Evaluation of Flathead Grey Mullets (Mugil cephalus) Immunization and Long-Term Protection against Vibrio harveyi Infection Using Three Different Vaccine Preparations.

In recent years, flathead grey mullets (Mugil cephalus) cultured in Eilat (Israel) have been highly affected by Vibrio harveyi, showing neurological signs such as uncoordinated circular swimming followed by high mortality rates. Despite the advances in and different approaches to control vibriosis associated with Vibrio harveyi, including commercial vaccines, most of them have not succeeded in long-term protection. In this study, we evaluated the effectiveness, long-term protection, and antibody production of three vaccine preparations: heat-killed bacteria (HKB), membrane proteins denaturation (BME PROT), and internal proteins (INT PROT) developed specifically against Vibrio harveyi for grey mullets. Our results show that fish immunized with heat-killed bacteria emulsified with adjuvant presented the most effective and long-lasting protection against the bacterium, and a cross-protection against other bacteria from the harveyi clade. The effectiveness of each immunization treatment correlated with the levels of specific antibody production against Vibrio harveyi in the serum of the immunized fish.

Sparus aurata and Lates calcarifer skin microbiota under healthy and diseased conditions in UV and non-UV treated water.

BACKGROUND: The welfare of farmed fish is influenced by numerous environmental and management factors. Fish skin is an important site for immunity and a major route by which infections are acquired. The objective of this study was to characterize bacterial composition variability on skin of healthy, diseased, and recovered Gilthead Seabream (Sparus aurata) and Barramundi (Lates calcarifer). S. aurata, which are highly sensitive to gram-negative bacteria, were challenged with Vibrio harveyi. In addition, and to provide a wider range of infections, both fish species (S. aurata and L. calcarifer) were infected with gram-positive Streptococcus iniae, to compare the response of the highly sensitive L. calcarifer to that of the more resistant S. aurata. All experiments also compared microbial communities found on skin of fish reared in UV (a general practice used in aquaculture) and non-UV treated water tanks. RESULTS: Skin swab samples were taken from different areas of the fish (lateral lines, abdomen and gills) prior to controlled infection, and 24, 48 and 72 h, 5 days, one week and one-month post-infection. Fish skin microbial communities were determined using Illumina iSeq100 16S rDNA for bacterial sequencing. The results showed that naturally present bacterial composition is similar on all sampled fish skin sites prior to infection, but the controlled infections (T1 24 h post infection) altered the bacterial communities found on fish skin. Moreover, when the naturally occurring skin microbiota did not quickly recover, fish mortality was common following T1 (24 h post infection). We further confirmed the differences in bacterial communities found on skin and in the water of fish reared in non-UV and UV treated water under healthy and diseased conditions. CONCLUSIONS: Our experimental findings shed light on the fish skin microbiota in relation to fish survival (in diseased and healthy conditions). The results can be harnessed to provide management tools for commercial fish farmers; predicting and preventing fish diseases can increase fish health, welfare, and enhance commercial fish yields.

First description of Vibrio harveyi as the causative agent of morbidity and mortality in farmed flathead grey mullet Mugil cephalus.

Flathead grey mullet Mugil cephalus is an important species in the aquaculture industry in the Mediterranean basin and throughout the world. During the last 10 yr, M. cephalus breeding stocks, larvae, and juveniles cultured in Eilat (Israel) have shown neurological signs such as uncoordinated circular swimming, while also presenting oral hemorrhages. Death follows days after the onset of the clinical signs, and mortality rates may reach 80% in some cases, causing high economical losses. Bacteriology isolations from different organs, including the brain, and a Koch's postulate experiment, confirmed Vibrio harveyi as the causative agent. Histological analyses showed the presence of the bacterium in different organs. However, in the brain, the bacterium was observed only within blood vessels and meninges. In some samples, mild to severe brain tissue damage was seen. In order to understand the virulence and lethality of V. harveyi, a median lethal dose was calculated, and the result was 106 colony-forming units fish-1. To the best of our knowledge, this is the first report that describes V. harveyi isolated from the brain of M. cephalus and validates it as an etiological agent causing neurological signs in this fish species.

Rapid onsets of warming events trigger mass mortality of coral reef fish.

Our study reveals a hitherto overlooked ecological threat of climate change. Studies of warming events in the ocean have typically focused on the events' maximum temperature and duration as the cause of devastating disturbances in coral reefs, kelp forests, and rocky shores. In this study, however, we found that the rate of onset (Ronset), rather than the peak, was the likely trigger of mass mortality of coral reef fishes in the Red Sea. Following a steep rise in water temperature (4.2 °C in 2.5 d), thermally stressed fish belonging to dozens of species became fatally infected by Streptococcus iniae Piscivores and benthivores were disproportionately impacted whereas zooplanktivores were spared. Mortality rates peaked 2 wk later, coinciding with a second warming event with extreme Ronset The epizootic lasted ∼2 mo, extending beyond the warming events through the consumption of pathogen-laden carcasses by uninfected fish. The warming was widespread, with an evident decline in wind speed, barometric pressure, and latent heat flux. A reassessment of past reports suggests that steep Ronset was also the probable trigger of mass mortalities of wild fish elsewhere. If the ongoing increase in the frequency and intensity of marine heat waves is associated with a corresponding increase in the frequency of extreme Ronset, calamities inflicted on coral reefs by the warming oceans may extend far beyond coral bleaching.

Vaccination of European sea bass Dicentrarchus labrax with avirulent Mycobacterium marinum (iipA::kan mutant).

Mycobacteriosis is a chronic progressive disease affecting teleost fishes all over the world. No vaccine is commercially available against its main etiological agent, Mycobacterium marinum. The mycobacterial gene responsible for invasion and intracellular persistence, iipA, is known to modulate M. marinum pathology. The innate and adaptive immune responses in sea bass (Dicentrarchus labrax) vaccinated with M. marinum iipA::kan mutant with (and without) the use of adjuvant, with (and without) a booster vaccination were monitored. The adjuvanted vaccine induced enhanced immune responses. TNF-α transcription levels were extremely high in spleen of the fish vaccinated with the addition of adjuvant in both fish vaccinated once and twice, followed by an IgM response highly specific for M. marinum. Also, histologically, granulomas started appearing in spleen and head-kidney tissues (but with no visible bacteria) within a month after vaccination, mainly with the adjuvanted vaccine. This was followed by reduction in pathology, as demonstrated by the lower number of granulomas (with visible bacteria), indicating that even heat-killed bacteria were able to elicit granulomatous formations. Adhesion of the internal organs and moderate pigmentation were observed in the perivisceral adipose tissue of nearly all vaccinated fish. Although the adjuvanted heat-killed avirulent iipA::kan mutant clearly induced a strong humoral and adaptive immune response, the booster treatment did not seem to have produced a significantly higher degree of protection from the disease compared to fish that received a single vaccination.

Co-existence of Myxobolus spp. (Myxozoa) in gray mullet (Mugil cephalus) juveniles from the Mediterranean Sea.

Gray mullet (Mugilidae) occur in all seas and are farmed widely around the world, and thus, the risk of their parasites spreading through transport of aquaculture seed is a serious concern. Among others, gray mullets typically host a diversity of myxosporeans, a group in which spore morphometrics of genera has been consistently shown to be inadequate for determination of species. In this study, we investigated Myxobolus Bütschli 1882 (Myxosporea) species found in two fingerling stocks of Mugil cephalus caught in the wild off the coasts of the eastern (Israel) and western (Spain) Mediterranean. Although we observed similar morphological features, significant dissimilarities in spore size and differences in Myxobolus species SSU rDNA sequences were noted. Genetic analyses demonstrated that multiple Myxobolus species, some with SSU rDNA sequences new to GenBank, infected the stock from Spain. In addition, Myxobolus DNA was found associated with several types of host tissue (gill, tail, and internal organs), and sequence analyses indicated that multiple species of Myxobolus were also present, sometimes in different tissues from the same fish. The results suggest that the gray mullets supported a collection of several different Myxobolus species with similar morphology.

Evaluation of guppy (Poecilia reticulata Peters) immunization against Tetrahymena sp. by enzyme-linked immunosorbent assay (ELISA).

Analysis of the effectiveness of guppy (Poecilia reticulata Peters) immunization based on measurements of antibody (Ab) titers suffers from a shortage of reagents that can detect guppy antibodies (Abs). To overcome this problem, we immunized mice with different preparations of guppy immunoglobulins (Igs) and used the mouse antisera to develop a quantitative enzyme-linked immunosorbent assay (ELISA). The most efficient immunogen for mouse immunization was guppy Igs adsorbed on protein A/G beads. Antisera from mice boosted with this immunoglobulin (Ig) preparation were highly specific and contained high Ab titers. They immunoreacted in a Western blot with Ig heavy and light chains from guppy serum, and Ig heavy chain from guppy whole-body homogenate. The mouse anti-guppy Ig was applied in an ELISA aimed at comparing the efficiency of different routes of guppy immunization against Tetrahymena: (i) anal intubation with sonicated Tetrahymena (40,000 Tetrahymena/fish in a total volume of 10 µL) mixed with domperidon, deoxycholic acid and free amino acids (valine, leucine, isoleucine, phenylalanine and tryptophan), or (ii) intraperitoneal (i.p.) injection of sonicated Tetrahymena in complete Freund's adjuvant (15,000 Tetrahymena/fish in total a volume of 20 µL). Negative control fish were anally intubated with the intubation mixture without Tetrahymena, or untreated. ELISA measurement of anti-Tetrahymena Ab titer revealed a significantly higher level of Abs in i.p.-immunized guppies, compared to the anally intubated and control fish. In addition, the efficiency of immunization was tested by monitoring guppy mortality following (i) i.p. challenge with Tetrahymena (900 Tetrahymena/fish) or (ii) cold stress followed by immersion in water containing 10,000 Tetrahymena/mL. Fish mortality on day 14 post-Tetrahymena infection by i.p. injection exceeded 50% in the control and anally intubated fish, compared to 31% in i.p.-immunized fish. Immunization did not protect from pathogen challenge by immersion. The results suggest a direct correlation between the anti-Tetrahymena Ab response and fish resistance to i.p.-injected Tetrahymena, but not to infection by immersion preceded by cold stress.