Formaldehyde contamination in seafood industry: an update on detection methods and legislations.

Seafood is abundant in high-quality protein, healthy fats (n-3 and n-6 PUFAs), minerals (calcium, magnesium, copper, selenium, and so on), and vitamin D. Functional compounds in seafood can protect against lifestyle-related diseases. Having had all the merits mentioned, it is also a highly putrefiable food item. To maintain quality and prolong seafood's shelf life, various chemicals have been added, including nitrite, sulfur dioxide, and formaldehyde. In this review, we summarize the uses, the incidence of added formalin contamination, and the approved limit for seafood products. Additionally, worldwide regulations or standards for the use of formalin in seafood products, as well as recent changes relevant to new methods, are highlighted. Although strict limits and regulations have been placed on the utilization of formaldehyde for seafood preservation, there are few incidences reported of formalin/formaldehyde detection in seafood products around Asian countries. In this context, various qualitative and quantitative detection methods for formaldehyde have been developed to ensure the presence of formaldehyde within acceptable limits. Besides this, different rules and regulations have been forced by each country to control formaldehyde incidence. Although it is not an issue of formaldehyde incidence in European countries, strict regulations are implemented and followed.

Proximity ligation-based sequencing for the identification of human papillomavirus genomic integration sites in formalin-fixed paraffin embedded oropharyngeal squamous cell carcinomas.

Human papillomavirus (HPV) infections are an increasing cause of oropharyngeal squamous cell carcinomas (OPSCC). Integration of the viral genome into the host genome is suggested to affect carcinogenesis, however, the correlation with OPSCC patient prognosis is still unclear. Research on HPV integration is hampered by current integration detection technologies and their unsuitability for formalin-fixed paraffin-embedded (FFPE) tissues. This study aims to develop and validate a novel targeted proximity-ligation based sequencing method (targeted locus amplification/capture [TLA/TLC]) for HPV integration detection in cell lines and FFPE OPSCCs. For the identification of HPV integrations, TLA/TLC was applied to 7 cell lines and 27 FFPE OPSCCs. Following preprocessing steps, a polymerase chain reaction (PCR)-based HPV enrichment was performed on the cell lines and a capture-based HPV enrichment was performed on the FFPE tissues before paired-end sequencing. TLA was able to sequence up to hundreds of kb around the target, detecting exact HPV integration loci, structural variants, and chromosomal rearrangements. In all cell lines, one or more integration sites were identified, in accordance with detection of integrated papillomavirus sequences PCR data and the literature. TLC detected integrated HPV in 15/27 FFPE OPSCCs and identified simple and complex integration patterns. In general, TLA/TLC confirmed PCR data and detected additional integration sites. In conclusion TLA/TLC reliably and robustly detects HPV integration in cell lines and FFPE OPSCCs, enabling large, population-based studies on the clinical relevance of HPV integration. Furthermore, this approach might be valuable for clonality assessment of HPV-related tumors in clinical diagnostics.

Acetaminophen effects upon formalin-evoked flinching, postformalin, and postincisional allodynia and conditioned place preference.

Introduction: We explored in mice, the analgesic, tolerance, dependency, and rewarding effects of systemic acetaminophen (APAP). Methods: Studies employed adult mice (C57Bl6). (1) Intraplantar formalin flinching + post formalin allodynia. Mice were given intraperitoneal APAP in a DMSO (5%)/Tween 80 (5%) or a water-based formulation before formalin flinching on day 1 and tactile thresholds assessed before and after APAP at day 12. (2) Paw incision. At 24 hours and 8 days after hind paw incision in male mice, effects of intraperitoneal APAP on tactile allodynia were assessed. (3) Repeated delivery. Mice received daily (4 days) analgesic doses of APAP or vehicle and tested upon formalin flinching on day 5. (4) Conditioned place preference. For 3 consecutive days, vehicle was given in the morning in either of 2 chambers and in each afternoon, an analgesic dose of morphine or APAP in the other chamber. On days 5 and 10, animals were allowed to select a "preferred" chamber. Results: Formalin in male mice resulted in biphasic flinching and an enduring postformalin tactile allodynia. Acetaminophen dose dependently decreased phase 2 flinching, and reversed allodynia was observed postflinching. At a comparable APAP dose, female mice showed similarly reduced phase 2 flinching. Incision allodynia was transiently reversed by APAP. Repeated APAP delivery showed no loss of effect after sequential injections or signs of withdrawal. Morphine, but not APAP or vehicle, resulted in robust place preference. Conclusions: APAP decreased flinching and allodynia observed following formalin and paw incision and an absence of tolerance, dependence, or rewarding properties.

Molecular and ultrastructural morphological analyses of highly metamorphosed Aspergillus fumigatus on human formalin-fixed paraffin-embedded tissue.

Invasive fungal infections including invasive pulmonary aspergillosis (IPA) generally have a poor prognosis, because the fungi spread throughout various organs. Therefore, it is important to accurately identify the fungal species for treatment. In this article, we present the results of pathological and molecular morphological analyses that were performed to elucidate the cause of respiratory failure in a patient who died despite suspicion of IPA and treatment with micafungin (MCFG). Pathological analysis revealed the existence of cystic and linear fungi in lung tissue. The fungi were identified as Aspergillus fumigatus (A. fumigatus) by partial sequencing of genomic DNA. Correlative light microscopy and electron microscopy (CLEM) analysis confirmed that fungi observed with light microscopy can also be observed with scanning electron microscopy (SEM) using formalin-fixed paraffin-embedded tissue sections. SEM revealed an atypical ultrastructure of the fungi including inhomogeneous widths, rough surfaces, and numerous cyst-like structures of various sizes. The fungi showed several morphological changes of cultured A. fumigatus treated with MCFG that were previously reported. Our results indicate that integrated analysis of ultrastructural observation by SEM and DNA sequencing may be an effective tool for analyzing fungi that are difficult to identify by conventional pathological analysis.

Mitochondrial DNA copy number and risk of papillary thyroid carcinoma.

BACKGROUND: Mitochondrial DNA (mtDNA) copy number is associated with tumor activity and carcinogenesis. This study was undertaken to investigate mtDNA copy number in papillary thyroid cancer (PTC) tissues and to evaluate the risk of PTC development. The clinicopathological features of patients and mtDNA copy number were correlated. The value of mtDNA copy number was evaluated as a biomarker for PTC. METHOD: DNA was extracted from 105 PTC tissues and 67 control thyroid tissues, and mtDNA copy number mtDNA oxidative damage were determined using qPCR techniques. RESULTS: Overall, the relative mtDNA copy number was significantly higher in PTC patients (p < 0.001). The risk of developing PTC increased significantly across the tertiles of mtDNA copy number (p trend < 0.001). The higher the mtDNA copy number tertile, the greater the risk of developing PTC. Patients with follicular variants had an odds ratio of 2.09 (95% CI: 1.78-2.44) compared to those with classical variants (p < 0.001). The level of mtDNA oxidative damage in PTC was significantly elevated compared to controls (p < 0.001). The ROC analysis of mtDNA copy number indicated an area under the curve (AUC) of 77.7% (95% CI: 0.71 to 0.85, p < 0.001) for the ability of mtDNA copy number z-scores in differentiate between PTC and controls. CONCLUSION: Our results indicated that the augmentation of mtDNA content plays a significant role during the initiation of thyroid cancer, and it might represent a potential biomarker for predicting the risk of PTC.

Effects of hippocampal damage on pain perception in a rat model of Alzheimer's disease induced by amyloid-ß and ibotenic acid injection into the hippocampus.

Patients with Alzheimer's disease (AD) present with a variety of symptoms, including core symptoms as well as behavioral and psychological symptoms. Somatosensory neural systems are generally believed to be relatively unaffected by AD until late in the course of the disease; however, somatosensory perception in patients with AD is not yet well understood. One factor that may complicate the assessment of somatosensory perception in humans centers on individual variations in pathological and psychological backgrounds. It is therefore necessary to evaluate somatosensory perception using animal models with uniform status. In the current study, we focused on the hippocampus, the primary site of AD. We first constructed a rat model of AD model using bilateral hippocampal injections of amyloid-ß peptide 1-40 and ibotenic acid; sham rats received saline injections. The Morris water maze test was used to evaluate memory impairment, and the formalin test (1 % or 4 % formalin) and upper lip von Frey test were performed to compare pain perception between AD model and sham rats. Finally, histological and immunohistochemical methods were used to evaluate tissue damage and neuronal activity, respectively, in the hippocampus. AD model rats showed bilateral hippocampal damage and had memory impairment in the Morris water maze test. Furthermore, AD model rats exhibited significantly less pain-related behavior in phase 2 (the last 50 min of the 60-minute observation) of the 4 % formalin test compared with the sham rats. However, no significant changes were observed in the von Frey test. Immunohistochemical observations of the trigeminal spinal subnucleus caudalis after 4 % formalin injection revealed significantly fewer c-Fos-immunoreactive cells in AD model rats than in sham rats, reflecting reduced neuronal activity. These results indicate that AD model rats with hippocampal damage have reduced responsiveness to persistent inflammatory chemical stimuli to the orofacial region.

Systematic review and feasibility study on pre-analytical factors and genomic analyses on archival formalin-fixed paraffin-embedded breast cancer tissue.

Formalin-fixed paraffin-embedded (FFPE) tissue represents a valuable source for translational cancer research. However, the widespread application of various downstream methods remains challenging. Here, we aimed to assess the feasibility of a genomic and gene expression analysis workflow using FFPE breast cancer (BC) tissue. We conducted a systematic literature review for the assessment of concordance between FFPE and fresh-frozen matched tissue samples derived from patients with BC for DNA and RNA downstream applications. The analytical performance of three different nucleic acid extraction kits on FFPE BC clinical samples was compared. We also applied a newly developed targeted DNA Next-Generation Sequencing (NGS) 370-gene panel and the nCounter BC360® platform on simultaneously extracted DNA and RNA, respectively, using FFPE tissue from a phase II clinical trial. Of the 3701 initial search results, 40 articles were included in the systematic review. High degree of concordance was observed in various downstream application platforms. Moreover, the performance of simultaneous DNA/RNA extraction kit was demonstrated with targeted DNA NGS and gene expression profiling. Exclusion of variants below 5% variant allele frequency was essential to overcome FFPE-induced artefacts. Targeted genomic analyses were feasible in simultaneously extracted DNA/RNA from FFPE material, providing insights for their implementation in clinical trials/cohorts.

The influence of fixing condition on myoglobin stainability of striated muscle as a tool for forensic diagnosis.

It is reported that immunostaining of Myoglobin (Mb) is useful for forensic diagnosis. In this study, we investigated the condition of fixation of striated muscle in 10 % neutral-buffered formalin to obtain appropriate stationarity of Mb in immunostaining. Firstly, criteria for staining intensity and definition of the stainability of examined were determined for sheep muscle tissue. Sheep myocardial tissue was fixed using 10 % neutral-buffered formalin under the 21 different conditions based on combinations of the following: three ratios of volume of formalin (mL) to weight of myocardium (g) (RFM) of 1, 4 or 9, 7 durations of fixation (DF) of 0.5, 3 or 6 h, and 1, 2, 5 or 7 days. Secondly, detection of Mb diffused form skeletal muscle from autopsy cases into formalin during fixation were confirmed by ELISA. Finally, the evaluation of stainability of Mb of striated muscle in routine autopsy examinations was confirmed using sheep staining intensity standards. From this experimental investigation, it has been demonstrated that the most suitable formalin fixation condition for using Mb staining in forensic diagnosis is RFM4 with a fixation time of at least DF 3 h up to 1 day. It was evident that staining intensity decreases with fixation durations exceeding 2 days, irrespective of the RFM. Thus, the fixation time was deemed the most influential factor affecting the staining properties of Mb staining in skeletal muscle tissue. When conducting Mb staining using striated muscle as an evaluation sample, particular attention should be paid to the fixation time.

Obstructive Hydrocephalus and Cardiomyopathy Secondary to Disseminated Protothecosis in a Boxer Dog.

Canine protothecosis is a rare disease caused by saprophytic unicellular achlorophyllous aerobic algae that are ubiquitous in the environment. We report a novel case of neurological and cardiological manifestations associated with disseminated protothecosis. An adult spayed female Boxer dog was presented with a 1-week history of anorexia, progressive central vestibular signs, and a Grade III/VI systolic heart murmur. Magnetic resonance (MR) imaging revealed obstructive hydrocephalus at the level of the mesencephalic aqueduct, while echocardiography and elevated troponin levels suggested an infiltrative cardiomyopathy. No obvious cause was identified. Cerebrospinal fluid (CSF) collection was not performed due to associated procedural risks. Despite receiving symptomatic treatment and maintaining stability for 3 weeks, the dog eventually suffered cardiorespiratory arrest. Postmortem examination revealed disseminated protothecosis, predominantly affecting the heart and brain. We recommend that in cases where the cause of obstructive hydrocephalus is unclear, especially when CSF collection is not feasible, a comprehensive diagnostic method should be implemented. This includes meticulous investigations to identify infected tissues, followed by sampling and performing cytology/histology and culture tests to confirm the presence of the algal organism. Early diagnosis may allow early treatment, although long-term prognosis remains largely unfavorable due to the absence of effective treatments.

Immune response and protection efficacy of formalin-killed vaccines against Streptococcus iniae in four-finger threadfin Eleutheronema tetradactylum.

Four-finger threadfin, Eleutheronema tetradactylum farming in southern Taiwan has been facing disease problems caused by Streptococcus iniae since 2018. The development of a vaccine against infectious S. iniae in the cultured threadfin industry is necessary. Thus, this study aimed to examine the efficacy of threadfin immunized formalin-killed cells (FKC) from S. iniae GSI-111 for 42 days post-vaccination (dpv) using two doses of FKC alone (a booster at 14 dpv) as group A, and FKC mixed with ISA763A adjuvant using a single dose as group B or double doses as group C. Immunoglobulin (Ig)-M was purified from threadfin, and rabbit anti-threadfin IgM polyclonal antibodies were used to detect antibody level in immunized fish; the vaccinated group A displayed higher levels at 3 dpv and all vaccinated treatments demonstrated high antibody levels between 14 and 42 dpv. All vaccine groups showed significantly higher values of lysozyme activity at 42 dpv compared with the control group; the vaccinated A group peaked at 14 dpv. The expression profiles of pro-inflammatory and immune-related genes, TNF-α, IL-12A, and C2 were upregulated at 3 dpv, while CD8A and chemokine receptor CXCR4 were upregulated at 42 dpv. Finally, the threadfins were challenged with S. iniae at 42 dpv. The average relative percent survival was 96% for vaccination A and B treatments, and 100% for vaccination C treatment. In summary, this study demonstrated that FKC vaccines whether formulated with an adjuvant could stimulate immune response and effective protect threadfins against S. iniae infection.

Evaluation of the long-term effects of formaldehyde on the physiology of the Mediterranean mussel, Mytilusgalloprovincialis.

Formalin baths are the most widely used treatment for ectoparasitic fish diseases. Nonetheless, their use in fish cages has been blamed for a number of problems. Although a considerable amount of literature has been produced on the short-term toxic effects of formaldehyde, there is virtually no data on the long-term side effects of the compound on non-target organisms. Therefore, the purpose of this research was to assess the long-term formaldehyde toxicity in Mediterranean mussel, Mytilus galloprovincialis, a common sentinel species that inhabits the area surrounding cage farms. Mussels were kept in a laboratory microenvironment at 20 ± 1 °C for 21 days and exposed to two different formaldehyde concentrations during experimentation: a low dose (L; 40 ppb) based on formaldehyde field measurements in the vicinity of Mediterranean cages, and a high dose (H; 400 ppb) generated by a factor of 10 of the previous dose. A multi-biomarker approach that included antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD), lipid peroxidation (MDA), lysosomal stability (NRRT), genotoxicity tests, condition index (CI), and stress on stress (SoS), was used to evaluate the toxicity of formaldehyde on mussels. The results of the selected tests indicate that formaldehyde does not cause chronic toxicity in mussels subjected to commonly measured concentrations in the aquatic environment following formalin bath treatments. Despite being defined as reversible, the stress brought by the high dose used seems to reduce the antioxidant activity of the tested organism. The significance of this research lies in its contribution to understanding the wider ecological effects of formaldehyde exposure. Moreover, the results highlight the need for further research on other non-target marine organisms to fully understand the cumulative effects of formaldehyde on marine ecosystems.

Teaching medical students ultrasound-guided needle aspiration of synthetic cysts: effect of a formalin-embalmed cadaver simulation model.

BACKGROUND: Standard training for ultrasound-guided cyst needle aspiration is currently performed on live patients during residency. This practice presents risk of iatrogenic injury to patients and provides a high-stress learning environment for medical trainees. Simulation training using synthetic cysts in a formalin-embalmed cadaver model may allow for realistic, practical, and effective training free from patient risk. METHODS: Thirty first-year medical students viewed an orientation video, then attended a skills workshop to perform cyst needle aspiration in formalin-embalmed cadaver tissue under ultrasound guidance. Participants were randomly assigned to one of three ultrasound-trained instructor-types which included a medical student, clinical anatomist, or an ultrasound fellowship trained emergency medicine physician. After training, participants underwent a 5-min skills test to assess their ability to drain a synthetic cyst independently. Pre- and post-training self-confidence surveys were administered. RESULTS: Ultrasound images of synthetic cysts in formalin-embalmed tissue were clear and realistic in appearance, and sonographic needle visualization was excellent. Participants took an average of 161.5 s and 1.9 attempts to complete the procedure. Two of the 30 participants could not complete the procedure within the time limit. Participants' self-reported confidence with respect to all aspects of the procedure significantly increased post-training. Mean confidence scores rose from 1.2 (95% CI 0.96 to 1.39) to 4.4 (95% CI 4.09 to 4.53) (P < 0.0001) Procedure time, number of attempts, performance scores, and self-confidence outcomes were not significantly affected by instructor type. CONCLUSIONS: The use of synthetic cysts in formalin-embalmed cadaveric tissue is feasible, realistic, and efficacious for the teaching of ultrasound-guided needle aspiration to novice medical trainees. This simulation training method can be delivered effectively by multiple instructor types and may allow medical trainees to increase their tactical skill and self-confidence prior to performing ultrasound-guided cyst needle aspiration on live patients.

Flow cytometric analysis for Ki67 assessment in formalin-fixed paraffin-embedded breast cancer tissue.

BACKGROUND: Pathologists commonly employ the Ki67 immunohistochemistry labelling index (LI) when deciding appropriate therapeutic strategies for patients with breast cancer. However, despite several attempts at standardizing the Ki67 LI, inter-observer and inter-laboratory bias remain problematic. We developed a flow cytometric assay that employed tissue dissociation, enzymatic treatment and a gating process to analyse Ki67 in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue. RESULTS: We demonstrated that mechanical homogenizations combined with thrombin treatment can be used to recover efficiently intact single-cell nuclei from FFPE breast cancer tissue. Ki67 in the recovered cell nuclei retained reactivity against the MIB-1 antibody, which has been widely used in clinical settings. Additionally, since the method did not alter the nucleoskeletal structure of tissues, the nuclei of cancer cells can be enriched in data analysis based on differences in size and complexity of nuclei of lymphocytes and normal mammary cells. In a clinical study using the developed protocol, Ki67 positivity was correlated with the Ki67 LI obtained by hot spot analysis by a pathologist in Japan (rho = 0.756, P < 0.0001). The number of cancer cell nuclei subjected to the analysis in our assay was more than twice the number routinely checked by pathologists in clinical settings. CONCLUSIONS: The findings of this study showed the application of this new flow cytometry method could potentially be used to standardize Ki67 assessments in breast cancer.

The nociceptive inputs of the paraventricular hypothalamic nucleus in formalin stimulated mice.

The paraventricular hypothalamic nucleus (PVH) is an important neuroendocrine center involved in pain regulation, but the nociceptive afferent routes for the nucleus are still unclear. We examined the profile of PVH receiving injurious information by a combination of retrograde tracing with Fluoro-Gold (FG) and FOS expression induced by formalin stimuli. The result showed that formalin injection induced significantly increased expression of FOS in the PVH, among which oxytocin containing neurons are one neuronal phenotype. Immunofluorescent staining of FG and FOS revealed that double labeled neurons were strikingly distributed in the area 2 of the cingulate cortex (Cg2), the lateral septal nucleus (LS), the periaqueductal gray (PAG), the posterior hypothalamic area (PH), and the lateral parabrachial nucleus (LPB). In the five regions, LPB had the biggest number and the highest ratio of FOS expression in FG labeled neurons, with main subnuclei distribution in the external, superior, dorsal, and central parts. Further immunofluorescent triple staining disclosed that about one third of FG and FOS double labeled neurons in the LPB were immunoreactive for calcitonin gene related peptide (CGRP). In conclusion, the present study demonstrates the nociceptive input profile of the PVH area under inflammatory pain and suggests that neurons in the LPB may play essential roles in transmitting noxious information to the PVH.

Improvement of the Diagnostic Method for the Detection of Parasite Eggs Using the Formalin Ethyl Acetate Concentration Technique.

PURPOSE: The formalin-ethyl acetate (FEA) concentration method is commonly used in routine clinical practice to detect parasite eggs in feces. This procedure involves extraction of oil with the organic solvent ethyl acetate (EA), which reduces fecal sediment and provides a cleaner background for microscopic analysis. However, clinically, some sediment failed to float after EA treatment. METHODS: Hexane, commonly used in the food oil extraction from oilseeds did not float the feces. Gas chromatography-mass spectrometry (GC-MS) analysis showed that neither the amount of the oil nor the classes of the oil determined was differed whether hexane or EA was used to float the feces. Oil red, Bodipy and Calcofluor staining showed that the unabsorbed oil droplets in the fecal sediment were trapped within the leaf structure. HCl or acetic acid was added to see if the acid residue could dissolve the cellulose of the leaf to promote the bulk float. RESULTS: Our result showed that the fecal bulk contained the loosened mesophyll cell wall. The addition of acid residues improved fecal bulk float. The proximity of cellulose fiber to EA, but not hexane, may enhance the efficacy of oil extraction from cellulose. CONCLUSION: This is the first report that the interaction of cellulose with ethyl acetate in fecal solution has an effect on bulk float. This study improves the understanding of fecal bulk flotation and may assist in the visualization of parasite eggs in clinical practice with non-floating fecal samples in the FEA concentration method.

Genome-scale mutational signature analysis in fixed archived tissues.

Mutation spectra and mutational signatures in cancerous and non-cancerous tissues can be identified by various established techniques of massively parallel sequencing (or next-generation sequencing) including whole-exome or whole-genome sequencing, and more recently by error-corrected/duplex sequencing. One rather underexplored area has been the genome-scale analysis of mutational signatures as markers of mutagenic exposures, and their impact on cancer driver events applied to formalin-fixed or alcohol-fixed paraffin embedded archived biospecimens. This review showcases successful applications of the next-generation sequencing methodologies in archived fixed tissues, including the delineation of the specific tissue fixation-related DNA damage manifesting as artifactual signatures, distinguishable from the true signatures that arise from biological mutagenic processes. Overall, we discuss and demonstrate how next-generation sequencing techniques applied to archived fixed biospecimens can enhance our understanding of cancer causes including mutagenic effects of extrinsic cancer risk agents, and the implications for prevention efforts aimed at reducing avoidable cancer-causing exposures.

[Recycled formalin: A new environmental mitigation tool in pathology?] / Le formol recyclé : un nouvel outil d'atténuation de l'impact environnemental de l'ACP ?

Formalin is the international gold-standard fixative in pathology laboratories. However it is not the ideal one considering its deleterious effects on individuals and the environment. Complete formalin removal or even substitution does not seem possible in the near future. In this update, we present various tools allowing to integrate the use of formalin into an ecocare approach. Among them, formalin recycling according to the protocol developed by the University Hospital of Bordeaux is simple to implement and delivers rapid and significant results, allowing pathology professionals to meet the sustainable development objectives included in the France 2030 agenda.

Characterization of Photobacterium damselae subsp. damselae isolated from diseased Asian seabass (Lates calcarifer) and the preliminary development of a formalin-killed cell vaccine.

Asian seabass (Lates calcarifer) is an economically important fish species that is widely cultivated in Thailand. However, aquaculture of Asian seabass is limited by infectious diseases. One of the most serious diseases is photobacteriosis, caused by Photobacterium damselae. Vaccination is recognized as an efficient disease prevention and pathogen control method for strengthening the aquaculture industry. To promote vaccine development, the characterization of pathogenic bacteria and their pathogenesis is required. In this study, isolates of P. damselae were obtained from commercial aquaculture farms in Thailand during 2019-2021. Analyses of 16S rRNA and the urease subunit alpha genes identified the isolates as P. damselae subsp. damselae (Phdd). Antibiotic susceptibility analyses showed that all Phdd isolates were resistant to amoxicillin (10 µg). Haemolysis and phospholipase activities were used to categorize P. damselae into three groups based on their biological activities. The pathogenicity of four candidates (SK136, PD001, PD002 and T11L) was tested in Asian seabass. Isolate SK136 showed the highest virulence, with a lethal dose (LD50) of 1.47 × 105 CFU/fish, whereas isolate PD001 did not show any virulence. Genotypic characterization, based on multi-locus sequence typing analysis, demonstrated that all candidates were novel strains with new sequence types (64, 65, 66 and 67). Preliminary vaccination using formalin-killed cells (FKCs) protected Asian seabass from artificial challenges. Taken together, these results provide fundamental knowledge for vaccine development against Phdd infection in Asian seabass.

Diagnostic Utility of Immunofluorescence on Formalin-Fixed Paraffin-Embedded Skin Biopsy.

Background: When immunofluorescence on the frozen section is insufficient or unavailable, salvage immunofluorescence techniques can be used on formalin-fixed, paraffin-embedded tissue. The goal of the current investigation was to evaluate the diagnostic value of paraffin immunofluorescence following proteinase K digestion on skin biopsy samples in comparison to fresh frozen immunofluorescence. Materials and Methods: It was standardized and compared to the immunofluorescence on fresh frozen tissue (IF-Frozen) for paraffin immunofluorescence by proteinase K digestion of formalin-fixed paraffin-embedded skin biopsies (IF-FFPE). The study included 50 native skin biopsy cases, and fluorescein isothiocyanate-labeled IgA, IgG, IgM, and C3 intensity levels were evaluated in each case. Results: A total of 50 cases of native skin biopsy were included in the study, and their intensities for IgA, IgG, IgM, and C3 antibodies were compared. The average staining intensities in each disease group for the antibodies had equal intensity or had a minor difference (1+)/significant difference (2+). Paraffin immunofluorescence, proteinase K digestion had the best correlation, that is, had either equal or minor difference (1+) with fresh frozen immunofluorescence. The difference of 2+ intensity of antibodies between IF-FFPE and IF-Frozen was noted mainly in C3 antibody on paraneoplastic pemphigus. IF-FFPE showed a sensitivity of 100%, 97.6%, 100%, and 81.6% for IgA, IgG, IgM, and C3, respectively, whereas the specificity was 100% for IgA, IgG, IgM, and C3. Limitations: Small sample size and and the employment of one method of antigen retrieval in IF-FFPE. Conclusion: Immunofluorescence techniques done on formalin-fixed paraffin-embedded tissue can serve as salvage techniques in cases where immunofluorescence on the frozen section may not be adequate or may not be available.