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.

An update and ecological perspective on certain sentinel helminth endoparasites within the Mediterranean Sea.

The Mediterranean Sea is recognized as a marine biodiversity hotspot. This enclosed basin is facing several anthropogenic-driven threats, such as seawater warming, pollution, overfishing, bycatch, intense maritime transport and invasion by alien species. The present review focuses on the diversity and ecology of specific marine trophically transmitted helminth endoparasites (TTHs) of the Mediterranean ecosystems, aiming to elucidate their potential effectiveness as 'sentinels' of anthropogenic disturbances in the marine environment. The chosen TTHs comprise cestodes and nematodes sharing complex life cycles, involving organisms from coastal and marine mid/upper-trophic levels as definitive hosts. Anthropogenic disturbances directly impacting the free-living stages of the parasites and their host population demographies can significantly alter the distribution, infection levels and intraspecific genetic variability of these TTHs. Estimating these parameters in TTHs can provide valuable information to assess the stability of marine trophic food webs. Changes in the distribution of particular TTHs species can also serve as indicators of sea temperature variations in the Mediterranean Sea, as well as the bioaccumulation of pollutants. The contribution of the chosen TTHs to monitor anthropogenic-driven changes in the Mediterranean Sea, using their measurable attributes at both spatial and temporal scales, is proposed.

Setting epidemiological cut-off values for Vibrio harveyi relevant to MIC data generated by a standardised microdilution method.

The lack of internationally harmonised criteria for interpreting the data generated by standardised susceptibility testing methods presents a serious obstacle for the development of prudent use of antimicrobials in aquaculture. The data required to set epidemiological cut-off values for minimum inhibitory concentrations for antibiotic agents against Vibrio harveyi was determined using a standard microdilution method that specified the use of cation-adjusted Mueller Hinton broth and incubation at 28°C for 24 to 28 h. In total, 120 observations were made in 4 independent laboratories from 109 unique isolates. The aggregated data from these laboratories were analysed by the normalised resistance method and by ECOFFinder to calculate epidemiological cut-off values. The data for chloramphenicol, meropenem and sulfamethoxazole were not considered as suitable for analysis. The data for ampicillin indicated that this species is innately resistant to this agent. No acceptable ranges for quality control strains have been set for ceftazidime and, therefore, only provisional cut-off values could be generated for this agent. The epidemiological cut-off values were, however, calculated for the other 6 agents. These values were ≤0.5 µg ml-1 for enrofloxacin, ≤1 µg ml-1 for florfenicol, oxolinic acid and oxytetracycline, ≤4 µg ml-1 for gentamicin and ≤0.5/9.5 µg ml-1 for trimethoprim/sulfamethoxazole. Evidence is presented demonstrating that the data for these 6 antimicrobial agents was of sufficient quantity and quality that they could be used by the relevant authorities to set internationally harmonised, consensus epidemiological cut-off values for V. harveyi.

Epidemiological cut-off values for Vibrio anguillarum MIC and disc diffusion data generated by standardised methods.

This work aims to generate the data needed to set epidemiological cut-off values for minimum inhibitory concentration (MIC) and disc-diffusion zone measurements of Vibrio anguillarum. A total of 261 unique isolates were tested, applying standard methods specifying incubation at 28°C for 24-28 h. Aggregated MIC distributions for a total of 247 isolates were determined in 9 laboratories for 11 agents. Data aggregations of the disc zone for the 10 agents analysed contained between 157 and 218 observations made by 4 to 7 laboratories. Acceptable ranges for quality control (QC) reference strains were available for 7 agents and the related multi-laboratory aggregated data were censored, excluding the data of a laboratory that failed to meet QC requirements. Statistical methods were applied to calculate epidemiological cut-off values. Cut-off values for MIC data were calculated for florfenicol (≤1 µg ml-1), gentamicin (≤4 µg ml-1), oxytetracycline (≤0.25 µg ml-1) and trimethoprim/sulfamethoxazole (≤0.125/2.38 µg ml-1). The cut-off values for disc zone data were calculated for enrofloxacin (≥29 mm), florfenicol (≥27 mm), gentamicin (≥19 mm), oxolinic acid (≥24 mm), oxytetracycline (≥24 mm) and trimethoprim/sulfamethoxazole (≥26 mm). MIC and disc-diffusion zone data for the other agents where not supported by QC, thus yielding only provisional cut-off values (meropenem, ceftazidime). Regardless of whether QC is available, some of the aggregated MIC distributions (enrofloxacin, oxolinic acid), disc zone (sulfamethoxazole), and MIC and disc-diffusion distributions (ampicillin, chloramphenicol) did not meet the statistical requirements. The data produced will be submitted to the Clinical Laboratory Standards Institute for their consideration in setting international consensus epidemiological cut-off values.

Survey on the presence of Leishmania sp. in peridomestic rodents from the Emilia-Romagna Region (North-Eastern Italy).

Leishmaniasis is a neglected vector-borne parasitic disease caused in Italy only by the species Leishmania infantum of the Leishmania donovani complex, which is the causative agent of the zoonotic visceral leishmaniasis (VL) and the sporadic cutaneous leishmaniasis (CL) in humans, and of the canine leishmaniasis (CanL). The disease is considered endemic in southern, central, and insular Italian regions and recognizes phlebotomine sand flies as vector and dogs as main reservoir. Among northern Italian region, Emilia-Romagna shows peculiar epidemiological situation and recent studies are questioning the role of dog as main reservoir of L. infantum. Due to their synanthropic relationship with humans, rodents have been tested for Leishmania spp. in several European countries. The aim of this study was to assess the presence of Leishmania spp. in peridomestic rodents in the Emilia-Romagna. The study was carried out on 136 peridomestic rodents collected by professional pest control services: 47 brown rats (Rattus norvegicus), 39 black rats (Rattus rattus) and 50 mice (Mus musculus). Specimens of earlobe skin, spleen, liver and prescapular lymph nodes were tested with a real-time PCR. Fifteen (11%) rodents, tested positive for Leishmania spp. in particular five brown rats (10.6%), five black rats (12.8%) and five mice (10%). Positivity was obtained from different target organs. These findings revealed the presence of Leishmania spp. in peridomestic rodents of Emilia-Romagna Region, also in two species never tested before in Italy, namely R. norvegicus and M. musculus.

Roe deer (Capreolus capreolus) are a novel potential reservoir for human visceral leishmaniasis in the Emilia-Romagna region of northeastern Italy.

Leishmaniasis is a complex human disease caused by intracellular parasites of the genus Leishmania, predominantly transmitted by the bite of sand flies. In Italy, leishmaniasis is caused exclusively by Leishmania infantum, responsible for the human and canine visceral leishmaniases (HVL and CVL, respectively). Within the Emilia-Romagna region, two different foci are active in the municipalities of Pianoro and Valsamoggia (both in the province of Bologna). Recent molecular studies indicated that L. infantum strains circulating in dogs and humans are different, suggesting that there is an animal reservoir other than dogs for human visceral leishmaniasis in the Emilia-Romagna region. In this work, we analyzed specimens from wild animals collected during hunts or surveillance of regional parks near active foci of human visceral leishmaniasis for L. infantum infection in the province of Bologna. Out of 70 individuals analyzed, 17 (24%) were positive for L. infantum. The infection prevalence in hedgehogs (Erinaceus europaeus), roe deer (Capreolus capreolus), badgers (Meles meles), and bank voles (Myodes glareolus) was 80, 33, 25, and 11%, respectively. To distinguish the two strains of L. infantum we have developed a nested PCR protocol optimized for animal tissues. Our results demonstrated that most (over 90%) of L. infantum infections in roe deer were due to the strain circulating in humans in the Emilia-Romagna region.

Detection of Leishmania sp. kDNA in questing Ixodes ricinus (Acari, Ixodidae) from the Emilia-Romagna Region in northeastern Italy.

To date, sand flies (Phlebotominae) are the only recognized biological vectors of Leishmania infantum, the causative agent of human visceral leishmaniasis, which is endemic in the Mediterranean basin and also widespread in Central and South America, the Middle East, and Central Asia. Dogs are the main domestic reservoir of zoonotic visceral leishmaniasis, and the role of secondary vectors such as ticks and fleas and particularly Rhipicephalus sanguineus (the brown dog tick) in transmitting L. infantum has been investigated. In the present paper, the presence of Leishmania DNA was investigated in questing Ixodes ricinus ticks collected from 4 rural areas included in three parks of the Emilia-Romagna Region (north-eastern Italy), where active foci of human visceral leishmaniasis have been identified. The analyses were performed on 236 DNA extracts from 7 females, 6 males, 72 nymph pools, and 151 larvae pools. Four samples (1.7%) (i.e., one larva pool, 2 nymph pools, and one adult male) tested positive for Leishmania kDNA. To the best of our knowledge, this is the first report of the presence of Leishmania kDNA in questing I. ricinus ticks collected from a rural environment. This finding in unfed larvae, nymphs, and adult male ticks supports the hypothesis that L. infantum can have both transstadial and transovarial passage in I. ricinus ticks. The potential role of I. ricinus ticks in the sylvatic cycle of leishmaniasis should be further investigated.

Development and diagnostic validation of a one-step multiplex RT-PCR assay as a rapid method to detect and identify Nervous Necrosis Virus (NNV) and its variants circulating in the Mediterranean.

Nervous Necrosis Virus (NNV) represents one of the most threatening pathogens for Mediterranean aquaculture. Several NNV strains are currently co-circulating in the Mediterranean Basin with a high prevalence of the RGNNV genotype and the RGNNV/SJNNV reassortant strain and a more limited diffusion of the SJNNV genotype and the SJNNV/RGNNV reassortant. In the present study, a one-step multiplex RT-PCR (mRT-PCR) assay was developed as an easy, cost-effective and rapid diagnostic technique to detect RGNNV and the reassortant RGNNV/SJNNV strain and to distinguish them from SJNNV and the reassortant SJNNV/RGNNV strain in a single RT-PCR reaction. A unique amplification profile was obtained for each genotype/reassortant enabling their rapid identification from cell culture lysates or directly from brain tissues of suspected fish. The method's detection limit varied between 102.3 and 103.4 TCID50 ml-1 depending on viral strains. No cross-reacitivty with viruses and bacteria frequently associated with gilthead seabream, European seabass and marine environment was observed. The mRT-PCR was shown to be an accurate, rapid and affordable method to support traditional diagnostic techniques in the diagnosis of VNN, being able to reduce considerably the time to identify the viral genotype or the involvement of reassortant strains.

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.

Autochthonous Trypanosoma spp. in European Mammals: A Brief Journey amongst the Neglected Trypanosomes.

The genus Trypanosoma includes flagellated protozoa belonging to the family Trypanosomatidae (Euglenozoa, Kinetoplastida) that can infect humans and several animal species. The most studied species are those causing severe human pathology, such as Chagas disease in South and Central America, and the human African trypanosomiasis (HAT), or infections highly affecting animal health, such as nagana in Africa and surra with a wider geographical distribution. The presence of these Trypanosoma species in Europe has been thus far linked only to travel/immigration history of the human patients or introduction of infected animals. On the contrary, little is known about the epidemiological status of trypanosomes endemically infecting mammals in Europe, such as Trypanosomatheileri in ruminants and Trypanosomalewisi in rodents and other sporadically reported species. This brief review provides an updated collection of scientific data on the presence of autochthonous Trypanosoma spp. in mammals on the European territory, in order to support epidemiological and diagnostic studies on Trypanosomatid parasites.

Absence of anisakis nematodes in smoked farmed Atlantic salmon (Salmo salar) products on sale in European countries.

The increase of global demand of aquaculture products as compensation for the lowering of fishery sustainability has shown a parallel awareness by the consumers on the importance of the safety and quality of fish products. Among these, salmon industry has reached a leading position demonstrating the negligible risk of presence of zoonotic helminths such as anisakis nematodes in farmed salmon. Despite the massive production of data in literature on parasitological surveys carried out on fresh salmon, no data are published on processed farmed salmon such as smoked products. In 2016, 270 slices of smoked farmed Atlantic salmon (Salmo salar) and 13 smoked slices from wild sockeye salmon (Oncorhynchus nerka) have been analyzed by visual inspection and UV-press method searching for the presence of anisakid nematodes. No parasites were detected in samples from farmed Atlantic salmon, while 10 out of 13 from wild salmon were positive for Anisakis simplex s.s. larvae. This first survey on the possible presence of anisakid nematodes in processed smoked salmon confirms that this risk in farmed Atlantic salmon products has to be considered negligible.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry identification of Vibrio (Listonella) anguillarum isolated from sea bass and sea bream.

Vibrio (Listonella) anguillarum is a pathogenic bacterium causing septicaemia in a wide range of marine organisms and inducing severe mortalities, thus it is crucial to conduct its accurate and rapid identification. The aim of this study was to assess MALDI-TOF MS as a method of choice for identification of clinical V. anguillarum isolates from affected marine fish. Since the method accuracy might be influenced by the type of the medium used, as well as by the incubation conditions, we tested V. anguillarum isolates grown on standard media with and without the addition of NaCl, cultured at three incubation temperatures, and at three incubation periods. The best scores were retrieved for V. anguillarum strains grown on NaCl-supplemented tryptone soy agar (TSA) at 22°C and incubated for 48h (100% identification to species level; overall score 2.232), followed by incubation at 37°C and 48h (100% to species level; score 2.192). The strains grown on non-supplemented TSA gave the best readings when incubated at 22°C for 72h (100% identification to species level; overall score 2.182), followed by incubation at 15°C for 72h (100% to species level; score 2.160). Unreliable identifications and no-identifications were growing with the incubation duration at 37°C, on both media, amounting to 88.89% for 7d incubation on supplemented TSA, and 92.60% for 7d incubation on non-supplemented TSA. The age of the cultured strains and use of media significantly impacted the mass spectra, demonstrating that for reliable identification, MALDI-TOF MS protein fingerprinting with the on-target extraction should be performed on strains grown on a NaCl-supplemented medium at temperatures between 15 and 22°C, incubated for 48-72 hours.