Identification of new amoebae strains in rainbow trout (Oncorhynchus mykiss, Walbaum) farms affected by nodular gill disease (NGD) in Northeastern Italy.

Nodular gill disease (NGD) is an emerging condition associated with amoeba trophozoites in freshwater salmonid farms. However, unambiguous identification of the pathogens still must be achieved. This study aimed to identify the amoeba species involved in periodic NGD outbreaks in two rainbow trout (Oncorhynchus mykiss) farms in Northeastern Italy. During four episodes (February-April 2023), 88 fish were euthanized, and their gills were evaluated by macroscopic, microscopic and histopathological examination. The macroscopic and microscopic severity of the lesions and the degree of amoebae infestation were scored and statistically evaluated. One gill arch from each animal was put on non-nutrient agar (NNA) Petri dishes for amoeba isolation, cultivation and subsequent identification with SSU rDNA sequencing. Histopathology confirmed moderate to severe lesions consistent with NGD and mild to moderate amoeba infestation. The presence of amoebae was significantly correlated with lesion severity. Light microscopy of cultured amoebae strains and SSU rDNA analysis revealed the presence of a previously characterized amoeba Naegleria sp. strain GERK and several new strains: two strains from Hartmannelidae, three vannelid amoebae from the genus Ripella and cercozoan amoeba Rosculus. Despite the uncertainty in NGD etiopathogenesis and amoebae pathogenic role, identifying known and new amoebae leans towards a possible multi-aetiological origin.

Pearl formation associated with gymnophallid metacercariae in Mytilus galloprovincialis from the Northwestern Adriatic coast: Preliminary observations.

Gymnophallids of the genus Parvatrema are small trematodes infecting waterbirds in their adult stage. Several species of clams and mussels have been found to act as first and second intermediate hosts, in which the trematode larval stages induce the formation of pearls. In this study, a wild population of Mytilus galloprovincialis was sampled along the Northwestern coast of the Adriatic Sea to evaluate the origin and extent of visible pearls. Parasitological investigations, including morphological and molecular analyses, and histopathology were carried out on a representative sample of mussels (n = 158) from June to September 2021. The overall prevalence of infection reached 75.3 %, and the intensity of infection ranged from a few trematodes to thousands per mussel, mostly occurring in the mantle and surrounded by variable numbers of conchiolin layers. Morphological studies allowed classification of the metacercariae as belonging to the genus Parvatrema, and the pairwise comparison of the obtained sequences, encompassing the internal transcribed spacer (ITS) region, revealed a genetic similarity (96.8 %) to Parvatrema duboisi. However, the phylogenetic analysis demonstrated the independent clustering of the obtained ITS sequences compared to other available Parvatrema species. For the relevant commercial impact that pearl formation may have on farmed mussels, ecological and epidemiological aspects of this infection would deserve further investigation in the area.

Nodular gill disease in Northeastern Italy: An investigation on the prevalence of the disease and the risks of introduction in rainbow trout farms.

Nodular Gill Disease (NGD) is an emerging pathogenic condition that causes gill damage and mainly affects farmed freshwater fish, rainbow trout (Oncorhynchus mykiss) in particular, and this inevitably generates noticeable economic losses for the industry. The present study aimed to assess the prevalence of NGD in the Autonomous Province of Trento, a highly productive area located in Northeastern Italy, traditionally suited to rainbow trout production, and to identify possible risk factors for the introduction of this disease in trout farms. The necessary data were obtained through a questionnaire and the collection of fish samples. According to the data analysis, 42% of the investigated farms tested positive for NGD. The two possible risk factors identified for its introduction in farms are the presence of other diseases in the same farm (OR = 17.5; 95% CI = 2.7; 111.5) and having farms located 5 km upstream (OR = 24.8; 95% CI = 2.9; 211.1). These results highlight (i) a possible impairment of the immune system caused by other diseases as a predisposing factor to the manifestation of the pathology and (ii) the role of water in spreading pathogens.

Severe gastroenteropathy associated with Clostridium perfringens isolation in starving juvenile sturgeons.

In November 2020 a mortality episode (30%) in juvenile Siberian and Russian sturgeons (Acipenser baerii, Brandt, and A. gueldenstaedtii, Brandt & Ratzeburg) and GUBA hybrid sturgeons (A. gueldenstaedtii × A. baerii) occurred in a hatchery in Northern Italy, associated with severe coelomic distension and abnormal reverse surface swimming. The fish were reared in concrete tanks supplied by well water, fed at 0.4% of body weight (b.w.) per day. Thirty sturgeon specimens were collected for necropsy, histological, bacteriological and virological examination. Macroscopic findings included diffuse and severe bloating of gastrointestinal tracts due to foamy contents with thinning and stretching of the gastrointestinal walls. Histological analysis revealed variable degrees of sloughing and necrosis of the intestinal epithelium, and the presence of bacterial aggregates. Anaerobic Gram-positive bacteria were investigated, and Clostridium perfringens was isolated from the gut. Specific PCRs identified the toxinotype A and the ß2 toxin gene. The daily feed administration was increased to 1.5% b.w. and after 5 days, the mortality ceased. A new animal cohort from the same groups was examined after 12 weeks, showing neither gut alterations nor isolation of C. perfringens. The imbalance of intestinal microbiota, presumably caused by underfeeding, favoured C. perfringens overgrowth and severe gas formation. The diet increase possibly restored the normal microbiota.

Histological assessment of systemic granulomatosis progression in meagre Argyrosomus regius during cage ongrowing phase.

Meagre Argyrosomus regius is a potential candidate for Mediterranean aquaculture diversification, although several health issues still represent important bottlenecks for its sustainable production, including systemic granulomatosis (SG). To evaluate the SG progression in meagre during a 10 mo period of cage ongrowing, a histopathological investigation was carried out on 108 meagre fed 3 different diets (commercial pellets, hydrated commercial pellets and defrosted sardines). Histological sections of the gills and visceral organs were examined and lesions referable to SG scored from 1-3 according to the severity of the granulomatosis. The kidney and liver were the most affected organs, showing the highest percentage of positivity for granulomas and severity of lesions along the whole observation period. Using a statistical mixed model (GLMM) followed by odds ratio analysis, an effect of diet and temperature was found: the severity of liver and digestive tract SG scores decreased in the Cage 3 group (defrosted sardines) and with increasing temperature (p < 0.05, negative estimates, odds ratio <1). These observations, in accordance with the literature, suggest that SG in meagre could be related to nutritional-metabolic factors with the possible influence of environmental factors such as temperature.

Haplosporidium pinnae associated with mass mortality in endangered Pinna nobilis (Linnaeus 1758) fan mussels.

The fan mussel, Pinna nobilis (Linnaeus 1758), is an endemic bivalve of the Mediterranean basin, protected by international legislation as an endangered species. In the early summer of 2018, a mass mortality event (MME) of P. nobilis was recorded in the Gulf of Taranto (Southern Italy, Ionian Sea). Moribund specimens of P. nobilis were collected by scuba divers and processed by bacteriological, parasitological, histopathological and molecular analyses to investigate the causes of this MME. Different developmental stages (i.e., plasmodia, spores and sporocysts) of a presumptive haplosporidian parasite were observed during the histological analysis in the epithelium and in the lumen of the digestive tubules, where mature spores occurred either free or in sporocysts. The spores presented an operculum and an ovoid shape measuring 4.4 µm (±0.232) in length and 3.6 µm (±0.233) in width. BLAST analysis of an 18SrRNA sequence revealed a high nucleotide similarity (99%) with the reference sequence of Haplosporidium pinnae available in GenBank database. Phylogenetic analysis clustered the sequence of the pathogen in a paraphyletic clade with the reference sequence of H. pinnae, excluding other haplosporidians (i.e., Bonamia and Minchinia genera). Based on data reported, H. pinnae was the causative agent of MME in the populations of P. nobilis sampled in the Ionian Sea, where the conservation of this endangered species is heavily threatened by such a protozoan infection. Further investigations should contribute to knowledge about the life cycle of H. pinnae in order to reduce spread of the pathogen and to mitigate the burden of the disease where P. nobilis is facing the risk of extinction.

Assessing the health status of farmed mussels (Mytilus galloprovincialis) through histological, microbiological and biomarker analyses.

The Gulf of La Spezia (northern Tyrrhenian Sea, Italy) is a commercially important area both as a shipping port and for mussel farming. Recently, there has been increased concern over environmental disturbances caused by anthropogenic activities such as ship traffic and dredging and the effects they have on the health of farmed mussels. This paper reports the results of microbiological and histological analyses, as well as of measurement of several biomarkers which were performed to assess the health status of mussels (Mytilus galloprovincialis) from four rearing sites in the Gulf of La Spezia. Mussels were collected between October 2015 and September 2016 and histological analyses (including gonadal maturation stage), as well as the presence of pathogenic bacteria (Vibrio splendidus clade, V. aestuarianus and V. harveyi), viruses (Herpes virus and ostreid Herpes virus 1) and protozoa (Marteilia spp., in the summer season only) were carried out on a monthly basis. Conversely, biomarker responses in haemocyte/haemolymph (total haemocyte count, haemocyte diameter and volume, lysozyme and lactate dehydrogenase activities in cell-free haemolymph, and micronuclei frequency) and in gills and digestive gland (cortisol-like steroids and lipid peroxidation levels), were evaluated bimonthly. Microbiological data indicated that mussels contain a reservoir of potentially pathogenic bacteria, viruses and protozoa that in certain environmental conditions may cause a weakening of the immune system of animals leading to mortality episodes. The percentage of parasites detected in the mussels was generally low (9.6% for Steinhausia mytilovum, that is 17 samples out of 177 examined females; 3.4% for Proctoeces maculatus; 0.9% for Mytilicola intestinalis and 2% for ciliated protozoa), while symbiont loads were higher (31% for Eugymnanthea inquilina and Urastoma cyprinae). Interestingly, a previously undescribed haplosporidian was detected in a single mussel sample (0.2%) and was confirmed by in situ hybridization. Cells morphologically similar to Perkinsus sp. trophozoites were observed in 0.7% of the mussels analysed; however, infection with Perkinsus spp. could neither be confirmed by ISH nor by PCR. Different pathological aspects, such as host defence responses and regressive/progressive changes were detected in the gills, digestive glands, gonads and mantle. Only one single case of disseminated neoplasia (0.2%) was observed. As for the biomarker evaluation, the MANOVA analysis revealed the statistically significant effect that the variable "sampling site" had on the biological parameter measured, thus suggesting that the multibiomarker approach was able to differentiate the rearing sites.

Induction of brown cells in Venerupis philippinarum exposed to benzo(a)pyrene.

Benzo(a)pyrene is an important polycyclic aromatic hydrocarbon (PAH) commonly present in the marine environment and responsible for carcinogenic, teratogenic and mutagenic effects in various animal species. In the present study, we investigated by both histochemical and immunohistochemical approaches the effect of an acute exposure to different concentrations of B(a)P in the Manila clam Venerupis philippinarum. The general morphology of the different clam tissues, which was investigated histologically, evidenced a significant increase in the number of intestinal brown cells after B(a)P exposure. An increasing trend response to B(a)P was detected. The histochemical analysis for lipofuscin revealed the presence of lipofuscin-like substances inside the cytoplasm of intestinal brown cells. The same cells exhibited a PAS positivity and a reactivity to Schmorl's solution for melanin pigment. Moreover, intestinal brown cells exhibited an immunopositivity to HSP70 antibody confirming the increasing trend response to B(a)P detected by the histochemical analysis. Our results suggest that histological tissue changes resulting from exposure to B(a)P can be an useful marker in biomonitoring studies.

Histopathology and stress biomarkers in the clam Venerupis philippinarum from the Venice Lagoon (Italy).

The aim of this study was to evaluate the histomorphology and the stress response in the bivalve Venerupis philippinarum sampled in four differently polluted sites of the Venice Lagoon (Palude del Monte, Marghera, Ca' Roman and Val di Brenta). This species is often used as bioindicator of environmental pollution since it can bioaccumulate a large variety of pollutants because of its filter feeding. Chemical analyses for heavy metals (Cd, Cu, Hg and Pb) and polycyclic aromatic hydrocarbons (PAHs) were performed on whole soft tissues of V. philippinarum. The histological evaluation of clams revealed the presence of Perkinsus sp. infection in animals from all sites, although a very high prevalence of parasites was evidenced in clams from Ca' Roman. Perkinsus sp. were systemically distributed in the mantle, in the intestine and digestive gland, in gonads and gills. The trophozoites of Perkinsus sp. were found isolated or in cluster surrounded by a heavy hemocitical response. Haemocytes always exhibited an immunopositivity to cytochrome P4501A (CYP1A), heat shock protein 70 (HSP70), 4-hydroxy-2-nonenal (HNE) and nitrotyrosine (NT) antibodies. The digestive gland of animals from Palude del Monte showed the highest malondialdehyde (MDA) concentration, whereas clams from Ca' Roman exhibited the highest quantity of metallothioneins.

Disseminated mycobacteriosis in a stranded loggerhead sea turtle (Caretta caretta).

A loggerhead sea turtle (Caretta caretta) was found stranded alive along the Adriatic coast close to Ancona, Italy, displaying obtundation, tachypnea, and increased respiratory effort. It died a few hours after admission, and a postmortem examination was immediately performed. Miliary yellowish nodules were evident in the liver, and a lower number in the heart, stomach, and gut wall. Hundreds of whitish nodules were scattered in the lungs, with the majority of the pulmonary parenchyma being replaced by the lesions. Histologically, all nodular lesions consisted of a small central area of necrosis with acid-fast bacilli surrounded by epithelioid cells, macrophages, and lymphocytes. Giant cells were found in the spleen and the liver. Kidneys, lungs, liver, spleen, brain, and skin lesions were inoculated aseptically onto general isolation media and selective isolation media for mycobacteria. The isolate showed a restriction pattern identical to Mycobacterium chelonae by polymerase chain reaction-restriction fragment length polymorphism. To the best of the authors' knowledge, this is the first description of a disseminated infection caused by a potentially pathogenic mycobacteria in a stranded, free-ranging loggerhead sea turtle. Veterinary staff and biologists who handle sea turtles with suspected mycobacterial disease should protect themselves appropriately.

Illegal fishing with electrofishing devices in the Po river basin, Emilia Romagna, Italy.

Electric fishing is an illegal hunting method, unfortunately widely used by poachers to paralyze fish and to catch many animals in a short time. In Italy, it is authorized only for scientific and conservative purposes. Between 2014 and 2018, the Ferrara section of the Experimental Zooprophylactic Institute of Lombardy and Emilia Romagna, Italy, received nine cases of potentially illegal electric fishing in Po river and its tributary rivers. Necropsies were performed following standard protocols and samples of different tissues were collected and examined using histochemical and immunohistochemical techniques. Gross lesions frequently observed were circulatory alteration phenomena (i.e. multi-organ hyperemia, hemorrhages and congestion, hemopericardium), also found histologically, in addition to multifocal degenerative and necrotic muscular processes that could be attributed to injuries from electric current, as already reported in literature. Immunohistochemical investigations confirmed degenerative and necrotic lesions with myoglobin depletion and a corresponding fibrinogen accumulation. Myoglobin globules were also detected in the renal parenchyma, as consequent of rhabdomyolysis. The results of this study allowed to correlate electric fishing to gross, histologic and immunohistochemical lesions, which together constitute a pathognomonic picture to be considered a reference standard in this type of illegal controversy.

The life cycle of Myxobolus lentisuturalis (Myxozoa: Myxobolidae), from goldfish (Carassius auratus auratus), involves a Raabeia-type actinospore.

We studied a natural infection of the oligochaete Branchiura sowerbyi Beddard, 1892 with the Raabeia-type actinosporean stage of Myxobolus lentisuturalis Dyková, Fiala et Nie, 2002 which infected goldfish Carassius auratus auratus (L.) in Italy, using molecular analysis of the SSU rRNA gene. The existence of intraoligochaete development shows that this parasite follows the life-cycle pattern described by Wolf and Markiw (1984) for Myxobolus cerebralis. Histological examinations of the goldfish infected by M. lentisuturalis showed at low magnification the presence of two bilateral crescent-shaped masses in the dorsal epaxial muscle. These lesions were not circumscribed, presented irregular edges and infiltrated the underlying bundles of skeletal muscle and interstitial tissue. At higher magnification, disappearance of muscle fibres and substitution of the muscle tissue with Myxobolus spores and plasmodia were observed.

Morphological and Molecular Characterization of Larval and Adult Stages of Eustrongylides excisus (Nematoda: Dioctophymatoidea) with Histopathological Observations.

The genus Eustrongylides includes nematodes known as the etiological agent of the "big red worm disease." The aim of this work was to identify Eustrongylides spp. larvae from fish and adults from great cormorant (Phalacrocorax carbo) sampled at Lake Trasimeno, Italy, by morphological and molecular analysis. Histopathological description of the lesions in birds was also provided. We described adults of Eustrongylides excisus for the first time in Italy, and we also linked larval stages 3 and 4 to adults. The use of molecular tools combined with the traditional taxonomy will help the identification of the species, including species inquirendae. Moreover, molecular analysis can also help to investigate the role of intermediate and paratenic hosts, to deepen the knowledge about geographical distribution of the different Eustrongylides spp. and to define the zoonotic potential of E. excisus, which has not yet been identified as causal agent of human cases.

Ocular glioneuroma with medulloepitheliomatous differentiation in a goldfish (Carassius auratus).

An intraocular mass in the left eye causing chronic severe exophthalmia in an adult female goldfish (Carassius auratus) is described. The fish shared an aquarium with another goldfish found dead with gross and microscopic lesions consistent with mycobacteriosis. Histological examination of the left eye, histochemical (periodic acid-Schiff [PAS], Alcian blue, Ziehl-Neelsen) and immunohistochemical tests (glial fibrillary acidic protein, human neuronal protein, vimentin, and cytokeratin AE1/AE3) were carried out on the intraocular mass. Neoplastic cells forming an unencapsulated highly cellular proliferation partially covered by an intact corneal epithelium were stained with Alcian blue, which demonstrated an abundant hyaluronic acid-rich extracellular matrix. Multifocally, there were cyst-like dilatations bordered by neuroepithelial cells, which were PAS-positive. The complex neoplastic proliferation was composed of glial-like cells, neuronal-like cells (immunoreactive to glial fibrillary acidic protein and human neuronal protein, respectively) and neuroepithelium, which suggested a retinal origin.