Researches on pathogenic role and potential implications for public health of protozoa and microsporidia parasites of marine fish and studies on fish mycobacteriosis

Scopo primario delle attività di ricerca condotte nell’ambito di questo dottorato è stato quello di studiare le infezioni da protozoi e microsporidi enterici in specie ittiche d’allevamento e le micobatteriosi ittiche in ambienti d’allevamento e selvatici per ampliare le conoscenze su eziopatogenesi e diagnosi di queste patologie. Spigole, orate, rombi e cefali da diversi sistemi d’allevamento e di differenti età sono stati sottoposti ad indagini parassitologiche, molecolari ed istopatologiche per evidenziare, identificare e studiare protozoi Apicomplexa e Microsporidi a livello enterico, applicando diverse colorazioni istochimiche e, sui campioni positivi per microsporidi, la colorazione immunoistochimica con anticorpo anti-Encephalitozoon cuniculi. Su mugilidi selvatici e su spigole allevate sono stati condotti studi sui quadri istopatologici associati alle infezioni da Micobatteri atipici al fine di valutare la progressione delle lesioni. Le attività di ricerca hanno permesso di: individuare e descrivere in orate d’allevamento episodi d’infezione enterica da Enterospora nucleophila, microsporidio responsabile di sindromi emaciative in giovanili; rilevare massive infezioni da Cryptosporidium scophthalmi in rombi allevati e di infezioni da Cryptosporidium sp. in post-larve ed avannotti di orata; studiare infezioni da Mycobacterium spp. (M. fortuitum, M. abscessus, M. flavescens, M. chelonae, M. septicum, M. nonchromogenicum) in mugilidi selvatici con lesioni granulomatose positive alla ZN, e da Photobacterium damselae subsp. piscicida in soggetti con lesioni granulomatose negative; approfondire le conoscenze sulla micobatteriosi nella spigola descrivendo la concomitante presenza di lesioni a diverso stadio in tutti gli organi associate a presenza di batteri alcool-acido resistenti identificati come M. abscessus, M. scrofulaceum e M. gordonae sia all’interno delle lesioni che liberi nei tessuti e nei vasi, ipotizzando una forma di generalizzazione ematogena. Il rilievo di infezioni da micobatteri atipici in pesci marini allevati e selvatici appare di rilievo anche per le loro implicazioni in Sanità Pubblica.Main aim of the research activities carried out within this PhD was to study the infections due to enteric protozoa and Microsporidia in farmed marine fish and mycobacteriosis by atypical mycobacteria in wild and farmed marine fish in order to increase the knowledge about etiopathogenesis and diagnosis of these diseases. European sea bass, gilthead sea bream, turbots and mullets from different breeding systems and at different ages were subjected to parasitological, molecular and histopathological analyses using different histochemical methods; moreover immunohistochemical stain with antibody for Encephalitozoon cuniculi on positive samples for microsporidiosis was performed. Wild mullets and farmed European sea bass were subjected to studies on histopathological features associated to atypical mycobacteria infections in order to evaluate the progression of lesions. The research activities have allowed to: identify and describe in sea bream episodes of enteric infection by Enterospora nucleophila, microsporidium causing emaciative syndromes in juveniles; detect Cryptosporidium scophthalmi massive infections in farmed turbots and Cryptosporidium sp. infection in post-larvae and fry of sea bream; study infections due to Mycobacterium spp. (M. fortuitum, M. abscessus, M. flavescens, M. chelonae, M. septicum, M. nonchromogenicum) in wild mullets with granulomatous lesions positive for ZN, and due to Photobacterium damselae subsp. piscicida in subjects with negative lesions; increase the knowledge on mycobacteriosis in sea bass describing in all organs the simultaneous presence of lesions at different stages associated with the presence of positive ZN bacteria identified as M. abscessus, M. scrofulaceum and M. gordonae, both within the lesions that free in tissues and vessels, allowing to hypothesize a form of hematogenous generalization. The finding of atypical mycobacteria in wild and farmed fish is also relevant for their implications for public health

Investigating a Prognostic Factor for Canine Hepatocellular Carcinoma: Analysis of Different Histological Grading Systems and the Role of PIVKA-II

SIMPLE SUMMARY: PIVKA-II is an aberrant form of vitamin K that is increased in human coagulation disfunctions and in some neoplastic diseases. In veterinary medicine, PIVKA-II concentrations can be useful to identify patients with coagulative disorders in plasma and tissues, but its role as marker for hepatocellular carcinoma has not been investigated previously. In this study we characterized ed the expression of PIVKA-II in canine hepatocellular carcinomas in relation with the prognosis and some histological grading systems with the aim of finding useful prognostic factors for this canine tumour. ABSTRACT: Background: Hepatocellular carcinoma (HCC) in dogs is uncommon and often associated with a good prognosis, although some cases prove to be aggressive. In human oncology HCC is often very aggressive and diagnostic methods and prognostic factors are widely used to predict its biological behaviour. These include the expression of PIVKA-II. Methods: in order to identify a prognostic factor for canine HCC, we applied different methods of histological grading and investigated PIVKA-II expression in 22 HCC of dogs treated surgically and followed clinically for at least 2 years. Results: Nineteen patients analysed have passed the observation period without tumour recurrence, while 3 died following the development of metastases. PIVKA-II was positive in 15/22 cases without correlation with prognosis or tumoural grading even if a trend of PIVKA-II negativity in low WHO grades as well as increased number of PIVKA-II positive cases in higher WHO grades weres observed. Conclusions: This work showed that, PIVKA-II cannot be considered either as a marker of malignancy or as a prognostic marker for canine HCC. The poor prognosis depends usually on the clinical presentation. Thus prognostic parameters in canine HCC able to predict its aggressive behaviour through histological examination are still missing. The most promising method, limited to our study, seems to be the WHO histological grading

Toxoplasma gondii Genetic Diversity in Mediterranean Dolphins

Toxoplasma gondii constitutes a major zoonotic agent but also has been frequently identified as an important cause of clinical disease (e.g., abortion, pneumonia, encephalitis) in wildlife; specifically, T. gondii has been associated with neurological disease in cetaceans. This study investigated the genetic diversity of T. gondii strains involved in infections in dolphins found stranded in the Mediterranean coastlines of Italy. Tissue samples from 16 dolphins (Stenella coeruleoalba and Tursiops truncatus species) positive for T. gondii-DNA presence by PCR were examined by histology and subjected to further genetic characterization of strains detected by PCR-RFLP and multilocus PCR-sequencing assays. According to fully genotyped samples, the genotypes ToxoDB#3 (67%) and #2 (22%) were detected, the latter being reported for the first time in cetaceans, along with a mixed infection (11%). Subtyping by PCR-seq procedures provided evidence of common point mutations in strains from southwestern Europe. Despite evidence of T. gondii as a cause of neurological disease in dolphins, sources of infections are difficult to identify since they are long-living animals and some species have vast migration areas with multiple chances of infection. Finally, the genetic diversity of T. gondii found in the dolphins studied in the Mediterranean coastlines of Italy reflects the main genotypes circulating inland in the European continent

Anthropic Pressure on Cetaceans Stranded Along the Ligurian Coast Within the Pelagos Sanctuary: A Case Series

Simple Summary: In recent decades, environmental changes consequent to human activities have become more important factors in the conservation of wild species. Despite the Ligurian sea being part of the Pelagos Sanctuary, which has been a Specially Protected Areas of Mediterranean Importance (SPAMI) and a Marine Protected Area (MPA) since 1999, several anthropogenic threats affecting the health of cetacean populations living in this area are known to be present. In particular, interaction with fishing activities, contamination with chemical compounds such as organochlorine compounds (OCs), interaction with marine debris (especially, plastic debris), and the emergence of infectious agents, many of which are of terrestrial origin as a result of contamination of the marine environment by agricultural, animal, and human waste, are just some of the threats. In this regard, cetaceans stranded along the Ligurian coast between 2020 and 2022 were evaluated by means of a comprehensive post mortem examination and thorough specific diagnostic investigations to assess the level of anthropic pressure in this area by the Italian National Reference Center for Diagnosis of stranded Marine Mammals (C. Re. Di. Ma). Abstract: Data collected by C. Re. Di. Ma over a 3-year period (2020–2022) were considered to assess anthropic pressure on cetaceans living in the Ligurian sea. Out of a total of 37 stranded cetaceans, a complete post mortem examination was performed on 23 cases. Of these, 14 were further selected considering at least one of these conditions: (i) confirmed, probable, or suspected interaction with fishing activities through the application of a standardized diagnostic framework (7/14; 50%), (ii) toxicological stress through the evaluation of OCs hazardous levels (14/14; 100%), and (iii) terrestrial pathogen-associated disease (systemic infection and/or associated lesions) (7/14; 50%). For 9 animals out of a total of 14 selected, the cause of death was classified as natural (6/14; 42,8%), anthropic (3/14; 21,4%), or not determined (5/14; 35,7%) based on gross and histological pathology and ancillary testing. These findings extend our knowledge of the anthropic pressure to which cetaceans stranded along the Ligurian coastline are subjected from a multidisciplinary point of view

Effect of autolysis on the specificity of bovine spongiform encephalopathy rapid tests

<p>Abstract</p> <p>Background</p> <p>Routine rapid testing for Bovine Spongiform Encephalopathy (BSE) has highlighted some problems with BSE rapid test performance, the most significant being the number of initially reactive samples and the false positive results on autolyzed tissue. This point is important for BSE active surveillance in risk populations, because tissue autolysis is often unavoidable in routine cases. A robust test suitable for use on field material is therefore needed. To date, very limited information regarding the effect of autolysis on the robustness of rapid tests has been documented; therefore, the National Reference Centre for Animal Encephalopathies (CEA) rapid test laboratory selected 450 autolyzed and negative brain stem samples from fallen stock bovines older than 24 months to assess the specificity of four tests approved for BSE active surveillance: Biorad TeSeE, Enfer TSE version 2.0, Prionics^®Check LIA, and IDEXX Herd Check BSE Antigen Kit EIA. The samples were graded according to the degree of autolysis and then dissected into five portions, four of which randomly assigned to processing by rapid tests and one to be available for confirmatory Western blot analysis.</p> <p>Findings</p> <p>The specificity of the four systems was 100% for all three grades of autolysis, while the percentage of initially reactive results was 0.00 (95%CI 0.00-0.82), 0.22 (95%CI 0.006-1.23), 0.44 (95%CI 0.05-1.60), and 0.89 (95%CI 0.24-2.26) for the Biorad TeSeE, the Prionics^®Check LIA, the IDEXX Herd Check BSE and the Enfer TSE tests, respectively. No association with the degree of autolysis could be drawn.</p> <p>Conclusions</p> <p>The present study demonstrates that the four rapid tests can be considered well-running diagnostic tools regardless of tissue quality; nevertheless, the number of initial reactive samples reported for some systems must not be underestimated in routine testing.</p> <p>Furthermore the compliance with the reported performance can be guaranteed only when an ongoing high careful batch quality control system is in place.</p

A revisited hemolytic assay for palytoxin detection: Limitations for its quantitation in mussels

7siPalytoxin (PLTX) and its analogues have been detected as seafood contaminants associated with a series of human foodborne poisonings. Due to a number of fatalities ascribed to the ingestion of PLTXcontaminated marine organisms, the development of methods for its detection in seafood has been recommended by the European Food Safety Authority (EFSA). Due to its feasibility, the spectrophotometric hemolytic assay is widely used to detect PLTX in different matrices, even though a standardized protocol is still lacking. Thus, on the basis of available assay procedures, a new standardized protocol was set up using purified human erythrocytes exposed to PLTX (working range: 3.9 1010e2.5 108 M) in a Kþ-free phosphate buffered saline solution, employing a 5 h incubation at 41 C. An intra-laboratory characterization demonstrated its sensitivity (limit of detection, LOD ¼ 1.4 1010 M and quantitation, LOQ ¼ 3.4 1010 M), accuracy (bias ¼ 0.8%), repeatability (RSDr ¼ 15% and 6% for intra- and inter-day repeatability, respectively) and specificity. However, the standardized method seems not to be suitable for PLTX quantitation in complex matrices, such as mussel (Mytilus galloprovincialis) extracts, at least below the limit suggested by EFSA (30 mg PLTXs/Kg shellfish meat). Thus, the hemolytic assay for PLTX quantitation in seafood should be used only after a careful evaluation of the specific matrix effects.partially_openembargoed_20170623Brovedani, V.; Sosa, S.; Poli, M.; Forino, M.; Varello, K.; Tubaro, A.; Pelin, M.Brovedani, Valentina; Sosa, Silvio; Poli, M.; Forino, M.; Varello, K.; Tubaro, Aurelia; Pelin, Marc

A revisited hemolytic assay for palytoxin detection: Limitations for its quantitation in mussels

Palytoxin (PLTX) and its analogues have been detected as seafood contaminants associated with a series of human foodborne poisonings. Due to a number of fatalities ascribed to the ingestion of PLTXcontaminated marine organisms, the development of methods for its detection in seafood has been recommended by the European Food Safety Authority (EFSA). Due to its feasibility, the spectrophotometric hemolytic assay is widely used to detect PLTX in different matrices, even though a standardized protocol is still lacking. Thus, on the basis of available assay procedures, a new standardized protocol was set up using purified human erythrocytes exposed to PLTX (working range: 3.9 1010e2.5 108 M) in a Kþ-free phosphate buffered saline solution, employing a 5 h incubation at 41 C. An intra-laboratory characterization demonstrated its sensitivity (limit of detection, LOD ¼ 1.4 1010 M and quantitation, LOQ ¼ 3.4 1010 M), accuracy (bias ¼ 0.8%), repeatability (RSDr ¼ 15% and 6% for intra- and inter-day repeatability, respectively) and specificity. However, the standardized method seems not to be suitable for PLTX quantitation in complex matrices, such as mussel (Mytilus galloprovincialis) extracts, at least below the limit suggested by EFSA (30 mg PLTXs/Kg shellfish meat). Thus, the hemolytic assay for PLTX quantitation in seafood should be used only after a careful evaluation of the specific matrix effects

A novel divergent group of Ostreid herpesvirus 1 μVar variants associated with a mortality event in Pacific oyster spat in Normandy (France) in 2016

International audienceThe acute course of disease in young oysters infected by OsHV-1 and the rapid tissue degradation often preclude histological examination of specimens collected during outbreaks in field. Herein, live spat originated from two geographical areas were sampled just at the onset of a mortality event that occurred in Normandy (France) in June 2016. The lesions, associated with high OsHV-1 DNA quantities, were characterized by severe and diffuse haemocytosis mainly involving blast-like cells, myocyte degeneration and large, irregularly shaped degenerate eosinophilic cells in the connective tissue. The herpesvirus was identified by negative staining TEM and real-time PCR. Sequencing of the C region and ORFs 42/43 confirmed that the variants met the definition of OsHV-1 μVar. We sequenced 30 other ORFs in twenty OsHV-1-positive individuals and compared them to the μVar specimens isolated between 2009 and 2011. The ORFs encoding putative membrane protein

Abnormal Prothrombin (PIVKA-II) Expression in Canine Tissues as an Indicator of Anticoagulant Poisoning

PIVKA-II is an aberrant form of vitamin K that has been demonstrated to be increased in human coagulation disorders and in some neoplastic diseases. In veterinary medicine, PIVKA-II levels have been demonstrated to be useful for distinguishing anticoagulant poisoning from other coagulopathies. In forensic pathology, there is the need to distinguish malicious poisoning from other causes of death and, in some cases, identifying poisoned dogs from dogs that died as a result of other coagulative disorders can be challenging. In this study, dogs that suddenly died underwent necropsy, histological examination, and toxicological analysis to establish cause of death. PIVKA-II immunohistochemical expression was evaluated on hepatic and renal tissues, and on neoplastic lesions when present. A total of 61 dogs were analyzed and anticoagulant substances were identified in 16 of the 61. Immunolabelling for PIVKA-II was observed in 27 of 61 cases in the liver and in 24 of 61 cases in the kidneys. Among the poisoned dogs, the PIVKA-II expression was present in the liver in 15 of 16 cases and in the kidneys in 16 of 16. Neoplastic lesions represented mainly by haemangiosarcomas were negative. This study highlights how the immunohistochemical expression of PIVKA-II in hepatic and renal tissues can be useful to identify patients with coagulative disorders due to clinical condition or the ingestion of anticoagulants substances