subsequently, FLOTAC, using zinc sulphate, was compared through a randomisation technique with McMaster, flotation in tube and Baermann-Wetzel technique. The mean larvae per gramme (LPG) obtained by the FLOTAC for both dogs was significantly higher (P < 0.05) than those obtained by the other three techniques (the means of the other techniques all lie below the 95% CI of the mean LPG of the FLOTAC technique). In addition, the FLOTAC results were consistent across replicates with only Poisson (or random) variation between individual replicates. The other techniques appear to be less consistent with evidence of extra-Poisson variation in at least one of the two dogs across the replicates within each technique. The FLOTAC technique may contribute to an improvement of the ability to diagnose canine lungworm infections and represent a valuable alternative for larval counting of A. vasorum in faecal samples, especially following transport or storage where there may be limited larvae viability, and larval migration techniques cannot be use

Genome wide analysis of the bovine mucin genes and their gastrointestinal transcription profile

<p>Abstract</p> <p>Background</p> <p>Mucins are large glycoproteins implicated in protection of all mucosal surfaces. In humans and rodents, the mucin gene family has been well described and previous studies have investigated the distribution and function of mucins in the gastrointestinal (GI) tract. In contrast, little data is available on the mucin gene family in polygastric species, such as cattle. The aim of the current study was to identify all members of the bovine mucin family by genome mining and subsequently investigate the transcription pattern of these mucins in the GI tract.</p> <p>Results</p> <p>Nine bovine membrane-associated mucins (<it>MUC1</it>, <it>MUC3A</it>, <it>MUC4</it>, <it>MUC12</it>, <it>MUC13</it>, <it>MUC15</it>, <it>MUC16</it>, <it>MUC20 </it>and <it>MUC21</it>) and six secreted mucins (<it>MUC2</it>, <it>MUC5AC</it>, <it>MUC5B</it>, <it>MUC6</it>, <it>MUC7 </it>and <it>MUC19</it>) were identified in the bovine genome. No homologues could be identified for <it>MUC3B</it>, <it>MUC8 </it>and <it>MUC17</it>. In general, domain architecture of the membrane-associated mucins was found to be similar between humans and cattle, while the protein architecture of the gel-forming mucins appeared to be less conserved. Further analysis of the genomic organization indicated that the previously reported bovine submaxillary mucin (<it>BSM</it>) may be part of a larger gene encoding for MUC19. Analysis of the transcription profile showed that the secreted mucins were transcribed from the abomasum onwards, whereas the membrane associated mucins <it>MUC1 </it>and <it>MUC20 </it>were transcribed throughout the whole GI tract. In contrast to humans, <it>MUC5B </it>transcript was found in both the small and large intestine, but was absent in oesophageal tissue.</p> <p>Conclusions</p> <p>This study provides the first characterization of the mucin gene family in cattle and their transcriptional regulation in the GI tract. The data presented in this paper will allow further studies of these proteins in the physiology of the GI tract in ruminants and their interactions with pathogens.</p

Genetic Immunization with CDR3-Based Fusion Vaccine Confers Protection and Long-Term Tumor-Free Survival in a Mouse Model of Lymphoma

Therapeutic vaccination against idiotype is a promising strategy for immunotherapy of B-cell malignancies. We have previously shown that CDR3-based DNA immunization can induce immune response against lymphoma and explored this strategy to provide protection in a murine B-cell lymphoma model. Here we performed vaccination employing as immunogen a naked DNA fusion product. The DNA vaccine was generated following fusion of a sequence derived from tetanus toxin fragment C to the VHCDR3109−116 epitope. Induction of tumor-specific immunity as well as ability to inhibit growth of the aggressive 38C13 lymphoma and to prolong survival of vaccinated mice has been tested. We determined that DNA fusion vaccine induced immune response, elicited a strong protective antitumor immunity, and ensured almost complete long-term tumor-free survival of vaccinated mice. Our results show that CDR3-based DNA fusion vaccines hold promise for vaccination against lymphoma

Infection with the gastrointestinal nematode Ostertagia ostertagi in cattle affects mucus biosynthesis in the abomasum

The mucus layer in the gastrointestinal (GI) tract is considered to be the first line of defense to the external environment. Alteration in mucus components has been reported to occur during intestinal nematode infection in ruminants, but the role of mucus in response to abomasal parasites remains largely unclear. The aim of the current study was to analyze the effects of an Ostertagia ostertagi infection on the abomasal mucus biosynthesis in cattle. Increased gene expression of MUC1, MUC6 and MUC20 was observed, while MUC5AC did not change during infection. Qualitative changes of mucins, related to sugar composition, were also observed. AB-PAS and HID-AB stainings highlighted a decrease in neutral and an increase in acidic mucins, throughout the infection. Several genes involved in mucin core structure synthesis, branching and oligomerization, such as GCNT3, GCNT4, A4GNT and protein disulphide isomerases were found to be upregulated. Increase in mucin fucosylation was observed using the lectin UEA-I and through the evaluation of fucosyltransferases gene expression levels. Finally, transcription levels of 2 trefoil factors, TFF1 and TFF3, which are co-expressed with mucins in the GI tract, were also found to be significantly upregulated in infected animals. Although the alterations in mucus biosynthesis started early during infection, the biggest effects were found when adult worms were present on the surface of the abomasal mucosa and are likely caused by the alterations in mucosal cell populations, characterized by hyperplasia of mucus secreting cells

Diagnosis of coccidiosis by Eimeria spp. in free-range chickens using Mini-FLOTAC and McMaster techniques - preliminary results

Mini-FLOTAC is emerging as a more sensitive and accurate tool to identify gastrointestinal parasites in faecal samples from domestic animals, in comparison with the McMaster method. However, research regarding its specific application in poultry samples, particularly from free-range chickens, is scarce. The current research aimed to test the use of Mini-FLOTAC for the identification of Eimeria spp. in free-range chickens and compare its results with McMaster. For this study, 40 faecal samples were collected from free-range chickens in a poultry farm located in North-Western Lisbon (Portugal). Each sample was processed with McMaster and Mini-FLOTAC techniques for the detection and count of coccidian Eimeria spp. oocysts. The resulting OPG (oocysts per gram of faeces) data obtained by the two techniques were compared using the Wilcoxon Test and correlated with the Spearman Test, and Mini-FLOTAC’s relative sensitivity was assessed, using a significance level of p<0.05. The average OPG was higher with Mini-FLOTAC and doubled the one obtained using the McMaster method (2669.3 OPG and 1220 OPG, respectively), although these results were not significant. Mini-FLOTAC’s relative sensitivity obtained in this study reached 86% (70.5-95.3%, 95%CI), although this result was not statistically significant. However, correlation of OPG counts between Mini-FLOTAC and McMaster, was significant. These preliminary results suggest the potential interest in the use of Mini-FLOTAC for the diagnosis of coccidiosis by Eimeria spp. in poultry, based on its assessment in a free-range poultry production system.info:eu-repo/semantics/publishedVersio

Implementation of Mini-FLOTAC in routine diagnosis of Coccidia and Helminth infections in domestic and exotic birds

Mini-FLOTAC (MF) has recently been proposed for the fecal quantification of gastrointestinal (GI) parasites in birds due to its higher sensitivity and precision in comparison with the McMaster method. The current research aimed to test the use of MF in routine diagnosis of coccidia and helminth infections in several domestic and exotic bird collections in Portugal. Between July 2020 and April 2021, a total of 142 fecal samples from organic layers, peacocks and ratites were collected in four Portuguese bird collections and processed using MF and fecal cultures to identify and calculate GI parasite shedding and prevalence. The McMaster method was also used to compare the shedding levels obtained for both quantitative techniques. MF’s relative sensitivity and specificity were also assessed, using McMaster as the reference technique. The implementation of MF resulted in an average Eimeria spp. shedding higher in peacocks from bird collection 2 (502 OPG), followed by peacocks from collection 1 (107 OPG) and organic layers (24 OPG) and peacocks from collection 3 (9 OPG). Peacocks were also positive for Capillaria spp., Trichostrongylus tenuis and Strongyloides pavonis, whereas ostriches and emus were infected by L. douglassii. The MF protocol for exotic animals and the McMaster method did not differ significantly for each parasitic agent and bird species, and MF achieved relative sensitivities and specificities higher than 70% for Galliform Eimeria spp., peacock helminths and ratites’ L. douglassii infections. Higher L. douglassii EPG values were identified using the MF protocol for exotic species (2 g of feces/38 mL of sucrose solution), followed by McMaster 2/28, MF 5/45 and MF 2/18. The use of MF allowed for obtaining different intestinal parasitic populations in several bird species and locations, and MF 2/38 is globally proposed as the most suitable protocol for bird fecal samples as an alternative to the McMaster method in the diagnosis of avian intestinal parasitic infections.info:eu-repo/semantics/publishedVersio