Immune effects of bathing European eels in live pathogenic bacteria,Aeromonas hydrophila

The specific and non‐specific immune parameters and protection of European eels (Anguilla anguilla) were evaluated after bathing eels with Aeromonas hydrophila. Two hundred eels were distributed into two equal groups and bathed with Phosphate‐buffered saline (Control group) or 1.0 × 10⁷ cfu mL^?1 A. hydrophila (Test group) for 1 h respectively. Then, eels were bled aseptically from the caudal sinus on 1, 4, 7, 14 and 28 days post treatment. The blood cells were used to evaluate the cellular immunity and the serum was used to determine the titres of specific antibody as well as the activities of superoxide dismutase (SOD) and lysozyme. Eels from both groups were challenged by intraperitoneal injection of 1.0 × 10⁷ cfu of A. hydrophila on 28 and 42 days post bathing. The results show that eels bathed in A. hydrophila significantly (P < 0.05) enhanced the proliferation of different types of blood cells and the serum titres of anti‐A. hydrophila antibody. The Relative Percent Survival (RPS) after challenge on 28 and 42 days post bathing in Test group vs. Control group was 40% and 50% respectively. These results suggest that bathing European eels in A. hydrophila would positively affect specific as well as non‐specific immune parameters and protect against A. hydrophila infection in freshwater farming.     

Phenotypical and genotypical characterization of Shewanella putrefaciens strains isolated from diseased freshwater fish

Between 2007 and 2012, a variety of disease outbreaks most often characterized by skin disorders were observed among different species of freshwater fish in Poland. In most cases, the clinical signs included focally necrotized gills, necrotic skin lesions or ulcers. Internally, haemorrhages, oedematous kidney and abnormal spleen enlargement were generally noted. The disorders were accompanied by increased mortality. Most of the problems concerned cultured common carp Cyprinus carpio L. and rainbow trout Oncorhynchus mykiss (Walbaum). Fish have been examined from a number of these farms, and additionally, the wild and ornamental fish with similar clinical signs of diseases were also tested. Bacteria were isolated consistently from lesions and internal organs. They had characteristic orange-pigmented colonies which grew in pure culture or constituted 55–95% of total bacterial flora. One hundred and eighteen isolates were collected and biochemically identified as Shewanella putrefaciens group, and this was confirmed by sequencing. Challenge tests confirmed the pathogenicity of these bacteria. This is the first report characterizing and describing S. putrefaciens as a pathogen of different species of freshwater fish in Europe.     

Mass marking of stocked European glass eels (Anguilla anguilla) with alizarin red S

This study describes a mass marking method suitable for European glass eel Anguilla anguilla, avoiding osmotic shock and reducing fish stress. During a single day, 360,000 glass eels intended for a stocking operation were bathed into an alizarin red S (ARS) solution, at a density of 346 ind.·l^?1 before being released into a freshwater marsh along with unmarked glass eels. After 15 days, examination of marked and unmarked eels kept in enclosures in the marsh showed 100% of otoliths had been successfully marked and that no excess deaths related to marking process had occurred. Seven months after stocking, results from biometric measurements and otolith analysis, carried out on a sample of 53 elvers caught by electrofishing and belonging to the size class of the 0⁺ cohort, suggest that: (i) the proportions of marked and unmarked remained unchanged, hence suggesting that the marking process had not caused excess mortality, (ii) there was no difference in length, body mass or condition factor between marked and unmarked individuals, demonstrating that marking had caused no harmful effect after 7 months, and (iii) ARS marks in otoliths remained clearly visible. The benefits of this ARS mass marking technique are discussed through a stocking operation.     

Bacterial septicaemia in prerecruit edible crabs,Cancer pagurus L.

Juvenile edible crabs, Cancer pagurus L., were surveyed from Mumbles Head and Oxwich Bay in South Wales, UK, and the number of heterotrophic bacteria and vibrios in the hemolymph was determined. The percentage of crabs with hemolymph containing bacteria was variable over the survey with higher numbers of animals affected in summer than in winter. Post‐moult crabs contained significantly higher numbers of heterotrophic bacteria in the hemolymph than pre‐ and intermoult animals. Crabs with cuticular damage to the gills also had significantly higher numbers of bacteria in the hemolymph. Crabs were found to have a high prevalence of infection by the dinoflagellate, Hematodinium. Such animals had significantly fewer bacteria in the blood in comparison with Hematodinium‐free animals. Of the 463 crabs surveyed, only 3 individuals had hemolymph containing 2000 + CFU mL^?1. Based on 16S rRNA gene sequences, two of these crabs contained a Vibrio pectenicida‐like isolate, while the other had a mixed assemblage of vibrios. Although 59% of the crabs surveyed had culturable bacteria in the hemolymph, the majority only had small numbers (<2000 CFU mL^?1), suggesting that such infections may be of limited importance to the sustainability of the crab fishery in this region.     

Phaeobacter grown in biofilters: a new strategy for the control of Vibrionaceae in aquaculture

Growth, biofilm formation, antagonism and residence time in green seawater tanks maintained under fish rearing conditions of Phaeobacter 27‐4 were studied in commercial biofilters made from plastic, sintered glass and ceramic. Phaeobacter reached 10⁸–10⁹ CFU cm^?3 and formed rosettes in all materials, but a multilayer biofilm was only observed in the ceramic biofilters. In sterile seawater, plastic and ceramic biofilters reduced Vibrio anguillarum and V. splendidus concentration in one‐two Log after 24–48 h, showing 10²–10³ CFU mL^?1. Sintered glass biofilters only inactivated V. anguillarum. In Marine Broth, sintered glass and ceramic biofilters inhibited V. anguillarum growth in two‐three Log, showing 10⁴–10⁵ CFU mL^?1 after 24 h. Plastic biofilters reduced V. anguillarum concentration in one Log after 48 h. V. splendidus growth was only inhibited by sintered glass and ceramic biofilters in one‐two Log, showing 10⁷ CFU mL^?1 after 24 h. Phaeobacter also diminished biofilters colonization by the pathogens, both in seawater and in MB. Phaeobacter residence time in green seawater tanks maintained under fish rearing conditions was longer with sintered glass and ceramic biofilters. The latest showed the lowest detachment and, after 11 days, Phaeobacter (10⁶ bacteria·cm^?3) covered more than 80% of biofilters total culturable bacteria. DGGE profiles showed that Phaeobacter biofilters stabilizes the green seawater bacterial microbiota.     

Growth and mortality of larval anchoveta Engraulis ringens,in northern Chile during winter and their relationship with coastal hydrographic conditions

Larval growth, age, growth effect and instantaneous mortality were estimated in anchoveta, Engraulis ringens, collected biweekly during the austral winter of 2014 in nearshore waters off Bay of Antofagasta (23°41′W–70°30′S), northern Chile. Through measuring standard length (SL) and sagitta microstructure analysis, it was estimated that the growth rate of E. ringens larvae decreased from June (0.85 mm day^?1) to August (0.50 mm day^?1). However, the water temperature was homogeneous during the sampling dates (14.6, 15.2, 14.4, and 14.6°C), suggesting that the decelerating larval growth was not linked to changes in sea temperature. Additionally, larvae with slow growth have larger otoliths compared with conspecifics with fast growth (growth effect). Larval mortality rates tended to decrease until the middle of July (0.18 per day) but increased to 0.25 per day in early August, which coincided with lower food availability (i.e., chlorophyll‐a, 2.7–5.6 mg m^?3) and a high occurrence of smaller larvae (1.58–11.5 mm). Partial least squares analysis indicates low covariance between the biological and oceanographic variables (PLS: 11.71%), suggesting that other factors, such as parental effects, may explain the abrupt decrease in the larval growth rates.     

Growth differences of Japanese eels Anguilla japonica between fresh and brackish water habitats in relation to annual food consumption in the Kojima Bay‐Asahi River system,Japan

To learn about the relationships between feeding and growth of temperate eels in freshwater and brackish water habitats, we analysed 533 yellow‐phase Japanese eels Anguilla japonica collected in both types of habitats in southeastern Japan. Because male eels were very rare in each habitat (FW, N = 1; BW, N = 20), characteristics of female eels were compared between the different habitats. Annual food consumption was evaluated with the consideration of instantaneous food consumption and annual activity period. Stomach fullness index (stomach content weight/body weight) was used as an indicator of instantaneous food consumption. The ratios of number of months with eel catch to those when eel sampling was conducted were used as an indicator of activity period. Female yellow eels tended to be older and slower growing in fresh water (N = 78; age, mean ± SD = 7.9 ± 2.4 years; growth rate, 59.8 ± 14.0 mm year^?1) than in brackish water (N = 229; age, 5.5 ± 1.8 years; growth rate, 90.1 ± 24.4 mm year^?1). Irrespective of sex, yellow eels in brackish water had a higher stomach fullness index and a greater ratio of months with eel catches, indicating greater annual food consumption by brackish water eels. These results indicate that greater annual food consumption contributes to the greater growth rates of Japanese eels in brackish water habitats.     

Mortality and pathology of hybrid catfish,Clarias macrocephalus (Günther) × Clarias gariepinus (Burchell), associated with Edwardsiella ictaluri infection in southern Thailand

Enteric septicaemia of catfish (ESC) caused by Edwardsiella ictaluri is becoming an increasing problem in aquaculture and has been reported worldwide in a variety of fish species. This study reports ESC in hybrid catfish, Clarias macrocephalus (Günther) × Clarias gariepinus (Burchell), cultured in southern Thailand. The bacteria were identified as E. ictaluri by conventional and rapid identification systems, as well as by genetic and phylogenetic characterization. Analysis of 16S rRNA indicated 100% homology to the 16S rRNA sequence of several E. ictaluri strains in GenBank. Plasmid profiles demonstrated 4.0‐ and 5.6‐kb plasmids, compared with the 4.8‐ and 5.6‐kb plasmids in the US isolates, and representative genes of three of the four known pathogenicity islands of US isolates were present. Serologically, lipopolysaccharide (LPS) purified from the Thai isolates was not recognized by a monoclonal antibody against the LPS of US isolates. Fish experimentally infected with E. ictaluri showed 23–100% mortality within 14 days with a 168‐h LD₅₀ of 6.92 × 10⁷ CFU mL^?1 by immersion and a 96‐h LD₅₀ of 1.58 × 10⁶ CFU fish^?1 by intraperitoneal injection. Examination of tissue sections obtained from both naturally and experimentally infected fish indicated that infection of hybrid catfish with E. ictaluri produced lesions in several organs including liver, kidney, spleen, heart and brain. Histopathology findings included cellular necrosis, focal haemorrhage, infiltration of lymphocytes and multifocal granulomatous inflammation in the infected organs.     

Methods of microbial control in marine fish larval rearing: clay‐based turbidity and passive larval transfer

This study focused on methods to reduce bacterial loads in the larval culture tanks of California yellowtail (Seriola lalandi). We conducted two trials to evaluate methods to minimize bacterial loads in the larval rearing water. The first trial examined the use of bentonite clay as a turbidity agent to replace algae in a green water‐type environment. This trial consisted of three treatments: (1) clay with continuous feeding (CCO), (2) clay with batch feedings (CBA) and (3) algae paste with batch feedings (ALG). The results showed that both clay treatments had significantly fewer Vibrio colonies in the water column (CBA – 180 ± 78; CCO – 377 ± 120 CFU mL^?1) than the ALG treatment (5692 ± 2396 CFU mL^?1) after 14 days of culture. Survival was significantly higher in the CCO treatment (14.1 ± 2.6%) than either the CBA (2.3 ± 0.5%) or ALG treatments (2.8 ± 1.5%). The second trial attempted to limit bacterial loading in the larval culture tank by passively transferring the larvae into an adjacent, clean tank at 1, 5 and 9 days post hatch during the first 2 weeks of culture. The results from this trial showed that after 12 days of culture, water in the transfertank had fewer Vibrio colonies (1025 ± 541 CFU mL^?1) than the water in the control tanks (1962 ± 1415 CFU mL^?1). Also, survival was significantly higher among larvae that were transferred (43.9 ± 13.5%) than in the control tanks (23.1 ± 6.3%).     

Effects of salinity on osmoregulation,growth and survival in Asian swamp eel (Monopterus albus) (Zuiew 1793)

The Asian swamp eel Monopterus albus is normally considered a freshwater species, but can also occasionally be found in brackish water. It is an obligate air‐breather with highly reduced gills, making its osmoregulation physiology interesting because the gills normally represent the primary site of osmoregulation in teleosts. Being a popular fish for human consumption, the swamp eel is cultured extensively in freshwater ponds in the Mekong Delta of Vietnam. However, very little is known about its salinity tolerance, which is becoming topical due to the increasing salt‐water intrusions into tropical deltas. We therefore studied how increased salinity affects survival, growth, blood pressure, heart rate, blood osmolality and plasma ions. All eels survived prolonged exposure to 5 and 10 g L^?1, although plasma osmolality increased significantly at 10 g L^?1. Further elevation to 15 and 20 g L^?1 was associated with significantly elevated mortality, with a corresponding increase in plasma osmolality and ion con‐centrations. Our results show that swamp eels thrive in 0–10 g L^?1 with an optimum growth between 0 and 9 g L^?1, indicating that utilization of low saline brackish waters for aquaculture is possible.     

Effects of Lactobacillus pentosus on the growth performance,digestive enzyme and disease resistance of white shrimp,Litopenaeus vannamei (Boone, 1931)

The objective of this study was to evaluate the probiotic properties of lactic acid bacteria (LAB) strains isolated from digestive tract of white shrimp Litopenaeus vannamei. Eighteen LAB colonies were isolated and one bacterium was found capable of producing three extracellular enzymes (protease, cellulose and lipase) simultaneously and exhibited antagonistic activity against shrimp pathogens (Vibrio vulnificus, V. rotiferianus and V. campbellii). The putative probiotic strain AS13 was identified as Lactobacillus pentosus based on 16S rRNA sequencing. The L. vannamei were fed diet containing 0 (control), 10⁶, 10⁷ and 10⁸ CFU g^?1 bacterial cells of AS13 for 28 days. The results showed that supplementation of L. pentosus significantly improved the growth performance and feed utilization in the treated groups over the control. Similarly, digestive enzyme activities were elevated in the intestines of treated groups. Moreover, feeding of supplemented diets containing AS13 significantly reduced the mortality rate caused by pathogenic Vibrio species (V. vulnificus, V. rotiferianus and V. campbellii). Our results indicated L. pentosus AS13 addition at 10⁷ CFU g^?1 can effectively enhance the growth performance, feed utilization, digestive enzymes and disease resistance of L. vannamei in the laboratory condition.     

Cellular and humoral immune responses of Senegalese sole,Solea senegalensis (Kaup), following challenge with two Photobacterium damselae subsp. piscicida strains from different geographical origins

The present study aimed to investigate leucocyte responses to inflammation as well as some innate immune parameters of Senegalese sole, Solea senegalensis, following challenge with two strains of Photobacterium damselae subsp. piscicida belonging to the European and Japanese clones described for this bacterium. Pathogenicity assays were performed to assess the virulence of each Photobacterium damselae subsp. piscicida strain for sole. Subsequently, fish were intraperitoneally injected with phosphate‐buffered saline (control) or two concentrations (2 × 10² and 2 × 10⁶ CFU mL^?1) of each bacterial strain and sampled after 6 and 24 h. Results showed that the European isolate induces a higher degree of response than the Japanese strain. While blood neutrophilia and monocytosis correlated well with the increase in neutrophil and macrophage numbers in the peritoneal cavity, fish infected with the European isolate presented higher peritoneal cell numbers than fish challenged with the Japanese strain. In addition, alternative complement pathway activity and respiratory burst of head kidney leucocytes increased significantly in fish infected with the European isolate. The enhanced innate immune response displayed by Senegalese sole challenged with the European isolate is probably due to the higher degree of virulence presented by this Photobacterium damselae subsp. piscicida strain.     

Aeromonas jandaei and Aeromonas veronii caused disease and mortality in Nile tilapia,Oreochromis niloticus (L.)

Diseases caused by motile aeromonads in freshwater fish have been generally assumed to be linked with mainly Aeromonas hydrophila while other species were probably overlooked. Here, we identified two isolates of non‐A. hydrophila recovered from Nile tilapia exhibiting disease and mortality after exposed to transport‐induced stress and subsequently confirmed their virulence in artificial infection. The bacterial isolates were identified as Aeromonas jandaei and Aeromonas veronii based on phenotypic features and homology of 16S rDNA. Experimental infection revealed that the high dose of A. jandaei (3.7 × 10⁶ CFU fish^?1) and A. veronii (8.9 × 10⁶ CFU fish^?1) killed 100% of experimental fish within 24 h, while a 10‐fold reduction dose killed 70% and 50% of fish, respectively. When the challenge dose was reduced 100‐fold, mortality of the fish exposed to A. jandaei and A. veronii decreased to 20% and 10%, respectively. The survivors from the latter dose administration were rechallenged with respective bacterial species. Lower mortality of rechallenged fish (0%–12.5%) compared to the control groups receiving a primary infection (37.5%) suggested that the survivors after primary infection were able to resist secondary infection. Fish exposed to either A. jandaei or A. veronii exhibited similar clinical signs and histological manifestation.     

Commercial Bacillus probiotic supplementation of rainbow trout (Oncorhynchys mykiss) and brown trout (Salmo trutta): growth,immune responses and intestinal morphology

Probiotic administration is associated with the enhancement of host resistance to environmental and nutritional stressors, improving survival and growth rates. This study was carried out to evaluate the effects of dietary supplementation of Bacillus subtilis and Bacillus cereus toyoi on growth performance, innate immune responses and gut morphology of two trout species feeding a commercial diet high in soybean meal. A commercial probiotic (4.2 × 10⁹ CFU g^?1 of additive) was supplemented to the experimental diets at 0% (control), 0.03% (P₁; 6 × 10³ CFU g^?1 of diet) or 0.06% (P₂; 1.5 × 10⁶ CFU g^?1 of diet) and fed to brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) for 9 and 20 weeks respectively. Rainbow trout showed significantly better growth performance than brown trout, regardless of the dietary treatment. No effect of dietary probiotic supplementation was detected on growth performance, body composition or innate immune parameters (plasma lysozyme, alternative complement and peroxidase activities). In both species, after 9 weeks, intestinal lamina propria and submucosa were widened, with increased presence of inflammatory cells, significantly higher in groups fed probiotics. This inflammatory process, with villi and enterocytes noticeably damaged compared with the control group, was more pronounced in brown trout. Under the current trial conditions, the B. subtilis + B. cereus toyoi had no positive impact in either trout species, on the contrary a harmful effect was observed.     

The influence of phytoestrogens and oestradiol-17β on growth and sex determination in the European eel (Anguilla anguilla)

Under aquaculture conditions, European eels (Anguilla anguilla) produce a high percentage of males (80–95%) that normally stop growing at 100–200 g. Females continue to grow to 500–750 g and obtain higher market value. Therefore, increasing the percentage of females in a population would be beneficial to the culture of eels. The present study was carried out in order to examine the effect of oestradiol and phytooestrogens on sex differentiation and growth rate of eels. Juvenile European eels with undifferentiated gonads were fed pellets containing oestradiol‐17β (E2) or phytooestrogens for 100–150 days. Feeding E2 resulted in 50–61% increase in body weight compared with the control. Oestradiol‐17β and phytooestrogens both elevated significantly the percentage of females in the population. Feeding E2 at 20 mg kg^?1 feed resulted in 70% females, while lower concentration of E2 (2 mg kg^?1) resulted in only 30% after 100 days (Experiment 1). The same dose given for 150 days (Experiment 2) resulted in 88% females, indicating that both, the concentration and duration of E2 treatments had a significant effect on sex differentiation. Fish fed genistein at 2 mg kg^?1 for 100 days, resulted in 55% of females, but at a higher dose of 20 mg kg^?1 there were only 15% females. These results demonstrate that phytooestrogens can be used as alternatives to gonadal steroids for sex manipulation in eels, but the optimal concentrations and duration are still to be determined.     

Genotypic diversity among Shewanella spp. collected from freshwater fish

Different Shewanella species are isolated both from healthy and from diseased fish. To date, contemporary methods do not provide sufficient insight to determine species and detail differentiation between tested strains. Bacteria isolated from cultured (n = 33), wild (n = 12) and ornamental (n = 6) fish, as well as several reference strains, were tested by 16S rRNA gene sequencing, ERIC‐PCR and pulsed‐field gel electrophoresis (PFGE) assays. Our study indicates that isolates collected from freshwater fish were genetically diverse. Based on 16S rRNA gene sequences, bacteria were clustered into groups S. putrefaciens, S. xiamenensis and S. oneidensis. Some isolates were classified only to genus Shewanella; thus, 16S rRNA gene analyses were not enough to determine the species. ERIC‐PCR revealed 49 different genotype profiles indicating that the method might be useful for differentiation of Shewanella isolates irrespectively to species identification, contrary to PFGE which is not suitable for Shewanella typing.     

The yellow European eel (Anguilla anguilla L.) may adopt a sedentary lifestyle in inland freshwaters

Abstract – We analysed the movements of the growing yellow phase using a long-term mark–recapture programme on European eels in a small catchment (the Frémur, France). The results showed that of the yellow eels (>200 mm) recaptured, more than 90% were recaptured at the original marking site over a long period before the silvering metamorphosis and downstream migration. We conclude that yellow European eels >200 mm may adopt a sedentary lifestyle in freshwater area, especially in small catchment.     

Isolation of a novel polyomavirus,related to Japanese eel endothelial cell‐infecting virus,from marbled eels,Anguilla marmorata (Quoy & Gaimard)

Marbled eels, Anguilla marmorata (Quoy & Gaimard), cultured in Taiwan exhibited haemorrhage and mortality in January 2012. The severely diseased eels bled from the gills and showed congestion of the central venous sinus of the gill filaments and haemorrhage throughout the body similar to viral endothelial cell necrosis of eel. In this study, a novel polyomavirus (AmPyV) was isolated from the diseased eels using the AMPF cell line established from the pectoral fin of healthy marbled eels. AmPyV was found to encode a long T‐antigen orthologous gene. Phylogenetic analysis showed that AmPyV was closely related to Japanese eel endothelial cell‐infecting virus. PCR assays revealed AmPyV infection throughout the systemic organs. AmPyV proliferated in the AMPF, EK‐1 and EO‐2 cells at temperatures 25–30 °C, and the progeny virus yields were 10^7.0, 10^7.4 and 10^7.7 TCID₅₀ mL^?1, respectively. The purified virions were icosahedral particles, 70–80 nm in diameter. No clinical signs or mortality was observed among the eels injected with the virus; however, the virus was reisolated from the brain, eyes, kidneys, fins and gills of infected eels 2 month after injection. Our results suggest that AmPyV exhibits a latent infection. Pathogen of the disease needs to study further.     

Studies on bacterial pathogens isolated from diseased torafugu (Takifugu rubripes) cultured in marine industrial recirculation aquaculture system in Shandong Province,China

In spring of 2011, an epidemic outbreak of torafugu with high mortality occurred in an aquafarm with marine industrial recirculation aquaculture system (MIRAS) in Yantai, Shandong Province, China. The diseased fish showed anorexia, haemorrhaging and festering fin and skin and swelling internal organs. Forty‐five dominant bacterial isolates were obtained from the diseased fish, and were found to belong to 12 species according to 16S rRNA gene sequences. One strain from each species was selected to test the pathogenicity, and five strains were showed to be virulent to zebrafish. Whereas Enterovibrio nigricans Fr42 was highly virulent with the LD₅₀ of 7.8 × 10⁴ CFU g^?1, Photobacterium swingsii Fr23, Vibrio owensii Fr40, V. harveyi Fr51 and V. rotiferianus Fr71 were moderately virulent with the LD₅₀ of 1.7 × 10⁶ to 8.4 × 10⁶ CFU g^?1. Both the bacteria and their extracellular products of the five strains were found to show phospholipase, caseinase, gelatinase, amylase and/or lipase activities. The production of N‐acyl homoserine lactones (AHLs) of the five strains was detected by three different AHLs biosensors, and three of them were found to produce AHLs by at least one kind of biosensor. This is the first study describing various opportunistic bacterial pathogens of fish cultured in MIRAS in China.     

Preliminary assessment of in vivo safety of potentially probiotic lactic acid bacteria for American bullfrog culture

The effect of the administration of beneficial lactic acid bacteria (LAB): Lactococcus lactis CRL 1584, L. lactis CRL 1827, Enterococcus gallinarum CRL 1826 and combined CRL 1584+ CRL 1826 on the development of bullfrog embryos from the hatching stage until 31 days (tadpole) was evaluated. In vitro assays indicated that 10³, 10⁶ and 10⁹ CFU mL^?1 single LAB strains remained viable until 24 h in 10% Ringer. Around 10⁹ CFU mL^?1 LAB (individually and combined) were used in an experimental design built to evaluate their effect when administrated at different intervals (three 7‐day cycles with 5‐day rest periods in between) to embryos until day 31. The highest potentially beneficial population (LAB) numbers were detected in the LAB‐treated groups. All the LAB‐treatments increased it and were significantly higher than the controls. Although the highest, potentially pathogenic, population (Red‐Leg Syndrome‐RLS‐related pathogens) numbers were detected in the control and the lowest in the CRL 1584+ CRL 1826‐treated group, they did not differ significantly. Stereoscopic studies showed no malformations in any LAB‐treated group and all the specimens reached the same stage of their biological cycle with a survival >94%. The histological structure of target organs for RLS‐associated pathogens (intestine and skin) and stomach was not affected and the spleen was developed. Only the LAB‐treated groups showed microorganisms associated with the intestinal mucus, without inflammatory reaction in the lamina propria. This is the first report on the evaluation of the absence of adverse effects after LAB administration to bullfrog embryos using indigenous microorganisms.