On the morphology of Anisakis pegreffii: a comparative analysis of three microscopic techniques used to build a new parasite atlas

BACKGROUND: Human anisakidosis is a parasitic anthropozoonosis caused by larval nematodes of the family Anisakidae. Here, we report a detailed description of the morphology of Anisakis pegreffii third-stage larva performed using a conventional light and confocal microscopy, and scanning electron microscopy (SEM) that provide a basis for both phenotypic studies and genetic mutations. METHODS: The collected larvae from fish were morphologically identified as Anisakis larvae Type I, and they were characterized by PCR-RFLP to identify the Anisakis pegreffii specie. Using NC5/NC2 primers, ribosomal genomic regions ITS1, 5.8 SrRNA and ITS2 of DNA were amplified and PCR products were sequenced. Fifteen larvae belonging to Anisakis pegreffii were fixed, sectioned, and examined with a light and confocal microscope and by SEM. RESULTS: In our studies, have been acquired detailed ultrastructural images, which have been integrated with those derived from the dissection of the parasite, obtained with light and confocal microscopy. The structural and ultrastructural images concerning the third stage larvae of Anisakis pegreffii have been studied, analyzed and compared among them. The derived overall view has allowed detecting new interesting details of a well-known parasite and has been schematically showed. CONCLUSIONS: The aim of this study is to furnish an updated atlas of Anisakis pegreffii. Confocal microscopy, as well as the light and electron microscopy have played a pivotal role in the accumulation of new scientific data regarding the anatomical structures of this nematode. This work is the result of one year of engagement by the Authors and the outcome is a comprehensive atlas on Anisakis pegreffii microscopy

Exercise-induced Modifications on Haematochemical and Electrophoretic Parameters During 1600 and 2000 Meters Trot Races in Standardbred Horses

Abstract Piccione, G., Casella, S., Giannetto, C., Monteverde, V. and Ferrantelli, V. 2009. Exercise-induced modifications on haematochemical and electrophoretic parameters during 1600 and 2000 meters trot races in Standardbred horses. J. Appl. Anim. Res., 35: 131–135. The aim of the present study was to evaluate the modifications of some haematochemical and electrophoretic parameters during two different workloads in Standardbred horses. Ten clinically healthy horses were divided into two equal groups, taking part in an official trot race at 1600 or 2000 meters course, respectively. Blood samples were collected from each horse at rest, after warm up, 0, 30 and 60 min after the end of the race. Serum concentrations of albumin, globulins, total proteins, total cholesterol, triglycerides, glucose, hydroxybutyrate, urea, creatinine, creatine kinase (CK), glutamate pyruvate transaminases (GPT) and glutamate oxalacetate transaminases (GOT) were assessed. Exercise increased triglyceride, glucose, creatinine and..

Effects of diclofenac on the gametes and embryonic development of Arbacia lixula

Diclofenac (DCF), a non-steroidal anti-inflammatory drug (NSAID), is among the main pollutants of aquatic environments. Nevertheless, even if several authors evaluated its effects on marine organisms, no work has ever analysed its impact on the sea urchin Arbacia lixula. The purpose of this study was to analyse, for the first time, the impact of DCF at different concentrations (50 mg/l, 5 mg/l, 0.5 mg/l, 0.05 mg/l) on A. lixula embryos and gametes to indicate possible safe minimum levels of release of this drug in marine environments. Our results showed significant concentration-dependent effects on embryonic development (decrease in the number of developed embryos and increase in the quantity of degenerated eggs) and significant levels of bioaccumulation at DCF concentrations of 50 mg/l in both gametes and embryos. DCF showed a significant impact on the fertilisation of A. lixula due to its effects on gametes, confirming the possibility of environmental risk and highlighting the need to improve wastewater treatment and drug disposal processes

In vitro effect of cadmium and copper on separated blood leukocytes of Dicentrarchus labrax.

The immunotoxic effects of heavy metals on blood leukocytes of sea bass (Dicentrarchus labrax) were examined. The cells, separated by a discontinuous Percoll-gradients, were exposed in vitro to various sublethal concentrations of cadmium and copper (10(-7) M, 10(-5) M, and 10(-3) M) and their immunotoxic effect was then evaluated by measuring neutral red uptake, MU assay, DNA fragmentation and Hsp70 gene expression. First of all, we demonstrated that the cells treated in vitro could incorporate Cd and Cu. A relationship between heavy metal exposure and dose-time-dependent alterations in responses of leukocytes from blood was found for both metals, but copper was more immunotoxic than cadmium in all assays performed. A significant reduction in the cells' ability to uptake neutral red and viability by MU assay was recorded, indicating that both cadmium and copper could change the membrane permeability, inducing cellular apoptosis when the concentration of metals reached 10(-3) M. The apoptotic effect may also explain the high level of cytotoxicity found when the leukocytes were exposed to higher concentration of metals. These results demonstrated that toxic effect of copper and cadmium affect on the mechanisms of cell-mediated immunity reducing the immune defences of the organism. (C) 2014 Elsevier Inc. All rights reserved

Aroclor 1254 inhibits the chemiluminescence response of peritoneal cavity cells from sharpsnout sea bream (Diplodus puntazzo)

Chronic exposure to polychlorinated biphenyls (PCBs) affect the immune system of fish and could lead to a decreased disease resistance. The effects of Aroclor 1254, PCB mixtures, on the Diplodus puntazzo innate immunity were examined by assaying the zymosan stimulated chemiluminescence response (CL) of peritoneal cavity cells (PCCs) at various times (1, 24, 48 h and 1-4 weeks) from intraperitoneal injection of the xenobiotic (1 mg kg(-1) body weight). Controls were performed by assaying cells from medium-treated fish. Since the kinetic of the chemiluminescence response showed the highest peak at 25 min after the zymosan stimulation of the cells, the values found at that time were considered. The CL enhancement observed at 1 h after the treatment with xenobiotic was followed by a decreased response at 24 h and appeared to be lower at 1-4 weeks when compared to the CL response of the control, suggesting a protracted effect of PCBs on the peritoneal cavity. Since PCCs incubated in vitro for 1 h with 0.05 and 0.1 mu g ml(-1) Aroclor showed an enhanced CL, the effect of the xenobiotic could be exerted on the cell responsiveness to zymosan. It is known that fish CL response of PCCs can be imputed to phagocyte (macrophages and neutrophils) activation, these cells and their responsiveness to zymosan can be used in immunotoxicology assay to monitor the fish health in polluted environment. (C) 2014 Elsevier Ltd. All rights reserved

Changes in eggshell ultrastructure of Falco naumanni and Tyto alba exposed to pesticides and polychlorinated biphenyls (PCBs)

Changes in the quality of eggs of birds exposed to environmental contaminants have been described, but few reports concern eggshell ultrastructure. In this study, infertile or addled Lesser Kestrels (Falco naumanni) and Barn owls (Tyto alba) eggs were collected from the polluted area of Gela plain (Sicily) during 2007, and compared in terms of organophosphate and organochlorine pesticides, and PCBs levels, and eggshell ultrastructure as determined by scanning electron microscopy. Pesticide and PCB residues in eggs were determined by Gas chromatography/ mass spectrometry (GC/MS) [GC Agilent 7890A/MS Agilent 5975C (Agilent technologies) using a DB-5 capillary column in the selected ion monitoring mode]. The GC/MS analysis revealed that eggs contained measurable amounts of some pesticides and PCBs. There was a low detection of organophosphate pesticides while the most abundant organochlorine residues detected were p,p’ DDT, p,p’ DDE, and Hexachlorobenzene. While, the most abundant PCBs detected congeners were PCB 138, 153, 170, 180, and 187. Although the general structure of the eggshell layers was maintained, the results showed ultrastructural differences in mammillary and palisade eggshell layers between high level and low level contaminated eggs in Tyto alba. Furthermore, mammillary cores of the eggshell had an increased distance between themselves with respect to well organized structures present in uncontaminated egg. In this paper we verify the presence of environmental contaminants in the eggs and document structural changes in bird of prey eggshells. The data could suggest that some contaminants can contribute to reduced reproductive performance (infertile or addled egg) by structural changes in the eggshell. The alteration in morphological disposition of mammillary cores could also suggest an impairment of gas exchange

Anisakis sensitization in different population groups and public health impact. A systematic review

Anisakis simplex spp. sensitization rates have increased worldwide, with a significant impact on health-care systems. To date, no clear-cut diagnostic criteria and laboratory algorithm have been established, so anisakiasis still represents an under-reported health problem whose clinical manifestations, when present, mimic the much more common allergic and digestive disorders. Aim of the study was to systematically review the available literature on the prevalence of sensitization against Anisakis in the general population and in specific population groups, taking into account the impact of the different available diagnostic techniques on the epidemiological data. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement, relevant papers reporting Anisakis sensitization epidemiological data were found covering a period ranging from 1996 to February 2017. Overall, 41 studies comprising 31,701 participants from eleven countries were included in the qualitative synthesis. General asymptomatic population resulted sensitized to Anisakis in 0.4 to 27.4% of cases detected by means of indirect ELISA or ImmunoCAP specific IgE detection, and between 6.6% and 19.6% of the samples by Skin prick test (SPT). Occupationally exposed workers (fishermen, fishmongers and workers of fish-processing industries) documented specific IgE between 11.7% and 50% of cases, whereas SPT positivity ranged between 8% and 46.4%. Symptomatic allergic patients to any kind of allergen were found to be positive to Anisakis specific IgE detection between 0.0% (in children with mastocytosis) to 81.3% (among adults with shellfish allergy). Results highlighted that hypersensitivity prevalence estimates varied widely according to geographical area, characteristics of the population studied, diagnostic criteria and laboratory assays. Further studies are needed to overcome the documented misdiagnosis by improving the diagnostic approach and, consequently, providing more affordable estimates in order to address public health interventions on populations at high risk of exposure to Anisakis and to tailor health services related to specific groups

Development of a rapid and eco-friendly UHPLC analytical method for the detection of histamine in fish products

We developed, validated, and confirmed with proficiency tests a fast ultra-high-performance liquid chromatography with diode array detector (UHPLC-DAD) method to determine histamine in fish and fishery products. The proposed method consists of two successive solid–liquid extractions: one with a dilute solution of perchloric acid (6%) and the second only with water. The instrumental analysis with UHPLC provides a very fast run time (only 6 min) with a retention time of approximately 4 min, a limit of quantification (LOQ) of 7.2 mg kg−1, a limit of detection (LOD) of 2.2 mg kg−1, a recovery around 100%, a relative standard deviation (RSD%) between 0.5 and 1.4, and an r2 of calibration curve equal to 0.9995. The method detected optimal values of the validation parameters and required a limited number of reagents in comparison to other methods reported in the literature. Furthermore, the method could detect histamine in a very short time compared with other methods. This method, in addition to being validated, precise, specific, and accurate, avoids wasting time, money, and resources, and limits the use of organic solvents

Trace elements in stomach oil of Scopoli's shearwater (Calonectris diomedea) from Linosa's colony

Calonectris diomedea is a colonial Procellariiform breeding on Mediterranean islands. The stomach oil produced during chick rearing is a peculiar trait of this species. The composition of the stomach oil is likely to reflect the composition of the prey ingested and might reveal the contaminants uptake with prey becoming a possible tool for the marine pollution monitoring. We examined the concentration of 15 trace elements by ICP-MS and direct mercury analyser. The principal component analysis revealed a heterogeneous pattern of metal concentration, showing a significant separation between samples collected 20 and 70 days after hatching. The data obtained in this work give preliminary information on the feeding habits and breeding ecology of Linosa's colony of Scopoli's shearwater. The trace metals variability found suggest that the stomach oil may have a role as trophic markers to understand predator-prey relationships and to have evidence on the accumulation of pollutants in the latter