Immunomodulation Evidence of Nanostructured Recombinant Proteins in Salmonid Cells.

Recent studies have demonstrated that immune-related recombinant proteins can enhance immune function, increasing host survival against infectious diseases in salmonids. This research evaluated inclusion bodies (IBs) of antimicrobial peptides (CAMPIB and HAMPIB) and a cytokine (IL1ßIB and TNFαIB) as potential immunostimulants in farmed salmonids. For this purpose, we produced five IBs (including iRFPIB as a control), and we evaluated their ability to modulate immune marker gene expression of three IBs in the RTS11 cell line by RT-qPCR. Additionally, we characterized the scale-up of IBs production by comparing two different scale systems. The results showed that CAMPIB can increase the upregulation of tnfα, il1ß, il8, and il10, HAMPIB significantly increases the upregulation of tnfα, inos, and il10, and IL1ßIB significantly upregulated the expression of tnfα, il1ß, and cox2. A comparison of IL1ßIB production showed that the yield was greater in shake flasks than in bioreactors (39 ± 1.15 mg/L and 14.5 ± 4.08 mg/L), and larger nanoparticles were produced in shake flasks (540 ± 129 nm and 427 ± 134 nm, p < 0.0001, respectively). However, compared with its shake flask counterpart, the IL1ßIB produced in a bioreactor has an increased immunomodulatory ability. Further studies are needed to understand the immune response pathways activated by IBs and the optimal production conditions in bioreactors, such as a defined medium, fed-batch production, and mechanical bacterial lysis, to increase yield.

Correlation of fetal heart rate dynamics to inflammatory markers and brain-derived neurotrophic factor during pregnancy.

OBJECTIVES: This study aims to show the relation between biomarkers in maternal and cord-blood samples and fetal heart rate variability (fHRV) metrics through a non-invasive fetal magnetocardiography (fMCG) technique. METHODS: Twenty-three women were enrolled for collection of maternal serum and fMCG tracings immediately prior to their scheduled cesarean delivery. The umbilical cord blood was collected for measurement of biomarker levels. The fMCG metrics were then correlated to the biomarker levels from the maternal serum and cord blood. RESULTS: Brain-derived neurotrophic factor (BDNF) had a moderate correlation with fetal parasympathetic activity (0.416) and fetal sympathovagal ratios (-0.309; -0.356). Interleukin (IL)-6 also had moderate-sized correlations but with an inverse relationship as compared to BDNF. These correlations were primarily in cord-blood samples and not in the maternal blood. CONCLUSIONS: In this small sample-sized exploratory study, we observed a moderate correlation between fHRV and cord-blood BDNF and IL-6 immediately preceding scheduled cesarean delivery at term. These findings need to be validated in a larger population.

Oxidative stress promotes cytotoxicity in human cancer cell lines exposed to Escallonia spp. extracts.

BACKGROUND: Standard cancer treatments show a lack of selectivity that has led to the search for new strategies against cancer. The selective elimination of cancer cells modulating the redox environment, known as "selective oxycution", has emerged as a viable alternative. This research focuses on characterizing the unexplored Escallonia genus plant extracts and evaluating their potential effects on cancer's redox balance, cytotoxicity, and activation of death pathways. METHODS: 36 plant extracts were obtained from 4 different species of the Escallonia genus (E. illinita C. Presl, E. rubra (Ruiz & Pav.) Pers., E. revoluta (Ruiz & Pav.) Pers., and E. pulverulenta (Ruiz & Pav.) Pers.), which were posteriorly analyzed by their phytoconstituents, antioxidant capacity, and GC-MS. Further, redox balance assays (antioxidant enzymes, oxidative damage, and transcription factors) and cytotoxic effects (SRB, ∆Ψmt, and caspases actives) of those plant extracts were analyzed on four cell lines (HEK-293T, MCF-7, HT-29, and PC-3). RESULTS: 36 plant extracts were obtained, and their phytoconstituents and antioxidant capacity were established. Further, only six extracts had EC50 values < 10 µg*mL- 1, indicating high toxicity against the tested cells. From those, two plant extracts were selective against different cancer cell lines: the hexane extract of E. pulverulenta´s stem was selective for HT-29, and the ethyl acetate extract of E. rubra´s stem was selective for PC-3. Both extracts showed unbalanced redox effects and promoted selective cell death. CONCLUSIONS: This is the first study proving "selective oxycution" induced by Chilean native plant extracts.

Exploring the Influence of Fetal Sex on Heart Rate Dynamics Using Fetal Magnetocardiographic Recordings.

Fetal sex has been associated with different development trajectories that cause structural and functional differences between the sexes throughout gestation. Fetal magnetocardiography (fMCG) recordings from 123 participants (64 females and 59 males; one recording/participant) from a database consisting of low-risk pregnant women were analyzed to explore and compare fetal development trajectories of both sexes. The gestational age of the recordings ranged from 28 to 38 weeks. Linear metrics in both the time and frequency domains were applied to study fetal heart rate variability (fHRV) measures that reveal the dynamics of short- and long-term variability. Rates of linear change with GA in these metrics were analyzed using general linear model regressions with assessments for significantly different variances and GA regression slopes between the sexes. The fetal sexes were well balanced for GA and sleep state. None of the fHRV measures analyzed exhibited significant variance heterogeneity between the sexes, and none of them exhibited a significant sex-by-GA interaction. The absence of a statistically significant sex-by-GA interaction on all parameters resulted in none of the regression slope estimates being significantly different between the sexes. With high-precision fMCG recordings, we were able to explore the variation in fHRV parameters as it relates to fetal sex. The fMCG-based fHRV parameters did not show any significant difference in rates of change with gestational age between sexes. This study provides a framework for understanding normal development of the fetal autonomic nervous system, especially in the context of fetal sex.

Regulation and distribution of European sea bass perforins point to their role in the adaptive cytotoxic response against NNV.

Cell-mediated cytotoxicity is a complex immune mechanism that involves the release of several killing molecules, being perforin (PRF) one of the most important effector players. Perforin is synthesized by T lymphocytes and natural killer cells in mammals and responsible for the formation of pores on the target cell membrane during the killing process. Although perforin has been extensively studied in higher vertebrates, this knowledge is very limited in fish. Therefore, in this study we have identified four prf genes in European sea bass (Dicentrarchus labrax) and evaluated their mRNA levels. All sea bass prf genes showed the typical and conserved domains of its human orthologue and were closely clustered by the phylogenetic analysis. In addition, all genes showed constitutive and ubiquitous tissular expression, being prf1.9 gene the most highly expressed in immune tissues. Subsequently, in vitro stimulation of head-kidney (HK) cells with phytohemagglutinin, a T-cell activator, showed an increase of all prf gene levels, except for prf1.3 gene. European sea bass HK cells increased the transcription of prf1.2 and prf1.9 during the innate cell-mediated cytotoxic activity against xenogeneic target cells. In addition, sea bass infected with nodavirus (NNV) showed a similar expression pattern of all prf in HK and brain at 15 days post-infection, except for prf1.3 gene and in the gonad. Finally, the use of a polyclonal antibody against PRF1.9 showed an increase of positive cells in HK, brain and gonad from NNV-infected fish. Taken together, the data seem to indicate that all prf genes, except prf1.3, appear to be involved in the European sea bass immunity, and probably in the cell-mediated cytotoxic response, with PRF1.9 playing the most important role against nodavirus. The involvement of the PRFs and the CMC activity in the vertical transmission success of the virus is also discussed.

Ghrelin-responsive mediobasal hypothalamic neurons mediate exercise-associated food intake and exercise endurance.

Previous studies have implicated the orexigenic hormone ghrelin as a mediator of exercise endurance and the feeding response postexercise. Specifically, plasma ghrelin levels nearly double in mice when they are subjected to an hour-long bout of high-intensity interval exercise (HIIE) using treadmills. Also, growth hormone secretagogue receptor-null (GHSR-null) mice exhibit decreased food intake following HIIE and diminished running distance (time until exhaustion) during a longer, stepwise exercise endurance protocol. To investigate whether ghrelin-responsive mediobasal hypothalamus (MBH) neurons mediate these effects, we stereotaxically delivered the inhibitory designer receptor exclusively activated by designer drugs virus AAV2-hSyn-DIO-hM4(Gi)-mCherry to the MBH of Ghsr-IRES-Cre mice, which express Cre recombinase directed by the Ghsr promoter. We found that chemogenetic inhibition of GHSR-expressing MBH neurons (upon delivery of clozapine-N-oxide) 1) suppressed food intake following HIIE, 2) reduced maximum running distance and raised blood glucose and blood lactate levels during an exercise endurance protocol, 3) reduced food intake following ghrelin administration, and 4) did not affect glucose tolerance. Further, HIIE increased MBH Ghsr expression. These results indicate that activation of ghrelin-responsive MBH neurons is required for the normal feeding response to HIIE and the usual amount of running exhibited during an exercise endurance protocol.

Evidence that fish death after Vibrio vulnificus infection is due to an acute inflammatory response triggered by a toxin of the MARTX family.

Vibrio vulnificus is an emerging zoonotic pathogen associated with fish farms that is capable of causing a hemorrhagic septicemia known as warm-water vibriosis. According to a recent transcriptomic and functional study, the death of fish due to vibriosis is more related to the inflammatory response of the host than to the tissue lesions caused by the pathogen. In this work, we hypothesize that the RtxA1 toxin (a V. vulnificus toxin of the MARTX (Multifunctional Autoprocessing Repeats in Toxin) family) is the key virulence factor that would directly or indirectly trigger this fatal inflammatory response. Our hypothesis was based on previous studies that showed that rtxA1-deficient mutants maintained their ability to colonize and invade, but were unable to kill fish. To demonstrate this hypothesis, we infected eels (model of fish vibriosis) by immersion with a mutant deficient in RtxA1 production and analyzed their transcriptome in blood, red blood cells and white blood cells during early vibriosis (0, 3 and 12 h post-infection). The transcriptomic results were compared with those obtained in the previous study in which eels were infected with the V. vulnificus parental strain, and were functionally validated. Overall, our results confirm that fish death after V. vulnificus infection is due to an acute, early and atypical inflammatory response triggered by RtxA1 in which red blood cells seem to play a central role. These results could be relevant to other vibriosis as the toxins of this family are widespread in the Vibrio genus.

Heritability of Immunity Traits and Resistance of Atlantic Salmon against the Sea Louse Caligus rogercresseyi.

The immune response of Atlantic salmon to sea lice has been extensively studied, but we still do not know the mechanisms by which some fish become resistant and others do not. In this study, we estimated the heritabilities of three key proteins associated with the innate immunity and resistance of Salmo salar against the sea louse Caligus rogercresseyi. In particular, we quantified the abundance of 2 pro-inflammatory cytokines, Tnfα and Il-8, and an antioxidant enzyme, Nkef, in Atlantic salmon skin and gill tissue from 21 families and 268 individuals by indirect ELISA. This covers a wide parasite load range from low or resistant (mean sea lice ± SE = 8.7 ± 0.9) to high or susceptible (mean sea lice ± SE = 43.3 ± 2.0). Our results showed that susceptible fish had higher levels of Nkef and Tnfα than resistant fish in their gills and skin, although gill Il-8 was higher in resistant fish, while no significant differences were found in the skin. Furthermore, moderate to very high heritable genetic variation was estimated for Nkef (h2 skin: 0.96 ± 0.14 and gills: 0.97 ± 0.11) and Tnfα (h2 skin: 0.53 ± 0.17 and gills: 0.32 ± 0.14), but not for Il-8 (h2 skin: 0.22 ± 0.12 ns and gills: 0.09 ± 0.08 ns). This work provides evidence that Nkef and Tnfα protein expressions are highly heritable and related to resistance against sea lice in Atlantic salmon.

Antiviral Activity of an Endogenous Parvoviral Element.

Endogenous viral elements (EVEs) are genomic DNA sequences derived from viruses. Some EVEs have open reading frames (ORFs) that can express proteins with physiological roles in their host. Furthermore, some EVEs exhibit a protective role against exogenous viral infection in their host. Endogenous parvoviral elements (EPVs) are highly represented in mammalian genomes, and although some of them contain ORFs, their function is unknown. We have shown that the locus EPV-Dependo.43-ODegus, an EPV with an intact ORF, is transcribed in Octodon degus (degu). Here we examine the antiviral activity of the protein encoded in this EPV, named DeRep. DeRep was produced in bacteria and used to generate antibodies that recognize DeRep in western blots of degu tissue. To test if DeRep could protect against exogenous parvovirus, we challenged cells with the minute virus of mice (MVM), a model autonomous parvovirus. We observed that MVM protein expression, DNA damage induced by replication, viral DNA, and cytopathic effects are reduced when DeRep is expressed in cells. The results of this study demonstrate that DeRep is expressed in degu and can inhibit parvovirus replication. This is the first time that an EPV has been shown to have antiviral activity against an exogenous virus.

Evidence of extraganglionic vagal mechanoreceptors in the mouse vagus nerve.

Vagal afferent neuronal somas are in the nodose and jugular ganglia. In this study, we identified extraganglionic neurons in whole-mount preparations of the vagus nerves from Phox2b-Cre-ZsGreen transgenic mice. These neurons are typically arranged in small clusters and monolayers along the cervical vagus nerve. Although infrequent, these neurons were sometimes observed along the thoracic and esophageal vagus. We performed RNAscope in situ hybridization and confirmed that the extraganglionic neurons detected in this transgenic mouse strain expressed vagal afferent markers (i.e., Phox2b and Slc17a6) as well as markers that identify them as potential gastrointestinal mechanoreceptors (i.e., Tmc3 and Glp1r). We also identified extraganglionic neurons in the vagus nerves of wild-type mice that were injected intraperitoneally with Fluoro-Gold, thereby ruling out possible anatomical discrepancies specific for transgenic mice. In wild-type mice, extraganglionic cells were positive for peripherin, confirming their neuronal nature. Taken together, our findings revealed a previously undiscovered population of extraganglionic neurons associated with the vagus nerve. Going forward, it is important to consider the possible existence of extraganglionic mechanoreceptors that transmit signals from the abdominal viscera in future studies related to vagal structure and function.

An Unfortunate Miss of Undiagnosed Arterial Ischemic Stroke (AIS) in the Setting of Diabetic Ketoacidosis in an Adult: A Case Report.

We present the case of a 43-year-old male with a history of poorly controlled type II diabetes who presented with altered mental status, urinary incontinence, and diabetic ketoacidosis (DKA). Initial brain imaging studies were negative for acute intracranial pathology; however, the next day, the patient was found to have left-sided paralysis. Repeat imaging studies revealed a right middle cerebral artery infarct with hemorrhagic conversion. Given that the rate of reported strokes in the setting of DKA in adults is limited, this case report affirms to discuss the importance of prompt recognition, evaluation, and adequate treatment of DKA to prevent neurological complications, as well as the pathophysiology behind the etiology of DKA-induced stroke. This case also underscores the importance of early recognition and missed strokes in the emergency department (ED) and emphasizes the need for stroke evaluation in patients with altered mental status even though an alternative diagnosis is apparent to avoid anchor bias.

The effect of maternal pregestational diabetes on fetal autonomic nervous system.

Heart rate variability assessment of neonates of pregestational diabetic mothers have shown alterations in the autonomic nervous system (ANS). The objective was to study the effect of maternal pregestational diabetes on ANS at the fetal stage by combining cardiac and movement parameters using a non-invasive fetal magnetocardiography (fMCG) technique. This is an observational study with 40 participants where fetuses from a group of 9 Type 1, 19 Type 2 diabetic, and 12 non-diabetic pregnant women were included. Time and frequency domain fetal heart rate variability (fHRV) and coupling of movement and heart rate acceleration parameters related to fetal ANS were analyzed. Group differences were investigated using analysis of covariance to adjust for gestational age (GA). When compared to non-diabetics, the Type 1 diabetics had a 65% increase in average ratio of very low-frequency (VLF) to low-frequency (LF) bands and 63% average decrease in coupling index after adjusting for GA. Comparing Type 2 diabetics to non-diabetics, there was an average decrease in the VLF (50%) and LF bands (63%). Diabetics with poor glycemic control had a higher average VLF/LF (49%) than diabetics with good glycemic control. No significant changes at p < 0.05 were observed in high-frequency (HF) frequency domain parameters or their ratios, or in the time domain. Fetuses of pregestational diabetic mothers exhibited some differences in fHRV frequency domain and heart rate-movement coupling when compared to non-diabetics but the effect of fHRV related to fetal ANS and sympathovagal balance were not as conclusive as observed in the neonates of pregestational diabetic mothers.

Evidence of the Autophagic Process during the Fish Immune Response of Skeletal Muscle Cells against Piscirickettsia salmonis.

Autophagy is a fundamental cellular process implicated in the health of the cell, acting as a cytoplasmatic quality control machinery by self-eating unfunctional organelles and protein aggregates. In mammals, autophagy can participate in the clearance of intracellular pathogens from the cell, and the activity of the toll-like receptors mediates its activation. However, in fish, the modulation of autophagy by these receptors in the muscle is unknown. This study describes and characterizes autophagic modulation during the immune response of fish muscle cells after a challenge with intracellular pathogen Piscirickettsia salmonis. For this, primary cultures of muscle cells were challenged with P. salmonis, and the expressions of immune markers il-1ß, tnfα, il-8, hepcidin, tlr3, tlr9, mhc-I and mhc-II were analyzed through RT-qPCR. The expressions of several genes involved in autophagy (becn1, atg9, atg5, atg12, lc3, gabarap and atg4) were also evaluated with RT-qPCR to understand the autophagic modulation during an immune response. In addition, LC3-II protein content was measured via Western blot. The challenge of trout muscle cells with P. salmonis triggered a concomitant immune response to the activation of the autophagic process, suggesting a close relationship between these two processes.

Priming European Sea Bass Female Broodstock Improves the Antimicrobial Immunity of Their Offspring.

Acquiring immunocompetence is essential in the development of fish embryos, as they are exposed to environmental pathogens even before they are fertilized. Despite the importance of the antimicrobial function as the first line of defense against foreign microorganisms, little knowledge is available about its role in larval development. In vertebrates, transgenerational immune priming influences the acquisition of immunocompetence of specimens, regulating the selective allocation of nongenetic resources to their progeny and modulating their development. In this work, we primed teleost European sea bass broodstock females with a viral protein expression vector in order to evaluate the innate immunity development of their offspring. Several antimicrobial functions, the pattern of expression of gene coding for different antimicrobial peptides (AMPs), and their protein levels, were evaluated in eggs and larvae during development. Our data determined the presence of antimicrobial proteins of maternal origin in eggs, and that female vaccination increases antimicrobial activities and the transcription and synthesis of AMPs during larval development.

A novel and practical technique to facilitate lead insertion at the His bundle region.

BACKGROUND: His bundle (HB) pacing techniques are challenging and time-consuming. This is primarily due to the limitations in locating the relatively small area of the HB body for pacing. METHODS: Permanent HB pacing was performed in 133 consecutive patients with symptomatic bradycardia. A right atrial septo-gram (RAS) was performed in all patients to locate the HB. Briefly, 8-10 cc of contrast was injected through the Medtronic C315HIS delivery sheath while fluoroscopy cine runs were obtained in the RAO 15-20° projection. The images obtained provided the visualization of an approximately 90° angle composed by the medial aspect of the tricuspid valve annulus (TVA) anteriorly and the superior aspect of the interatrial septum superiorly. The apex of this angle coincides with the tip of the triangle of Koch (TK), where the HB body is usually located. A Medtronic SelectSecure™ MRI SureScan™ Model 3830 lead was then advanced and directed towards this area. The HB was mapped using pace mapping and unipolar recordings from the lead tip. RESULTS: Localization of the apex of the TK/HB body with the RAS was successful in all patients. The overall acute success of inserting the lead at the HB was 95%. CONCLUSION: This study demonstrated that our method of utilizing a RAS to facilitate the localization the HB body proved to be safe and efficient in achieving permanent HB pacing with a success rate higher than previously reported.

Immunity elicited by AMP-encoding plasmids fails to increase the protection of European sea bass against nodavirus.

Antimicrobial peptides (AMPs) are a potent arm of the innate immune system that can directly kill pathogens and induce immunomodulation. In the marine aquaculture, European sea bass (Dicentrarchus labrax L.) is one of the most prosperous species but is highly susceptible to nodavirus (NNV), which produces high rates of mortality in larvae and juvenile stages. Thus, we aimed to evaluate whether AMPs exert immunomodulatory and/or NNV-preventive actions in sea bass. To do this, plasmids encoding the sea bass AMPs dicentracin (pDIC), beta-defensin (pDB1), hepcidin (pHAMP2) or NK-lysin (pNKL) were generated and intramuscularly injected into sea bass juveniles to evaluate their immunomodulatory and anti-NNV roles. Sea bass muscle transcribes the AMPs and produces an increase in their circulating levels, along with an increase of the antibacterial activity. Immune-related gene analysis revealed a great activation of the inflammatory response and the recruitment of neutrophilic granulocytes at the site of injection. However, AMP-encoding plasmids, namely pHAMP2, negatively affected to NNV disease by increasing fish mortality. In conclusion, plasmids encoding AMPs show immunostimulatory effects on European sea bass but do not improve the resistance to NNV.

Young adult male LEW.1WR1 rats have reduced beta cell area and develop glucose intolerance.

Prediabetes affects 1 in 3 American adults and is characterized by insulin resistance, insulin hypersecretion, and impaired glucose tolerance. Weanling LEW.1WR1 (1WR1) rats have increased blood insulin concentrations, so we hypothesized that young adult 1WR1 rats would develop impaired glucose tolerance due to the poor regulation of insulin. We monitored glucose tolerance, insulin tolerance, and weight gain for 10 weeks to assess if there was a decline in glucose processing over time. 1WR1 rats were significantly more glucose intolerant after 8 weeks. 1WR1 rats had increased body mass, yet abdominal fat mass was not significantly increased. Although the 1WR1 rats had increased circulating insulin and glucagon protein levels, 1WR1 rat beta cell area was significantly reduced. There may be underlying insulin resistance as evidenced by dysfunctional insulin regulation during fasting. Understanding the metabolic phenotype of this rat model can provide insight into the human pathophysiological changes that increase susceptibility to glucose intolerance and prediabetes.

Why vaccines fail against Piscirickettsiosis in farmed salmon and trout and how to avoid it: A review.

Piscirickettsiosis is the most severe, persistent, and damaging disease that has affected the Chilean salmon industry since its origins in the 1980s. As a preventive strategy for this disease, different vaccines have been developed and used over the last 30 years. However, vaccinated salmon and trout frequently die in the sea cages and the use of antibiotics is still high demonstrating the low efficiency of the available vaccines. The reasons why the vaccines fail so often are still debated, but it could involve different extrinsic and intrinsic factors. Among the extrinsic factors, mainly associated with chronic stress, we can distinguish: 1) biotic including coinfection with sea lice, sealions attacks or harmful algal blooms; 2) abiotic including low oxygen or high temperature; and 3) farm-management factors including overcrowding or chemical delousing treatments. Among the intrinsic factors, we can distinguish: 1) fish-related factors including host's genetic variability (species, population and individual), sex or age; 2) pathogen-related factors including their variability and ability to evade host immune responses; and 3) vaccine-related factors including low immunogenicity and poor matches with the circulating pathogen strain. Based on the available evidence, in order to improve the development and the efficacy of vaccines against P. salmonis we recommend: a) Do not perform efficacy evaluations by intraperitoneal injection of pathogens because they generate an artificial protective immune response, instead cohabitation or immersion challenges must be used; b) Evaluate the diversity of pathogen strains in the field and ensure a good antigenic match with the vaccines; c) Investigate whether host genetic diversity could be improved, e.g. through selection, in favor of better and longer responses to vaccination; d) To reduce the stressful effects at the cage level, controlling the co-infection of pathogens and avoiding fish overcrowding. To date, we do not know the immunological mechanisms by which the vaccines against P. salmonis may or may not generate protection. More studies are required to identify what type of response, cellular or molecular, is required to develop effective vaccines.

Commercial Vaccines Do Not Confer Protection against Two Genogroups of Piscirickettsia salmonis, LF-89 and EM-90, in Atlantic Salmon.

In Atlantic salmon, vaccines have failed to control and prevent Piscirickettsiosis, for reasons that remain elusive. In this study, we report the efficacy of two commercial vaccines developed with the Piscirickettsia salmonis isolates AL100005 and AL 20542 against another two genogroups which are considered highly and ubiquitously prevalent in Chile: LF-89 and EM-90. Two cohabitation trials were performed to mimic field conditions and vaccine performance: (1) post-smolt fish were challenged with a single infection of LF-89, (2) adults were coinfected with EM-90, and a low level coinfection of sea lice. In the first trial, the vaccine delayed smolt mortalities by two days; however, unvaccinated and vaccinated fish did not show significant differences in survival (unvaccinated: 60.3%, vaccinated: 56.7%; p = 0.28). In the second trial, mortality started three days later for vaccinated fish than unvaccinated fish. However, unvaccinated and vaccinated fish did not show significant differences in survival (unvaccinated: 64.6%, vaccinated: 60.2%, p = 0.58). Thus, we found no evidence that the evaluated vaccines confer effective protection against the genogroups LF-89 and EM-90 of P. salmonis with estimated relative survival proportions (RPSs) of -9% and -12%, respectively. More studies are necessary to evaluate whether pathogen heterogeneity is a key determinant of the lack of vaccine efficacy against P. salmonis.