The involvement of Pseudoterranova decipiens fish infestation on the shelf-life of fresh Atlantic cod (Gadus morhua) fillet.

Zoonotic nematodes of the family Anisakidae are highly common in many marine fish species, which act as paratenic hosts for the third larval stage. In the fish, these parasites may migrate from the fish's gastro-intestinal tract (GI-tract) further to the coelomic cavity and muscles, making them a possible contamination source of bacteria they carry on their cuticle and in their GI-tract. A previous study revealed no apparent effect of Anisakis simplex on spoilage of fish, but the equally common anisakid species Pseudoterranova decipiens has a larger body surface potentially increasing the bacterial load brought into the fish muscle upon migration. As the presence of shelf-life reducing spoilage bacteria in the microbiome of this anisakid species has been demonstrated, the objective of the present study was to assess the potential shelf-life reducing effect of P. decipiens in fresh fish fillets stored in a domestic refrigerator. Atlantic cod was used as a model since members of the cod family are the third most consumed marine fish globally and it has the highest prevalence of P. decipiens infections. Infected and non-infected codfish fillet portions were collected and microbiologically analyzed at day 0 and day 4 of storage in a domestic fridge. Three isolation media were used to enhance maximum bacterial recovery and isolates were identified using MALDI-TOF MS and 16S rRNA gene sequencing. In parallel to the microbiological examination, sensory analysis was performed daily on the cod fillets to evaluate the freshness of the fish. Results revealed the presence of typical spoilage bacteria (e.g., Pseudomonas sp., Photobacterium sp.) in all fish, but based on the total viable counts, total H2S-producing bacteria, and sensory analysis, there were no objective indications to assume an increased fish spoilage rate by the presence and migration P. decipiens. Additionally, a beta-diversity comparison revealed no significant differences in microbiota composition between infected and non-infected fish parts, though individual heterogeneity in microbiome composition among Atlantic codfish individuals was found. As total viable counts did, however, exceed the guideline limits for fresh fish, further research should now focus on the role of the candling step as a potential source of post-harvest contamination. As such, anisakid infection might still accelerate fish spoilage, though now in an indirect way.

Systematic review and critical reflection on the isolation and identification methods for spoilage associated bacteria in fresh marine fish.

Consumers demand more fresh, safe, and high-quality food. As this is partiallycorrelated to the microbial profile, several microbiological examination tools are available. Incontrast to meat, no microbiological normalized methods to assess the microbiological quality of fresh marine fish have been agreed on. As a result, studies on the detection and diversity of spoilage associated organisms (SAOs) in fish often apply various detection, isolation, and identification techniques. This complicates the comparison and interpretation of data reported, and often results in different or inconclusive results. Therefore, the present review aimed to present a critical overview of the isolation/cultivation and detection techniques currently applied in fish microbiology. After a comprehensive search in the PubMed, Web of Science and Scopus databases, a total of 111 studies fulfilled the review selection criteria. Results revealed that when relying on culture media for the isolation of SAOs in fish, it is essential to include a salt-containing medium next to plate count agar that is currently used as the reference medium for the enumeration of bacteria on fish. In terms of identification, MALDI-TOF MS and 16S rRNA gene sequencing are currently the most promising tools, though other housekeeping genes should be targeted as well, and, the biggest challenge at this point is still the lack of comprehensive proteomic and sequence databases for SAOs. A full replacement of cultivation by next generation sequencing is difficult to recommend due to the absence of a standardized experimental methodology, especially for fish, and the relatively high sequencing costs. Additionally, a discrepancy between culture-dependent and independent methods in revealing the bacterial diversity, and abundancy, from marine fish was demonstrated by several authors. It is therefore recommended to consider both approaches as complements of one another, rather than substitutes, and to include them simultaneously to yield more complete results regarding the SAOs in fresh marine fish. As such, a thorough understanding of the biology of spoilage organisms and process will be obtained to prolong the shelf-life and deliver a high-quality product.

Preliminary evaluation of different methods to detect and quantify Taenia eggs in sludge and water samples: A spiking experiment to assess recovery efficiency.

An improved understanding of the environmental transmission of Taenia spp. is key to control of the parasite. Methods to detect and quantify Taenia eggs in different environmental matrices, including sludge and water, currently lack performance validation with regard to the recovery efficiency and process ease of use. Therefore, this study aimed to assess the recovery efficiency and process duration of commonly used methods for the detection of Taenia eggs in sludge and water samples. Ten detection methods for Taenia spp. eggs were selected from a systematic review. Sludge and water samples were spiked with a high dose of Taenia saginata eggs, i.e., around 200 eggs/g sludge and 50 eggs/ml water, and were tested using five methods each. The two methods with the highest egg recovery efficiencies were selected per matrix for assessment with a lower spiking dose, i.e., 4 eggs/g sludge and 1 egg/ml water. Each time five replicates were used. Recovery efficiency was defined as the proportion of the number of eggs recovered to the total number of eggs spiked. Using the high spiking dose, all samples tested positive for all the methods. The mean egg recovery efficiency varied from 4% to 69% for sludge samples and from 3% to 68% for water samples. Using the lower spiking dose, one of the methods performed on sludge samples was able to detect all replicates, whereas only one replicate was positive using the other method. For water, all low dose samples tested positive using both methods. In conclusion, most methods performed inadequately in recovering Taenia eggs from sludge and water, with half of the methods performed on the high dose samples having a mean egg recovery efficiency of approximately 10% or less. The assessed recovery methods were generally time-consuming and labourious. A more thorough validation of existing recovery methods and improvement of method protocols to increase recovery efficiency is thus urgently needed.

Targeted proteomics and specific immunoassays reveal the presence of shared allergens between the zoonotic nematodes Anisakis simplex and Pseudoterranova decipiens.

The family Anisakidae, mainly represented by Anisakis simplex s.l. and Pseudoterranova decipiens, encompasses zoonotic nematodes infecting many marine fish. Both are responsible for gastrointestinal disease in humans after ingestion of a live larva by consumption of undercooked fish, and, in the case of A. simplex, an allergic reaction may occur after consuming or even handling infected fish. Due to its phylogenetic relatedness with A. simplex, few studies investigated the allergenic potential of P. decipiens, yet none of them focused on its excretory/secretory (E/S) proteins that easily get missed when working solely on extracts from crushed nematodes. Moreover, these E/S allergens remain behind even when the larva has been removed during fish quality processing. Therefore, the aim was to investigate if Anisakis-like allergens could also be detected in both crushed and E/S P. decipiens protein extract using targeted mass spectrometry analysis and immunological methods. The results confirmed that at least five A. simplex allergens have homologous proteins in P. decipiens; a result that emphasizes the importance of also including E/S protein extracts in proteomic studies. Not only A. simplex, but also P. decipiens should therefore be considered a potential source of allergens that could lead to hypersensitivity reactions in humans.

Unraveling the microbiota of the fish parasite Pseudoterranova decipiens in codfish (Gadus morhua) reveals a fish-related bacterial community.

Anisakidae, mainly represented by the species Anisakis simplex and Pseudoterranova decipiens, are one of the most commonly zoonotic nematodes present in marine fish species. Apart from public health risks directly linked to the parasite itself, little is known on the effects of the migrating nematodes on the hygienic quality of the fish fillet due to bacteria it carries. In the present study, the cultivated bacterial community on and in individual P. decipiens larvae deriving from codfish is reported. Four isolation media were included and evaluated to increase the bacterial diversity isolated, and identification of the bacterial growth was performed by a combination of Matrix-Assisted Laser Desorption Ionization Time-Of-Flight mass spectrometry and 16S rRNA gene sequencing. Results revealed that the microbiota of P. decipiens larvae comprises both potential spoilage bacteria and human opportunistic pathogens, and that a combined isolation on the general isolation medium tryptone soy agar and a medium supplemented with artificial seawater resulted in the highest bacterial recovery in terms of diversity and enumeration. Dissimilarity analysis also revealed similar, though unique, bacterial communities between nematodes originating from the same fish suggesting that anisakid microbiota compositions are reflections of the microbial assemblages in the fish host as an individual, and that the gut microbiome is diverse within gadoid fish species originating from the same geographical habitat. Future research should, based on the results in the present study, further elaborate on the comparison of the bacterial communities of both the larva and the codfish from which it was isolated, and, explore the extrapolation potential towards other fish and nematode species. Also, the actual degree of risk beyond the simple presence of the parasite due to carriage of opportunistic bacteria should be examined, as well as the nematode's true effect on spoilage.

Diagnostic tools for the detection of taeniid eggs in different environmental matrices: A systematic review.

The cestode family Taeniidae consists of the genera Echinococcus and Taenia, both of which include zoonotic tapeworms of serious public health importance. Various environmental matrices have been identified from which parasite transmission to animals and humans can occur, and many techniques for detecting taeniid eggs in different environments have been developed. However, the majority lack appropriate validation, and standardized egg isolation procedures are absent. This hampers interstudy comparisons and poses a challenge for future researchers when deciding which technique to implement for assessing taeniid egg contamination in a particular matrix. Therefore, the aim of this systematic review was to present an overview of the detection methods for taeniid eggs in the environment, to discuss and compare them, and to provide recommendations for future studies. In total, 1814 publications were retrieved from scientific databases, and, ultimately, data were systematically reviewed from 90 papers. The results provide an overview of numerous diagnostic tests for taeniid egg detection in (or on) water, food, soil, insects, objects, and air. These tools could be categorized as either conventional (light microscopy), molecular, or immunodetection tools. The relatively cheap microscopy techniques often lack sensitivity and are unable to identify a taeniid egg at the genus level. Nevertheless, several records ascribed a genus, or even species, to taeniid eggs that had been detected by light microscopy. Molecular and immunodetection tools offer better specificity, but still rely on the preceding egg recovery steps that also affect overall sensitivity. Finally, the majority of the methods lacked any attempt at performance evaluation and standardization, especially at the earlier stages of the analysis (e.g., sampling strategy, storage conditions, egg recovery), and viability was rarely addressed. As such, our review highlights the need for standardized, validated detection tools, that not only assess the extent of environmental contamination, but also the egg genus or species, and address viability.

Foodborne Parasites and Their Complex Life Cycles Challenging Food Safety in Different Food Chains.

Zoonotic foodborne parasites often represent complex, multi host life cycles with parasite stages in the hosts, but also in the environment. This manuscript aims to provide an overview of important zoonotic foodborne parasites, with a focus on the different food chains in which parasite stages may occur. We have chosen some examples of meat-borne parasites occurring in livestock (Taenia spp., Trichinella spp. and Toxoplasma gondii), as well as Fasciola spp., an example of a zoonotic parasite of livestock, but transmitted to humans via contaminated vegetables or water, covering the 'farm to fork' food chain; and meat-borne parasites occurring in wildlife (Trichinella spp., Toxoplasma gondii), covering the 'forest to fork' food chain. Moreover, fish-borne parasites (Clonorchis spp., Opisthorchis spp. and Anisakidae) covering the 'pond/ocean/freshwater to fork' food chain are reviewed. The increased popularity of consumption of raw and ready-to-eat meat, fish and vegetables may pose a risk for consumers, since most post-harvest processing measures do not always guarantee the complete removal of parasite stages or their effective inactivation. We also highlight the impact of increasing contact between wildlife, livestock and humans on food safety. Risk based approaches, and diagnostics and control/prevention tackled from an integrated, multipathogen and multidisciplinary point of view should be considered as well.

Currently Available Monitoring and Surveillance Systems for Taenia spp., Echinococcus spp., Schistosoma spp., and Soil-Transmitted Helminths at the Control/Elimination Stage: A Systematic Review.

An increasing global focus on neglected tropical diseases (NTDs) has resulted in the set up of numerous control and elimination activities worldwide. This is partly true for Taenia solium taeniasis/cysticercosis, the most important foodborne parasitic infection. Despite substantial progress, adequate monitoring and surveillance (M&S) are required to sustain a status of control/elimination. This is often lacking, especially for T. solium. Therefore, the objective was to conduct a systematic literature review of the currently available M&S systems at the control/elimination stage of the four top-ranked helminth NTDs. Specifically, Taenia spp., Echinococcus spp., Schistosoma spp., and soil-transmitted helminths (STHs) were considered to determine if there are any similarities between their M&S systems and whether certain approaches can be adopted from each other. The systematic review demonstrated that rigorous M&S systems have been designed for the control/elimination stage of both STHs and schistosomiasis, particularly in China. On the other hand, a concept of M&S for Taenia spp. and Echinococcus spp. has not been fully developed yet, due to a lack of epidemiological data and the fact that many endemic countries are far away from reaching control/elimination. Moreover, accurate diagnostic tools for all four diseases are still imperfect, which complicates proper M&S. Finally, there is an urgent need to develop and harmonize/standardize M&S activities in order to reliably determine and compare the epidemiological situation worldwide.