First report of recurrent parthenogenesis as an adaptive reproductive strategy in the endangered common smooth-hound shark Mustelus mustelus.

Parthenogenesis, or virgin birth, describes a mode of reproduction where an egg develops into an offspring without fertilization, and is observed across various vertebrate taxa, excluding mammals. Obligate parthenogenesis, found in around 100 vertebrate species and 1000 invertebrate species, is relatively rare. Conversely, facultative parthenogenesis, where females can reproduce both sexually and parthenogenetically, is observed in some vertebrates, including elasmobranchs. Notably, this phenomenon in elasmobranchs is mainly documented in captivity, allowing for detailed long-term observation. Specifically, this study reports the first case of facultative parthenogenesis in the common smooth-hound shark Mustelus mustelus, a species classified by IUCN as endangered. Here we show that the juvenile M. mustelus were born through parthenogenesis, exhibiting homozygosity at each genetic marker, consistent with terminal fusion automixis. Remarkably, this finding reveals that parthenogenesis can occur annually in these sharks, alternating between two females, and conclusively excludes long-term sperm storage as a cause. Consequently, this enhances our understanding of parthenogenesis in elasmobranchs and highlights the reproductive flexibility of M. mustelus. Overall, these results contribute to our broader understanding of reproductive strategies in elasmobranchs, which could inform conservation efforts for endangered species.

A Case of Food-Borne Salmonellosis in a Corn Snake (Pantherophis guttatus) after a Feeder Mouse Meal.

Reptiles are usually asymptomatic carriers of Salmonella, with the manifestation of typical clinical signs of acute forms in adult and non-immunocompromised animals being considered exceptions. In the present case, an adult male corn snake (Pantherophis guttatus) was found dead due to septic shock 48 h after consuming a feeder mouse purchased online. The snake's tissue samples and faeces were cultured for bacteria isolation. Microbiological examinations of the snake and mouse livers revealed the presence of Salmonella enterica subsp. enterica serovar Midway. A whole-genome analysis of these two isolates showed a high correlation between them: they belonged to the strain type ST-357 for the classic MLST scheme and to the strain type ST 171322 for the cgMLST scheme. Also, a virulence gene analysis revealed the presence of stdB and STM3026 genes. This report conveys a case of food-borne salmonellosis in a pet snake, transmitted from a feeder mouse, likely responsible for the snake's death due to septic shock. It highlights the relevance of feeder mice as a source of Salmonella infections in snakes and the associated risks to human health.

First Report of Human Urinary Tract Infection Caused by Lactococcus petauri.

Lactococcus petauri is a recently described species of the genus Lactococcus. It was reported as an etiological agent of piscine lactococcosis together with Lactococcus garvieae. L. garvieae was already described as an opportunistic pathogen in human infections, with a potential zoonotic role. This paper represents the first report of a human urinary tract infection caused by L. petauri. A 91-year-old man was admitted to the emergency department for a femur fracture consequent to a domestic accident. The fracture was reduced by surgery and a catheterized specimen urine culture revealed a high bacterial load sustained by Gram-positive cocci, identified by Vitek 2 compact as L. garvieae, and subsequently as L. petauri through Internal Transcribed spacer 16S-23S r-RNA amplification. The number of L. petauri infections in humans is expected to rise in the near future mainly due to diagnostic improvement. A dedicated survey on L. garvieae and L. petauri infections in humans should be performed to better understand their role as pathogens and as zoonotic agents.

Disclosing Frauds in Herbal Food Supplements Labeling: A Simple LC-MS/MS Approach to Detect Alkaloids and Biogenic Amines.

Food supplements are a category of products perceived safe and therefore commonly used by different categories of consumers without any particular attention or precaution. However, health risks associated with the consumption of supplements containing undeclared substances cannot be excluded. A variety of analytical methods are used to control supplement quality composition, but usually, these procedures are complex and time-consuming. Here, we report the results of a simple and fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, to detect and quantify simultaneously different categories of active molecules, such as biogenic ammines and natural alkaloids that at high doses can produce negative health effect in consumers. Three categories of products intended for body weight loss, energy boosting, and erectile dysfunction treatment, purchased through e-commerce sites and from local supermarkets, were analyzed (n = 91). The caffeine, synephrine, agmatine sulfate, yohimbine, phenethylamine, and icariin were correctly separated and identified with good precision (RSD < 20%) and recovery (89-109%). The identification and quantification of the analytes in real samples highlighted that the 26% of the samples were not compliant with labeling, confirming that frauds are very common also in the natural supplements market. This LC-MS/MS method could be easily used to test natural supplements in order to check the correct labeling and to protect consumers from potential health risks and food frauds.

16S-23S rRNA Internal Transcribed Spacer Region (ITS) Sequencing: A Potential Molecular Diagnostic Tool for Differentiating Lactococcus garvieae and Lactococcus petauri.

Lactococcus garvieae is the etiological agent of lactococcosis, a clinically and economically significant infectious disease affecting farmed rainbow trout. L. garvieae had been considered the only cause of lactococcosis for a long time; however, L. petauri, another species of the genus Lactococcus, has lately been linked to the same disease. The genomes and biochemical profiles of L. petauri and L. garvieae have a high degree of similarity. Traditional diagnostic tests currently available cannot distinguish between these two species. The aim of this study was to use the transcribed spacer (ITS) region between 16S rRNA and 23S rRNA as a potential useful molecular target to differentiate L. garvieae from L. petauri, saving time and money compared to genomics methods currently used as diagnostic tools for accurate discrimination between these two species. The ITS region of 82 strains was amplified and sequenced. The amplified fragments varied in size from 500 to 550 bp. Based on the sequence, seven SNPs were identified that separate L. garvieae from L. petauri. The 16S-23S rRNA ITS region has enough resolution to distinguish between closely related L. garvieae and L. petauri and it can be used as a diagnostic marker to quickly identify the pathogens in a lactococcosis outbreak.

What Can Genetics Do for the Control of Infectious Diseases in Aquaculture?

Infectious diseases place an economic burden on aquaculture and a limitation to its growth. An innovative approach to mitigate their impact on production is breeding for disease resistance: selection for domestication, family-based selection, marker-assisted selection, and more recently, genomic selection. Advances in genetics and genomics approaches to the control of infectious diseases are key to increasing aquaculture efficiency, profitability, and sustainability and to reducing its environmental footprint. Interaction and co-evolution between a host and pathogen can, however, turn breeding to boost infectious disease resistance into a potential driver of pathogenic change. Parallel molecular characterization of the pathogen and its virulence and antimicrobial resistance genes is therefore essential to understand pathogen evolution over time in response to host immunity, and to apply appropriate mitigation strategies.

Discrimination between Wild and Farmed Sea Bass by Using New Spectrometry and Spectroscopy Methods.

European sea bass (Dicentrarchus labrax L.) is one of the most economically important fish species in the Mediterranean Sea area. Despite strict requirements regarding indications of production method (wild/farmed), incorrect labelling of sea bass is a practice still frequently detected. The aim of this study was to evaluate the capabilities of two techniques, Near-InfraRed (NIR) spectroscopy and mass spectrometry, to discriminate sea bass according to the production method. Two categories were discriminated based on the docosahexaenoic and arachidonic fatty acid ratio by using a Direct Sample Analysis (DSA) system integrated with a time-of-flight (TOF) mass spectrometer. The cut-off value of 3.42, of fatty acid ratio, was able to discriminate between the two types of fish with sensitivity and specificity of 100%. It was possible to classify fish production by using multivariate analysis with portable NIR. The results achieved by the developed validation models suggest that this approach is able to distinguish the two product categories with high sensitivity (100%) and specificity (90%). The results obtained from this study highlight the potential application of two easy, fast, and accurate screening methods to detect fraud in commercial sea bass production.

Isolation and Genetic Characterization of Streptococcus iniae Virulence Factors in Adriatic Sturgeon (Acipenser naccarii).

The first case of infection of Streptococcus iniae in Adriatic sturgeon (Acipenser naccarii) was recently reported in a raceway system located in Northern Italy. A second episode of infection in sturgeons with absence of mortality and evident clinical signs, was registered in November 2020 in the same farm and is reported in this study. Histopathological changes observed in infected organs are described. The strains isolated in the two episodes were compared using molecular analysis based on PCR, phylogeny and virulence factors analysis. Not all the major virulence factors were detected for the two strains; in particular the strains 78697, isolated in November, lacks cpsD, compared to the strains 64844, isolated in September. Moreover, genetic variations were reported for lctO and pmg genes. These findings let us hypothesize a different virulence of the strains in accordance with clinical findings related to the sturgeons.

Development of a screening method to rapidly discriminate extravirgin olive oil from other edible vegetable oil by means of direct sample analysis with high resolution mass spectrometry.

Extra virgin olive oil is the highest quality olive oil mainly due to its beneficial constituents and nutritional properties. However, olive oil adulteration is a common fraudulent practice by deliberate mislabelling of less expensive oil categories and admixing expensive olive oils with low oils. To protect consumers from such commercial frauds, an easy and fast method to detect the real composition of oil is needed. For this study we used direct sampling analysis (DSA) coupled with a high-resolution mass spectrometer (AxION2 TOF Perkin Elmer) to analyse the fatty acid composition of three types of edible oil: extra virgin olive oil, refined olive oil and seed oil (EVOO, ROO, and SO respectively) to find a marker that could distinguish between them. Good precision in repeatability and reproducibility (RSD% < 15%) was obtained. The fatty acid ratio between the oleic acid/oleic acid dimer was able to distinguish EVOO from the other two types of oil, while the ratio between linoleic and oleic acid was found to discriminate refined oil from seed oil. The development of an easy, fast and cost-effective method can help to limit commercial frauds, increase the number of controlled samples, and enhance food control along the commercial chain. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13197-021-05063-y.

Wild or Farmed Gilthead Seabream (Sparus aurata)? How To Distinguish between Them by Two-Dimensional Gel Electrophoresis.

ABSTRACT: Because the world's wild fish stocks are limited and the market demand is increasing, fish farming has become an alternative food source and a way to reduce costs for consumers. The sale of farmed as wild fish is a fraudulent practice; it is, therefore, important to find new and alternative tools that can help in the fight against fraud to protect consumers and to ensure food traceability. The proteomic profiles of farmed and wild fish differ. With this study we wanted to identify liver protein markers via two-dimensional electrophoresis that would allow us to distinguish wild from farmed gilthead seabream. The liver samples from 32 gilthead seabream, wild and farmed, were stored at -80°C before protein extraction. The samples were subjected to two-dimensional electrophoresis to detect qualitative and quantitative differences. Proteomic analysis showed a protein spot (molecular weight of ∼34 kDa and isoelectric point of ∼6.9) only in the samples from the wild gilthead seabream; liquid chromatography-tandem mass spectrometry identified the spot as ubiquitin. Ubiquitin could be a valid marker to differentiate wild from farmed gilthead seabream; it could be used to ensure continuous monitoring throughout the entire commercial chain and to fight commercial fraud.

Development of a Novel Method for Rapid Discrimination between Wild and Farmed Sea Bream.

A simple method based on direct sampling analysis, coupled with a time of flight mass spectrometer, was developed to discriminate between wild and farmed sea bream on the basis of the docosahexaenoic and arachidonic fatty acid ratio. Good precision in repeatability and reproducibility (relative standard deviation < 15%) was obtained. The fatty acid ratios of the two types of fish were statistically significant (Student's t < 0.001). The use of a simple, rapid, and cost-effective tool could aid in the detection of commercial fish fraud, increase the number of controlled samples, and strengthen control along the entire commercial chain.

Two-dimensional gel and shotgun proteomics approaches to distinguish fresh and frozen-thawed curled octopus (Eledone cirrhosa).

The substitution and sale of frozen-thawed fish labeled as fresh is a widespread, difficult to unmask commercial fraud and a potential risk for consumer health. Proteomics could help to identify markers for the rapid screening of food samples and the identification of frozen-thawed seafood. Using two-dimensional electrophoresis (2-DE) and high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS), we identified biomarkers that are able to discriminate between fresh and frozen-thawed tissue samples of curled octopus (Eledone cirrhosa). The 2-DE analysis showed a significant reduction in two protein spots (molecular weight of 45-50 kDa, isoelectric point of 6.5-7) identified as transgelin. At shotgun analysis, nine proteins resulted modulated and transgelin was confirmed as down-regulated, making it a potentially useful marker for differentiating between fresh and frozen-thawed fish product samples. BIOLOGICAL SIGNIFICANCE: This work, based on two different proteomics approaches, investigated differentially expressed proteins in the tentacles of the curled octopus (E. cirrhosa) after freezing-thawing processes. We were able to characterize the proteome of the tentacles, increasing our knowledge on this species, and a common down-regulated protein was identified by 2-DE and shotgun analysis, a calponin-like protein called transgelin, suggesting a potential use as a marker to distinguish different states of conservation in this species.

Rapid Screening Technique To Identify Sudan Dyes (I to IV) in Adulterated Tomato Sauce, Chilli Powder, and Palm Oil by Innovative High-Resolution Mass Spectrometry.

Sudan dyes are synthetic azo dyes used by industry in a variety of applications. Classified as carcinogenic, they are not allowed in foodstuffs; however, their presence as adulterants in food products has been regularly reported. Here, we describe an innovative screening method to detect Sudan I, II, III, and IV in tomato sauce, palm oil, and chilli powder. The method entails minimal sample preparation, completely avoiding the liquid chromatography phase, followed by detection and identification through atmospheric pressure chemical ionization time-of-flight mass spectrometry, in positive ionization mode. Analytes were efficiently identified and detected in samples, fortified both with individual analytes and with their mixture, with an error in mass identification less than 5 ppm. Limits of identification of the analytes in the fortified samples were 0.5 to 1 mg/kg, depending on the dye and matrix. The method had a linear range of 0.05 to 5 mg/kg and good linear relationships (R2 > 0.98). Repeatability was satisfactory, with a coefficient of variation lower than 20%. The method was applied to detect the dyes in real adulterated chilli samples, previously found positive by confirmatory high-performance liquid chromatography-mass spectrometry and ELISA, and in commercial products purchased from supermarkets. In all positive samples, analytes were correctly identified with an error in mass identification lower than 5 ppm, while none of the 45 commercial samples analyzed were found to be contaminated. The proposed new assay is sensitive, with a limit of identification, for all the three matrices, complying with the limits defined by the European Union (0.5 to 1 mg/kg) for analytical methods. Compared with conventional methods, the new assay is rapid and inexpensive and characterized by a high throughput; thus, it could be suitable as screening technique to identify Sudan dyes in adulterated food products.

Lysine at position 222 of the goat prion protein inhibits the binding of monoclonal antibody F99/97.6.1.

Prion protein (PrP) is encoded by the PRNP gene, which is highly polymorphic in goats, with polymorphisms encoding amino acid substitutions at the protein level. In the current study, the reactivity of monoclonal antibody (mAb) F99/97.6.1 in binding PrP from goats polymorphic at PRNP codon 222 was investigated. Nervous tissue from 30 scrapie-negative goats with 3 different genotypes (222Q/Q, 222Q/K, and 222K/K) was analyzed by Western blot using mAbs P4 and F99/97.6.1. Although PrP was detected in all 30 samples by mAb P4, detection of PrP by mAb F99/97.6.1 was limited to 222Q/Q (12/12). No PrP was detected by mAb F99/97.6.1 in the 222K/K samples (n = 6), and the signal intensity of mAb F99/97.6.1 for PrP was lower for the 222Q/K samples (12/12 samples). To further investigate these results, additional Western blot analyses were performed, and the PrP signals detected by mAbs F99/97.6.1 and SAF84 were then quantified. The mean F99/SAF84 ratio (± standard deviation) calculated for the 222Q/Q group was 0.73 ± 1.26, and the mean for the 222Q/K group was 0.27 ± 1.31. Statistical analysis of these values evidenced statistically significant differences between the 222Q/Q and 222Q/K samples. The results of the study thus revealed an inhibition by lysine at position 222 on the binding of mAb F99/97.6.1 to goat PrP. This has implications for the use of mAb F99/97.6.1 for diagnostic purposes. Because the 222K allele could be a target for genetic selection in goats, the differential reactivity of mAb F99/97.6.1 could be exploited with a genotyping test setup.