Antimicrobial susceptibility of Peruvian Yersinia ruckeri isolates to oxytetracycline and florfenicol using MIC testing.

Yersiniosis, caused by Yersinia ruckeri, has become the most common disease in farmed rainbow trout Oncorhynchus mykiss in Peru, affecting Puno and Junín Regions, important aquaculture areas in the country. Florfenicol (FLO) and oxytetracycline (OXY) are the antimicrobials most frequently used to mitigate losses attributed to this pathogen. This study presents an analysis of the susceptibility patterns of 60 Y. ruckeri isolates (30 isolates each from Puno and Junín), including the type strain CECT 4319T and the strains CECT 955 and CECT 956, against FLO and OXY. Minimum inhibitory concentrations (MICs) were determined following the guideline for standard broth dilution method published by the Clinical and Laboratory Standards Institute. MIC results ranged from 4.0 to 8.0 µg ml-1 for FLO and 0.5 to 4.0 µg ml-1 for OXY. Normalized resistance interpretation (NRI) analysis identified epidemiological cut-off values of ≤16.0 µg ml-1 for FLO and ≤4.0 µg ml-1 for OXY. All Peruvian isolates, including the collection strains, were categorized as wild-type for both antimicrobials. Even though the number of Y. ruckeri isolates with MIC values of 8 µg ml-1 for FLO is more than double in Puno than in Junín (15 vs. 7 isolates), the NRI analysis showed the same epidemiological cutoff of 16 µg ml-1; while for OXY, it was 4.0 µg ml-1 for Puno and 2.0 µg ml-1 for Junín. This study establishes the basis for monitoring susceptibility to FLO and OXY in new Y. ruckeri isolates in Peruvian rainbow trout farming.

Characterization of Tenacibaculum maritimum isolated from diseased salmonids farmed in Chile reveals high serological and genetic heterogeneity.

The diversity of Tenacibaculum maritimum in Chile remains poorly understood, particularly in terms of antigenic and genetic diversity. This information is crucial for the future development of a vaccine against tenacibaculosis and would increase understanding of this important fish pathogen. With this aim, the biochemical, antigenic, and genetic characteristics were analysed for 14 T. maritimum isolates, recovered from diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) farmed in Chile between 1998 and 2022. Biochemical analysis showed a homogeneity among all the Chilean T. maritimum isolates and all four other strains included for comparison purposes. Serological characterization using dot-blot assaying revealed antigenic heterogeneity with the use of unabsorbed antisera. The majority of isolates showed cross-reactions, identifying three main serological patterns. When the PCR-based serotyping scheme was performed, the existence of antigenic heterogeneity was confirmed. Four Atlantic salmon isolates were 4-0; and most isolates, including the rainbow trout isolate, were 3-1 (n = 9). A turbot (Scophthalmus maximus) isolate was 1-0. Using an existing Multilocus Sequence Typing system, two newly identified sequence types (ST193 and ST198) in the database were detected. ST193 encompassed nine isolates obtained from Atlantic salmon and rainbow trout, while ST198 regrouped four isolates, all retrieved from diseased Atlantic salmon in 2022. These findings highlight significant antigenic and genetic diversity among the Chilean isolates. This information is useful for epizootiology and the selection of suitable candidate strain(s) for vaccine development against tenacibaculosis caused by T. maritimum in Chilean salmon farming.

Ancestry and genetic structure of resident and anadromous rainbow trout (Oncorhynchus mykiss) in Argentina.

Since the first introduction from North America more than a century ago, rainbow trout (Oncorhynchus mykiss) have rapidly established self-sustaining populations in major river basins of Patagonia. Many generations later, only the freshwater resident life history is expressed in the Chubut and Negro rivers of northern Argentinian Patagonia, whereas both the resident and anadromous life histories are found in the Santa Cruz River of southern Argentina. Despite previous studies that have tried to identify the sources of these introduced populations, uncertainty still exists. Here we combined data from many single-nucleotide polymorphisms and microsatellite loci in O. mykiss populations from Argentina and North America to evaluate putative source populations, gene flow between Argentinian river basins, and genetic diversity differences between Argentinian and North American populations. We found that populations from northern and southern Patagonia are highly differentiated and have limited gene flow between them. Phylogeographic analysis also confirmed that they have separate origins, with the northern populations most closely related to the domesticated rainbow trout strains that are raised worldwide and the Santa Cruz River populations most closely related to North American populations from California and Oregon that have an anadromous component. In addition, fish with different life histories in the Santa Cruz River were found to constitute a single interbreeding population. No evidence was found of reduced genetic variation in introduced rainbow trout, suggesting multiple contributing sources. In spite of these advances in understanding, significant questions remain regarding the origins and evolution of the introduced O. mykiss in Patagonia.

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.

Skin Microbial Community Associated to Strawberry Disease in Farmed Rainbow Trout (Oncorhynchus mykiss Walbaum, 1792).

Aquaculture plays a crucial role in addressing the growing global demand for food. However, diseases associated with intensive aquaculture practices, especially those affecting the skin, can present significant challenges to both fish health and the industry as a whole. Strawberry disease (SD), also known as red-mark syndrome, is a persistent and non-lethal skin condition observed in Rainbow Trout (Oncorhynchus mykiss) in the United States and various European countries. SD is a nonlethal skin condition of an unclear etiology that affects rainbow trout reared in freshwater close to the harvest period. We used a RNA-based approach to examine active microbiota in the SD skin lesions and compared to non-injured skin. Our results, based on using 16S rRNA gene next-generation sequencing, showed that the skin microbiota was dominated by the phyla Firmicutes, Proteobacteria, and Actinobacteria. The comparisons of the skin microbiota between injured and non-injured samples showed differences in the alpha diversity (Fisher index) and beta diversity metrics (ANOSIM). At the genus level, both Pseudomonas and Candidatus Midichloria were highlighted as the most abundant taxa detected in samples obtained from fish affected with strawberry diseases. In contrast, the most abundant taxa in non-injured skin were Escherichia-Shigella, Streptococcus, and Pseudoalteromonas. In conclusion, our study on SD revealed distinct differences in the microbiota composition between skin lesions and non-injured skin. This is the first description of microbiota associated with SD-injured skin samples using an RNA approach.

Assessing the Effectiveness of Sex-Linked Molecular Markers to Identify Neomale Breeders for the Production of All-Female Progenies of Rainbow Trout.

A cultured stock of masculinized rainbow trout was diagnosed with Y-linked markers (sdY and OmyY1) aiming to detect neomales before their use at the production level. To achieve a reliable diagnosis, the following steps were considered: (1) PCR amplification of the housekeeping ß-actin gene to determine the DNA quality of samples, (2) validation of the Y-linked markers by their PCR amplification in male and female samples with known sex, and (3) molecular sexing of the masculinized juveniles based on male-specific (XY genotype) and neomale-specific (XX genotype) PCR product band patterns visualized on agarose gel. The validity and concordance of the markers were assessed. The housekeeping gene identified samples with negative PCR amplification revealing a poor DNA quality. The OmyY1 marker presented a more distinctive PCR product band pattern between males and females than the sdY marker and identified a higher proportion of true males (sensitivity = 1.0 and 0.91, respectively). The OmyY1 marker accurately identified 105 neomales of the 198 masculinized individuals on account their consistent and distinctive PCR product band pattern. Among both markers, there was a medium high positive concordance (γ index = 0.7). It is concluded that the OmyY1 marker shows the best performance to reliably detect neomales, a step that is essential to have certified breeders for the production of all-female progenies in fish farming.

Tenacibaculum dicentrarchi produce outer membrane vesicles (OMV) that are associated with the cytotoxic effect in rainbow trout head kidney macrophages.

Tenacibaculum dicentrarchi is the second most important pathogen in Chilean salmon farming. This microorganism causes severe skin lesions on the body surface of farmed fish. The bacterium can also adhere to surfaces and form biofilm, survive in fish skin mucus, and possess different systems for iron acquisition. However, the virulence mechanisms are still not fully elucidated. Outer membrane vesicles (OMV) are nanostructures released by pathogenic Gram-negative bacteria during growth, but none has been described yet for T. dicentrarchi. In this study, we provide the first reported evidence of the fish pathogen T. dicentrarchi producing and releasing OMV from 24 h after incubation, increasing thereafter until 120 h. Analyses were conducted with T. dicentrarchi TdCh05, QCR29, and the type strain CECT 7612T . The OMV sizes, determined via scanning electron microscopy, ranged from 82.25 nm to 396.88 nm as per the strain and incubation time point (i.e., 24 to 120 h). SDS-PAGE revealed that the number of protein bands evidenced a drastically downward trend among the T. dicentrarchi strains. In turn, the OMV shared five proteins (i.e., 22.2, 31.9, 47.7, 56.3, and 107.1 kDa), but no protein pattern was identical. A heterogeneous amount of protein, RNA, and DNA were obtained, depending on the time at which OMV were extracted. Purified OMV were biologically active and induced a cytotoxic effect in macrophage-enriched cell cultures from rainbow trout (Oncorhynchus mykiss) head kidneys. This is the first step towards understanding the role that OMV could play in the pathogenesis of T. dicentrarchi.

Histological examination of the young Oncorhynchus mykiss intestines using the feed with chelated and probiotic supplements / Exame histológico do intestino jovem de Oncorhynchus mykiss utilizando a ração com suplementos quelatados e probióticos

Macronutrient and micronutrient deficiencies in the fish diet can affect fish growth rate, ability to resist disease, and fillet quality. Determination of effective dose of developed feed additives consisting of chelate compounds of biogenic elements and probiotic preparation based on Bacillus subtilis. Young rainbow trout were divided into four groups consisting of 100 fish each. The control group received standard food; the experimental groups received food with chelates and probiotics in different concentrations. At the end of the experiment (30 days), the middle intestine histology and morphometric parameters were studied. The use of chelated compounds alone affected the state of the intestinal villi and the infiltration of the epithelium by lymphocytes. The addition of a probiotic had a positive effect on the migration of immune cells in the intestinal villi; however, the probiotic in high concentrations contributed to a decrease in the number and area of goblet cells. Synergistic effects of chelated compounds and probiotic preparations on the morphometric parameters of the medium intestine were observed. In the proper doses, the studied feed additives might be recommended for rainbow trout farming under industrial aquaculture conditions.

As deficiências de macronutrientes e micronutrientes na dieta dos peixes podem afetar a taxa de crescimento dos peixes, a capacidade de resistir a doenças e a qualidade do filé. Determinação da dose efetiva de aditivos alimentares desenvolvidos constituídos por compostos quelatos de elementos biogênicos e preparação probiótica à base de Bacillus subtilis. As trutas arco-íris jovens foram divididas em quatro grupos de 100 peixes cada. O grupo controle recebeu ração padrão; os grupos experimentais receberam alimentos com quelatos e probióticos em diferentes concentrações. Ao final do experimento (30 dias), a histologia do intestino médio e os parâmetros morfométricos foram estudados. O uso de compostos quelados sozinho afetou o estado das vilosidades intestinais e a infiltração do epitélio por linfócitos. A adição de um probiótico teve um efeito positivo na migração de células imunes nas vilosidades intestinais; entretanto, o probiótico em altas concentrações contribuiu para a diminuição do número e da área das células caliciformes. Efeitos sinérgicos de compostos quelados e preparações probióticas sobre os parâmetros morfométricos do intestino médio foram observados. Nas doses adequadas, os aditivos alimentares estudados podem ser recomendados para a criação de trutas arco-íris em condições de aquacultura industrial.

Chitosan Microparticles Enhance the Intestinal Release and Immune Response of an Immune Stimulant Peptide in Oncorhynchus mykiss.

The aquaculture industry is constantly increasing its fish production to provide enough products to maintain fish consumption worldwide. However, the increased production generates susceptibility to infectious diseases that cause losses of millions of dollars to the industry. Conventional treatments are based on antibiotics and antivirals to reduce the incidence of pathogens, but they have disadvantages, such as antibiotic resistance generation, antibiotic residues in fish, and environmental damage. Instead, functional foods with active compounds, especially antimicrobial peptides that allow the generation of prophylaxis against infections, provide an interesting alternative, but protection against gastric degradation is challenging. In this study, we evaluated a new immunomodulatory recombinant peptide, CATH-FLA, which is encapsulated in chitosan microparticles to avoid gastric degradation. The microparticles were prepared using a spray drying method. The peptide release from the microparticles was evaluated at gastric and intestinal pH, both in vitro and in vivo. Finally, the biological activity of the formulation was evaluated by measuring the expression of il-1ß, il-8, ifn-γ, Ifn-α, and mx1 in the head kidney and intestinal tissues of rainbow trout (Oncorhynchus mykiss). The results showed that the chitosan microparticles protect the CATH-FLA recombinant peptide from gastric degradation, allowing its release in the intestinal portion of rainbow trout. The microparticle-protected CATH-FLA recombinant peptide increased the expression of il-1ß, il-8, ifn-γ, ifn-α, and mx1 in the head kidney and intestine and improved the antiprotease activity in rainbow trout. These results suggest that the chitosan microparticle/CATH-FLA recombinant peptide could be a potential prophylactic alternative to conventional antibiotics for the treatment of infectious diseases in aquaculture.

New Evidence for the Role of Pituitary Adenylate Cyclase-Activating Polypeptide as an Antimicrobial Peptide in Teleost Fish.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is a multifunctional neuropeptide that is widely distributed and conserved across species. We have previously shown that in teleost fish, PACAP not only possesses direct antimicrobial properties but also immunomodulatory effects against the bacterial pathogens Flavobacterium psychrophilum and Pseudomonas aeruginosa using in vitro and in vivo experiments. These previous results suggest PACAP can be used as an alternative to antibiotics to prevent and/or treat bacterial infections in the aquaculture industry. To accomplish this goal, more studies are needed to better understand the effect of PACAP on pathogens affecting fish in live infections. In the present study, the transcripts PACAP, PRP/PACAP, and VPAC2 receptor were examined in rainbow trout (Oncorhynchus mykiss) naturally infected with Yersinia ruckeri, which exhibited an increase in their expression in the spleen when compared to healthy fish. Synthetic Clarias gariepinus PACAP-38 has direct antimicrobial activity on Y. ruckeri and inhibits up to 60% of the bacterial growth when the peptide is at concentrations between 50 and 100 µM in TSB. The growth inhibition increased up to 90% in the presence of 12.5 µM of PACAP-38 when salt-free LB broth was used instead of TSB. It was also found to inhibit Y. ruckeri growth in a dose-dependent manner when the rainbow trout monocyte/macrophage-like cell line (RTS11) was pre-treated with lower concentrations of the peptide (0.02 and 0.1 µM) before going through infection. Differential gene expression was analyzed in this in vitro model. Overall, the results revealed new evidence to support the role of PACAP as an antimicrobial and immunomodulatory peptide treatment in teleosts.

Human Health Risk Assessment for Exposure to Heavy Metals via Dietary Intake of Rainbow Trout in the Influence Area of a Smelting Facility Located in Peru.

Abandoned mining-metallurgical sites can significantly impact the environment and human health by accumulating heavy metals in aquatic ecosystems. The water in the sub-basin near an abandoned smelting facility in the city of La Oroya, Peru, is primarily used for pisciculture. The objective of this study was to assess the risk to human health from exposure to heavy metals via dietary intake of rainbow trout (Oncorhynchus mykiss) in the influence area of a smelting facility located in the central Andean region of Peru. The bioconcentration factor, biosediment accumulation factor, and consumption risks were evaluated using the Monte Carlo method. The results showed that the concentrations of elements (Zn > Pb > Cu > As) in rainbow trout muscle did not exceed the maximum limit (ML). However, the water significantly exceeded the ML for Pb in all sectors and As in the lower and middle parts of the river. The concentration of Pb in sediments also significantly exceeded the ML in the upper and lower parts of the river. Consequently, rainbow trout consumption in the study area presents risks to human health due to the bioaccumulation of heavy metals, with a 1.27% carcinogenic risk in samples from the lower part of the river.

Use of antioxidants and time of cold storage: effects over viability parameters and enzymatic levels in semen of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) / Uso de antioxidantes e tempo de armazenamento a frio: efeitos sobre parâmetros de viabilidade e níveis enzimáticos no sêmen da truta arco-íris (Oncorhynchus mykiss, Walbaum, 1792)

The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.(AU)

O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.(AU)

Use of antioxidants and time of cold storage: effects over viability parameters and enzymatic levels in semen of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) / Uso de antioxidantes e tempo de armazenamento a frio: efeitos sobre parâmetros de viabilidade e níveis enzimáticos no sêmen da truta arco-íris (Oncorhynchus mykiss, Walbaum, 1792)

The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.

O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.

Use of antioxidants and time of cold storage: effects over viability parameters and enzymatic levels in semen of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792)

Abstract The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.

Resumo O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.

Use of antioxidants and time of cold storage: effects over viability parameters and enzymatic levels in semen of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) / Uso de antioxidantes e tempo de armazenamento a frio: efeitos sobre parâmetros de viabilidade e níveis enzimáticos no sêmen da truta arco-íris (Oncorhynchus mykiss, Walbaum, 1792)

Abstract The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.

Resumo O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.

Detection of pathogenic Aeromonas hydrophila from two rainbow trout (Oncorhynchus mykiss) farms in Peru.

Fish farms are one of the principal food industries located in peri-urban and rural communities that use available resources to ensure the quality of their products. However, trout can suffer from bacterial infections affecting the sector and being a key component of human health risk. We aimed to identify and characterize Enterobacteriaceae in 46 trout (Oncorhynchus mykiss) in two fish farms in Lima, Peru. Adult trouts older than seven weeks (> 200 grams weight) were included. Cultures were performed in duplicate (n=192 trials) with swabs from the squamous surface and visceral cavity. The isolates were identified with the Vitek® 2 Compact system, and the minimum inhibitory concentrations (MIC) were interpreted with the CLSI VET 03-A guide. At El Molino and El Angelito fish farms, 66 (68.8%) and 57 (59.4%) isolates were obtained. The most frequently isolated species were Escherichia coli (56.8%), Proteus sp. (4.2%) and Klebsiella pneumoniae (2.6%). E. coli was present in all sampling areas, and Aeromonas hydrophila was only present in one open viscera sample at El Angelito fish farm. A. hydrophila showed antibiotic resistance to Ampicillin/Sulbactam (≥32 MIC), Oxytetracycline (>8 MIC), Imipenem (8 MIC), Levofloxacin (>8 MIC), and Ceftazidime (≥64 MIC). Our results suggest the presence of multi-resistant A. hydrophila in O. mykiss. Further studies are needed to understand the developmental context of A. hydrophila, which is crucial to the food industry, aquaculture and public health.

As pisciculturas são uma das principais indústrias alimentícias localizadas em comunidades peri-urbanas e rurais que utilizam os recursos disponíveis para garantir a qualidade de seus produtos. No entanto, a truta pode sofrer de infecções bacterianas que afetam o setor e são um componente chave do risco para a saúde humana. Nosso objetivo foi identificar e caracterizar Enterobacteriaceae em 46 trutas (Oncorhynchus mykiss) em duas pisciculturas em Lima, Peru. Foram incluídas trutas adultas com mais de sete semanas (> 200 gramas de peso). As culturas foram realizadas em duplicata (n=192 tentativas) com swabs da superfície escamosa e da cavidade visceral. Os isolados foram identificados com o sistema Vitek® 2 Compact, e as concentrações inibitórias mínimas (CIM) foram interpretadas com o guia CLSI VET 03-A. Nas pisciculturas El Molino e El Angelito foram obtidos 66 (68,8%) e 57 (59,4%) isolados. As espécies mais frequentemente isoladas foram Escherichia coli (56,8%), Proteus sp. (4,2%) e Klebsiella pneumoniae (2,6%). E. coli estava presente em todas as áreas de amostragem, e Aeromonas hydrophila estava presente apenas em uma amostra de vísceras abertas na piscicultura El Angelito. A. hydrophila mostrou resistência a antibióticos para Ampicilina/Sulbactam (≥32 MIC), Oxitetraciclina (>8 MIC), Imipenem (8 MIC), Levofloxacina (>8 MIC) e Ceftazidima (≥64 MIC). Nossos resultados sugerem a presença de A. hydrophila multirresistente em O. mykiss. Mais estudos são necessários para entender o contexto de desenvolvimento de A. hydrophila, que é crucial para a indústria de alimentos, aquicultura e saúde pública.

CXCL9-11 chemokines and CXCR3 receptor in teleost fish species.

The coordinated migration of immune cells from lymphoid organs to in or out of the bloodstream, and towards the site of infection or tissue damage is fundamental for an efficient innate and adaptive immune response. Interestingly, an essential part of this movement is mediated by chemoattractant cytokines called chemokines. Although the nature and function of chemokines and their receptors are well documented in mammals, much research is needed to accomplish a similar level of understanding of the role of chemokines in fish immunity. The first chemokine gene identified in teleosts (rainbow trout, Oncorhynchus mykiss) was CK1 in 1998. Since then, the identification of fish chemokine orthologue genes and characterization of their role has been more complex than expected, primarily because of the whole genome duplication processes occurring in fish, and because chemokines evolve faster than other immune genes. Some of the most studied chemokines are CXCL9, CXCL10, CXCL11, and the CXCR3 receptor, all involved in T cell migration and in the induction of the T helper 1 (Th1) immune response. Data from the zebrafish and rainbow trout CXCL9-11/CXCR3 axis suggest that these chemokines and the receptor arose early in evolution and must be present in most teleost fish. However, the pieces of knowledge also indicate that different numbers of gene copies can be present in different species, with distinct regulatory expression mechanisms and probably, also with different roles, as the differential expression in fish tissues suggest. Here, we revised the current knowledge of the CXCL9-11/CXCR3 axis in teleost fishes, identifying the gaps in knowledge, and raising some hypotheses for the role of CXCL9, CXCL10 CXCL11, and CXCR3 receptor axis in fish, which can encourage further studies in the field.

Protective Effect of [Cu(NN1)2](ClO4) Complex in Rainbow Trout Challenged against Flavobacterium psychrophilum.

Previously, we reported an in vitro evaluation regarding antibacterial effects against F. psychrophilum by a new Cu (I) complex, [Cu(NN1)2](ClO4). This study presents the results of an in vivo evaluation of [Cu(NN1)2](ClO4) added as a dietary supplement against F. psychrophilum in rainbow trout. The results showed that the administration of [Cu(NN1)2](ClO4) at 29 and 58 µg/g of fish for 15 days does not affect the growth of rainbow trout. On the other hand, the amount of copper present in the liver, intestine, and muscle of rainbow trout was determined. The results showed that the amount of copper in the liver, when compared between treated fish and control fish, does not change. While, in the intestine, an increase in the fish fed at 58 µg/g of fish was observed. In muscle, a slight decrease at 29 µg/g was obtained. Additionally, copper concentrations in the pond water after 15 days of feeding with the [Cu(NN1)2](ClO4) complex showed the highest levels of copper. Finally, the effect of the administration of [Cu(NN1)2](ClO4) for 15 days at 58 µg/g of fish was evaluated against F. psychrophilum, where a 75% survival was obtained during 20 days of challenge.

Selenium Nanoparticle-Enriched and Potential Probiotic, Lactiplantibacillus plantarum S14 Strain, a Diet Supplement Beneficial for Rainbow Trout.

Lactic acid bacteria (LAB), obtained from rainbow trout (Oncorhynchus mykiss) intestine, were cultured in MRS medium and probiotic candidates. Concurrently, producers of elemental selenium nanoparticles (Se0Nps) were selected. Probiotic candidates were subjected to morphological characterization and the following tests: antibacterial activity, antibiotic susceptibility, hemolytic activity, catalase, hydrophobicity, viability at low pH, and tolerance to bile salts. Two LAB strains (S4 and S14) satisfied the characteristics of potential probiotics, but only strain S14 reduced selenite to biosynthesize Se0Nps. S14 strain was identified, by 16S rDNA analysis, as Lactiplantibacillus plantarum. Electron microscopy showed Se0Nps on the surface of S14 cells. Rainbow trout diet was supplemented (108 CFU g-1 feed) with Se0Nps-enriched L. plantarum S14 (LABS14-Se0Nps) or L. plantarum S14 alone (LABS14) for 30 days. At days 0, 15, and 30, samples (blood, liver, and dorsal muscle) were obtained from both groups, plus controls lacking diet supplementation. Fish receiving LABS14-Se0Nps for 30 days improved respiratory burst and plasmatic lysozyme, (innate immune response) and glutathione peroxidase (GPX) (oxidative status) activities and productive parameters when compared to controls. The same parameters also improved when compared to fish receiving LABS14, but significant only for plasmatic and muscle GPX. Therefore, Se0Nps-enriched L. plantarum S14 may be a promising alternative for rainbow trout nutritional supplementation.

Comparative polyphasic characterization of Weissella strains isolated from beaked whale and rainbow trout (Oncorhynchus mykiss): confirmation of Weissella ceti sp. nov. and description of the novel Weissella tructae sp. nov. isolated from farmed rainbow trout.

The weissellosis agent bacterium (WS08T = CBMAI 2730) was isolated from diseased rainbow trout (Oncorhynchus mykiss) in Brazil. The whole genome sequence of this strain was compared with the Mexican W-1 strain, also isolated from diseased rainbow trout, and with the Weissella ceti type strain CECT 7719 T (= 1119-1A-09 T = CCUG 59653 T), recovered from the beaked whale. Digital DNA-DNA hybridization pairwise analyses scored 98.7% between the Mexican W-1 and Brazilian WS08T but just 24.4% for both fish isolates compared to the W. ceti type strain CECT 7719 T. The 16S rRNA gene sequence comparisons with isolates of W. ceti, available at GenBank, were conducted. All rainbow trout-pathogenic isolates grouped close (97% bootstrap confirmation), but when this group was compared to the W. ceti type strain CECT 7719 T the similarity varied from 78.9 to 79.1%. Phenotypic assays were also conducted, and the W. ceti type strain diverged from WS08T and W-1 in the hydrolysis of aesculin, D-mannose, and potassium gluconate and in the hydrolysis of hippurate. Moreover, WS08T and W-1 showed weak growth at 5 °C whereas no growth was observed for W. ceti CECT 7719 T. The major fatty acids (> 10% total fatty acids) presented by WS08T and W-1 were summed feature 8 (C18:1 ω7c/C18:1 ω6c), summed feature 3 (C16:1 ω6c/C16:1ω7c), and C16:0. The results of phylogenetic and phenotypic analyses clearly differentiated the W. ceti CECT 7719 T type strain from the assessed pathogenic strains obtained from rainbow trout. Therefore, Weissella strains isolated from rainbow trout, here represented by strain WS08T (= CBMAI 2730), should be known as members of a novel species for which the name Weissella tructae sp. nov. is proposed.