Molecular identification and prevalence of plerocercoid larvae (Cestoda: Diphyllobothriidae) in some commercial fish species from Peru.

Diphyllobothriosis, a fish-borne zoonosis in South America, is mainly caused by the Pacific broad tapeworm Adenocephalus pacificus Nybelin, 1931, a parasite of considerable concern in fishery resources due to its impact on public health. A new diphyllobothrid, Diphyllobothrium sprakeri Hernández-Orts et al. Parasites Vectors 14:219, 2021, was recently described from sea lions from the Pacific Coast, but marine fish acting as intermediate hosts are unknown. The objective of this study was to confirm the presence of plerocercoid larvae of Diphyllobothriidae Lühe, 1910 (Cestoda: Diphyllobothriidea) in nine fish species of commercial importance in Peru. Of a total of 6999 fish (5861 Engraulis ringens, 853 Sciaena deliciosa, 6 Sciaena callaensis, 171 Scomber japonicus, 40 Trachurus murphyi, 40 Ariopsis seemanni, 18 Merluccius peruanus, 5 Sarda chiliensis, and 5 Coryphaena hippurus), 183 were infected with plerocercoid larvae, representing a total prevalence of 2.61% and a mean intensity of 3.2. Based on mtDNA cox1 sequences of 43 plerocercoids, a phylogenetic analysis revealed that 41 belong to A. pacificus and two to D. sprakeri. These findings are first molecular data for D. sprakeri larvae, and the infections of E. ringens and T. murphyi by plerocercoid larvae represent the first records of intermediate/paratenic hosts for this species. Hence, the findings of the current study enhance our understanding of the presence of diphyllobothriid species in commercial fish from the Southeastern Pacific Ocean and their potential impact on seafood safety for local human populations.

Susceptibility of the cultured Amazonian fish, Colossoma macropomum, to experimental infection with Aeromonas species from ornamental fish.

The global ornamental fish trade carries important risk factors for spreading pathogens between different countries and regions, not only for ornamental fish but also for cultured fish and even other animal species. In the current study, we reported the capacity of Aeromonas veronii and A. hydrophila isolated from ornamental fish to experimentally infect the reared Amazonian fish Colossoma macropomum. For this, those bacteria were identified, and a primary characterization was performed. Fish were inoculated with 0.1 mL of increasing concentrations of A. hydrophila or A. veronii (C1 = 1 × 102; C2 = 1.8 × 104; C3 = 2.1 × 106; C4 = 2.4 × 108 bacterial cells per mL) in the coelomic cavity. In the control group, fish received the same volume of sterile saline solution (0.9 %). Fish presented petechiae, skin suffusions, and mortality rates up to 100 % according to the inoculum concentration. Histopathologically, fish presented necrosis with karyolysis, loss of the cytoplasmic delimitation of cells of the renal tubules and hepatocytes, hemorrhage, cellular edema, and the presence of bacterial cells. The LD50-96h of A. veronii on C. macropomum was estimated at 2.4 × 106 CFU mL-1 and of A. hydrophila at 1.408 × 105 CFU mL-1. The results demonstrated that it is possible that Aeromonas species isolated from ornamental fish affect C. macropomum, causing similar clinical signs and lesions. This shows the importance of promoting risk control measures worldwide regarding the trade of ornamental fish.

Co-existence of two Yersinia ruckeri biotypes and serotype O1a retrieved from rainbow trout (Oncorhynchus mykiss) farmed in Puno, Peru.

Yersinia ruckeri causes important economic losses for rainbow trout (Oncorhynchus mykiss) farms worldwide. This bacterial disease is likely the most common among trout in Peru; however, no commercial vaccine is available nationally, which is, in part, due to a lack of information on the bacterium. The aim of the current study was to characterize 29 Y. ruckeri isolates sampled from seven cage-reared farms in the Puno Region, the focal point for aquaculture activities in Peru. For this, samples were taken from fish with clinical signs (i.e. haemorrhages, uni- or bilateral exophthalmia, hyphaemia and/or melanosis). Notable among our findings was the existence of both Y. ruckeri biotype 1 (9 isolates) and biotype 2 (20 isolates; negative for sorbitol and Tween 80). The isolates further differed in API profiles 5307100 (21 isolates), 1307100 (4 isolates), 1305100 (2 isolates), 1307120 (1 isolate) and 5305100 (1 isolate), with the main differences being in the tests for lysine decarboxylase, gelatine hydrolysis and D-saccharose fermentation. Despite these differences, all isolates shared identical ERIC-PCR and REP-PCR profiles and belonged to the O1a serotype. Fingerprints were identical to the reference strain CECT 955 (serotype O1a). The information obtained will be used for epidemiological purposes by health authorities and for the development of a vaccine against Y. ruckeri, a prominent request made by fish farmers in Peru.

Testing Novel Inactivation Methods and Adjuvants for Vaccines Against Streptococcus agalactiae in Nile Tilapia Oreochromis niloticus.

Inactivation by hydrogen peroxide and pH manipulation are two novel methods used recently in experimental vaccines against Streptococcus agalactiae in Nile tilapia. Here we describe in detail inactivation using novel methods as well as the classical method of inactivation. These vaccines showed similar moderate efficacy when compared to the conventional formaldehyde vaccine. In addition, we describe the inclusion of adjuvants in a hydrogen peroxide vaccine.

Immunogenicity in Oreochromis niloticus vaccinated with sonicated antigens against streptococcosis.

Streptococcosis causes great economic losses in intensive culture of tilapia. Vaccination is the most effective and safest way to tackle infectious diseases. Thus, this study sought the more effective and safer antigenic fraction after sonication of Streptococcus agalactiae to elaborate a vaccine against streptococcosis in Nile tilapia. For this, twenty-one days after vaccination with different fractions (soluble and insoluble) of S. agalactiae, the fish were challenged with the homologous strain (LD50). Then, samples were taken at zero, 14, 28, 60 and 90 days post-vaccination (DPV, n = 7). Blood and organs (cranial kidney, spleen and liver) were collected from vaccinated and unvaccinated fish. Finally, insoluble fraction vaccine presented the best effect, resulting in a 100% relative percent of survival (RPS) and without clinical manifestations. In view of the results, it was to evaluate the role of the insoluble fraction of the antigen in the protective immunity against streptococcosis. The results indicate that the spleen might be the main organ in the vaccine response in Nile tilapia due to the great morphological and immunological differences in vaccinated fish, evidenced by the greater of melanomacrophage centers (MMC) and IgM + lymphocytes in relation to the non-vaccinated fish. At 60 DPV, it was observed the peak of the protective immunity related to the maximum concentration of proteins, circulating leukocytes, antibody titers in the serum and tissue changes with greater expression of IgM + and MMC number in the spleen and kidney of Oreochromis niloticus. Vaccination with insoluble fraction of S. agalactiae was safe and provided effective protection against streptococcosis with maximum protective response at 60 DPV.

Efficacy of two adjuvants administrated with a novel hydrogen peroxide-inactivated vaccine against Streptococcus agalactiae in Nile tilapia fingerlings.

Streptococcus agalactiae is considered the main bacterial pathogen in cultured Nile tilapia. Formaldehyde-inactivated vaccines are the most accepted method for prevention and control of the disease. However, alternative inactivation methods for S. agalactiae vaccines have not been fully explored. Recently, we developed a hydrogen peroxide-inactivated vaccine against S. agalactiae with moderate efficacy, with the possibility to improve vaccine efficacy by adding adjuvants. The current study compared the efficacy of aluminum hydroxide and Freund's incomplete adjuvant (FIA) incorporated into a novel hydrogen peroxide-inactivated intraperitoneal vaccine against S. agalactiae for Nile tilapia fingerlings. The relative percentage survival (RPS) for aluminum hydroxide-adjuvanted vaccine (59.3%), and FIA-adjuvanted vaccine (77.8%) were higher than the vaccine without adjuvant (40.7%). In addition, fish immunized with aluminum hydroxide-adjuvanted vaccine had significantly higher levels of specific antibodies than control fish at 4 weeks post vaccination (wpv). Blood lymphocytes counts showed a decrease in vaccinated groups when compared to control fish, suggesting white cells migration to the tissues where antigen presentation is ongoing. Fish that received FIA-adjuvanted vaccine exhibited persistence of adjuvant deposits on intraperitoneal surfaces for at least 4 wpv that may be related to its superior performance compared to aluminum hydroxide adjuvanted vaccine, which did not evidence any type of deposit at any sampling times. The results observed in this study demonstrate that hydrogen peroxide-inactivated vaccine administered with either aluminum hydroxide or FIA induce optimal levels of protection, with a superior performance for FIA vaccine, which could be a good alternative to conventional formaldehyde-inactivated vaccines against S. agalactiae, due to its shorter manufacture time, and less toxicity.

Role of neuroendocrine modulation and biochemistry in the sepsis in Piaractus mesopotamicus.

Sepsis is a systemic process with multifactorial pathophysiology that affects most animal species. It is responsible for high rates of morbidity and mortality. This work aimed to study the biochemical and neuroendocrine changes of the sepsis process in Piaractus mesopotamicus after Aeromonas hydrophila inoculation analyzing changes in blood leukocyte and differences in neuroendocrine-biochemical modulation using RNA-seq. Fish showed hypercortisolemia, inhibition of glucose absorption, followed by hypocortisolemia and then hyperglycemia. Thyroid hormones (T3 and T4) showed immediate decrease in serum and T4 increased 6 h post-inoculation (HPI). Sepsis-induced hormonal alterations triggered changes in the metabolic pathways increasing protein and lipid catabolism, use of transient anaerobic glycolysis and liver injury. A reference transcriptome was constructed based on blood leukocytes from P. mesopotamicus. The assembly resulted in total 266,272 contigs with a N50 of 2786 bp. There was a reorganization of plasma membrane of leukocytes at the beginning of the septic process with increased expression of neuroendocrine receptors and with continuous flow of neurotransmitters, hormones and solutes with compensatory regulation at 6 HPI. Three and nine HPI seemed to be critical, the expression of a number of transcription factors was increased, including the modulatory DEGs related to glucocorticoid and thyroid hormones induced and suppressed (FDR < 0.05). Neuroendocrine modulation can regulate leukocytes and biochemical parameters of peripheral blood, being important sources for the study of the pathophysiology of sepsis. These finding highlights the importance of further studies focusing on biochemical-neuroendocrine changes in blood leukocytes and systemic sepsis.

Morphological, microbiological and ultrastructural aspects of sepsis by Aeromonas hydrophila in Piaractus mesopotamicus.

Aeromonas bacteria can cause an infection characterized by septicemia and is one of the most common pathogens in tropical fish. This disease is responsible for high morbidity and mortality rates, causing considerable losses in aquaculture. Thus, the understanding of its pathophysiology is crucial to develop control strategies of this bacterial infection in farmed fish. This study aimed to characterize early pathological aspects of acute sepsis in pacu (Piaractus mesopotamicus) experimentally infected with Aeromonas hydrophila. A total of 160 juvenile pacus were inoculated intraperitoneally with A. hydrophila (1.78 x 109 CFU/mL) and at 0 (control), 1, 3, 6, and 9 hours post-inoculation (hpi), animals were anesthetized and samples were collected for microbiological, light microscopy and transmission electron microscopy (TEM) analyzes. The results showed the occurrence of hemodynamic alterations, such as hemorrhage and congestion, which were observed mainly after 6 and 9 hpi. It was possible to re-isolate Aeromonas at all sampling times except in control group. However, just after 9 hpi it was possible to find the bacteria in all fish and tissues. Light microscopy analyses revealed a degenerative process, necrosis and vascular damage mainly at 6 and 9 hpi. According to the ultrastructural examination, areas of cellular death were identified in all examined tissues, especially at 6 and 9 hpi. However, the most severe, related to necrosis, were observed after 6 and 9 hpi. The findings suggested that this bacterium spreads in the first hpi through the fish organs, mainly affecting spleen, liver and kidney, causing irreversible lesions at the molecular level.

Hematological and immune changes in Piaractus mesopotamicus in the sepsis induced by Aeromonas hydrophila.

The pathogenesis of sepsis involves complex systems and multiple interrelationships between the host and pathogen producing high mortality rates in various animal species. In this study, hematological disturbances, innate immunity and survival during the septic process in Piaractus mesopotamicus inoculated with Aeromonas hydrophila were studied. For this aim, fish blood samples were taken from control and infected groups 1, 3, 6, and 9 h post-inoculation (HPI). Leukogram showed reduction in the number of leukocytes and thrombocytes, followed by cessation of leukocyte chemotaxis 6 HPI and severe morphological changes in leukocytes and erythrocytes. At 3 HPI production of reactive oxygen species increased and at 6 HPI decreased. There was no change in serum lysozyme concentration and lytic activity of the complement system, despite the progressive increase in serum lytic activity and bacterial agglutination. Finally, the changes in clinical signs due to aeromonosis and increasing septicemia resulted in a reduction in survival to 57.14% after 36 HPI. It was possible concluded that these hematological and immune are crucial event in the worsening of sepsis in P. mesopotamicus, and these findings are utility for diagnosing and understanding the pathophysiology sepsis in pacu induced by A. hydrophila.

Influence of dexamethasone and levamisole on macrophage recruitment, giant cell formation and blood parameters in the tropical fish Piaractus mesopotamicus / Influência da dexametasona e levamisol no recrutamento de macrófagos, formação de células gigantes e parâmetros hematológicos no peixe tropical Piaractus mesopotamicus

The aim of this study was to evaluate the effect of parenteral administration of dexamethasone and levamisole on the accumulation of macrophages and formation of giant cells in chronic inflammation by foreign body and blood parameters in pacu (Piaractus mesopotamicus). It was used 50 mg/kg of levamisole and 2.0 mg/kg of dexamethasone and the combination of both drugs. Coverslips were implanted under the skin. After 2, 7, and 15 days postimplantation (DPI), fish were anesthetized for removal of coverslips and the number of macrophages and giant cells were count. It was also collected blood from the caudal vessel to evaluate: red blood cell count, hematocrit, hemoglobin concentration, leukocytes count, thrombocytes count, MCV, and MCHC. We observed that dexamethasone affects negatively the formation of giant cells in the chronic inflammation for foreign body. Levamisole, despite being immunostimulatory in several species, showed limited action. However, it was enough to counteract the effect of dexamethasone; the association of the drugs did not interfere significantly in erythrocyte and leukocyte number in most of the treatments and times studied. In dexamethasone group there was a reduction in the number of erythrocytes and hemoglobin associated with increased mean corpuscular volume, suggesting slight macrocytic anemia. At 15 DPI, most groups showed the recovery of hematologic response. As in mammals, dexamethasone affects negatively the inflammatory response. Levamisole showed little effect by itself. However, in some parameters the association of both drugs causes similar response to control and naïve groups, showing the antagonistic effect of these drugs.

O objetivo deste estudo foi avaliar o efeito da administração parenteral de dexametasona e levamisol na acumulação de macrófagos e formação de células gigantes na inflamação crônica por corpo estranho e avaliação de parâmetros sanguíneos em pacu (Piaractus mesopotamicus). Utilizou-se 50 mg/kg de levamisol e 2,0 mg / kg de dexametasona e a combinação de ambos fármacos. As lamínulas foram implantadas sob a pele. Depois de 2, 7 e 15 dias pós-implantação (DPI), os peixes foram anestesiados para a remoção das lamínulas e contagem do número de macrófagos e células gigantes. Foi coletado sangue da veia caudal para realizar contagem de células vermelhas, leucócitos, trombócitos, concentração de hemoglobina, MCV e MCHC. Observou-se que a dexametasona afeta negativamente a formação de células gigantes na inflamação crónica por corpo estranho. O levamisol, apesar de ser considerado imunoestimulante em várias espécies, mostrou ação limitada. No entanto, foi suficiente para neutralizar o efeito da dexametasona; a associação das drogas não interferiu significativamente no número de eritrócitos e de leucócitos na maioria dos tratamentos e períodos estudados. No grupo da dexametasona, houve redução do número de eritrócitos e concentração de hemoglobina associado ao aumento do volume corpuscular médio sugerindo leve anemia macrocítica. Aos 15 DPI, a maioria dos grupos mostrou recuperação na resposta hematológica. Como em mamíferos, a dexametasona afeta negativamente a resposta inflamatória. O levamisol mostrou pouco efeito por si só. No entanto, em alguns parâmetros a associação das duas drogas provoca resposta similar ao grupo controle e naive, mostrando o efeito antagonista de estas drogas.

Wound healing in Piaractus mesopotamicus supplemented with chromium carbochelate and Saccharomyces cerevisiae / Cicatrização cutânea em Piaractus mesopotamicus suplementados com carboquelato de cromo e Saccharomyces cerevisiae

Wound healing and tissue repair are necessary to ensure survival and health of any organism. The aim of this study was to investigate the impact of supplementation with chromium carbochelate (CC) and Saccharomyces cerevisiae (SC) on wound healing in tropical teleost fish Piaractus mesopotamicus. Thus, fish were distributed into four groups: a) control (without supplementation); b) supplemented with 18 mg/kg of chromium carbochelate; c) supplemented with 0.3% of S. cerevisiae and d) supplemented with an association of both supplements. After 105 days of feeding, full-thickness skin incisions (2.0 x 1.0 x 0.25 cm) were performed removing epidermis and dermis. Macroscopic and histologic observations were carried out at 1, 3, 7, 14, 21, 28, and 35 days after wounding to monitor the healing rate. Opposing fronts advanced gradually and faster each day demonstrating a progressive increase in the healing process over time. The inflammatory process was exacerbated and expansive, with an increase in mucous cells and chromatophores. Although no significant differences were observed between groups on wound retraction and microscopic parameters, fish supplemented with CC and SC showed faster re-epithelialization, greater degree of organization of collagen fibers, and higher neovascularization. We concluded that supplementation with S. cerevisiae and chromium carbochelate improves specific aspects of cutaneous healing process in pacu.(AU)

A cicatrização e reparação de tecidos são mecanismos essenciais para garantir a sobrevivência e saúde de qualquer indivíduo. O objetivo deste trabalho foi avaliar o impacto da suplementação com carboquelato de cromo (CC) e Saccharomyces cerevisiae (SC) sobre a cicatrização no peixe tropical Piaractus mesopotamicus. Para isto, os peixes foram distribuídos em quatro grupos: controle (sem tratamento), suplementados com 18 mg/kg de carboquelato de cromo, 0,3% de S. cerevisiae e associação de ambos os suplementos. Após 105 dias de alimentação, foram realizadas incisões na pele de espessura completa (2,0 x 1,0 x 0,25 cm) removendo epiderme e derme. Avaliações macroscópicas e microscópicas foram realizadas 1, 3, 7, 14, 21, 28 e 35 dias após a indução das feridas, para monitorar a taxa de cicatrização. As bordas opostas das feridas avançaram gradualmente a cada dia, demonstrando o aumento progressivo do processo de cicatrização ao longo do tempo. O processo inflamatório foi exacerbado e expansivo, com aumento no número de células mucosas e cromatóforos. Apesar deste processo, não foram observadas diferenças significativas na retração das feridas e nos parâmetros microscópicos entre os grupos. Peixes suplementados com CC ou Sc apresentaram rápida reepitelização, maior grau de organização de fibras colágenas e de neovascularização inicial. Concluiu-se que a suplementação com S. cerevisiae ou carboquelato de cromo melhora aspectos específicos do processo cicatricial no pacu.(AU)

Dietary camu camu, Myrciaria dubia, enhances immunological response in Nile tilapia.

Camu camu, Myrciaria dubia, is an Amazon plant that presents high levels of vitamin C in its composition. Several studies in animals and humans have demonstrated their efficiency in the prevention and treatment of various diseases. However, there are no reports of its properties in fish. The aim of this study was to evaluate the effect of the oral administration of the extract of this plant in the immune parameters in Nile tilapia, Oreochromis niloticus. 400 Nile tilapia (80 ± 5 g) were randomly distributed into 20 tanks with 1500 L capacity each (20 fish/tank). After a week of adaptation to environmental conditions, it was provided a diet for 5 weeks, using different levels of inclusion of camu camu extract: 0, 50, 100, 250, and 500 mg/kg of feed. Each treatment consisted of four replicates. It was obtained 40.5 mg of vitamin C/g of camu camu pulp powder by high-performance liquid chromatography. At the end of the trial period, fish were inoculated with Aeromonas hydrophila in the swim bladder. Samples were taken after 6; 24 and 48 h of the challenge. Results revealed that fish supplemented with this herb showed significant increase (P < 0.05) in white blood cells counts in blood and exudate, burst respiratory activity, lysozyme activity, serum bactericidal activity, direct agglutination, and melanomacrophage centers count. Red blood cells count, hemoglobin, hematocrit, and biochemical profile of fish supplemented with the herb presented no statistical differences compared to control group (P > 0.05). No histopathological lesions were observed in intestine, kidney, spleen, and gills. It can be concluded that the addition of Myrciaria dubia in tilapia feed improves the immune response and the growth after 5 weeks, especially, at a dose of 500 mg/kg.

Uncaria tomentosa increases growth and immune activity in Oreochromis niloticus challenged with Streptococcus agalactiae.

Cat's claw (Uncaria tomentosa) is an Amazon herb using in native cultures in Peru. In mammals, it has been described several effects of this herb. However, this is the first report of its use on the diet of fish. The aim of this study was to determinate the effect of this plant on the growth and immune activity in Oreochromis niloticus. Nile tilapia (81.3 ± 4.5 g) were distributed into 5 groups and supplemented with 0 (non-supplement fish), 75, 150, 300, and 450 mg of U. tomentosa.kg(-1) of diet for a period of 28 days. Fish were inoculated in the swim bladder with inactivated Streptococcus agalactiae and samples were taken at 6, 24, and 48 h post inoculation (HPI). Dose dependent increases were noted in some of the evaluated times of thrombocytes and white blood cells counts (WBC) in blood and exudate, burst respiratory activity, lysozyme activity, melanomacrophage centers count (MMCs), villi length, IgM by immunohistochemistry in splenic tissue, and unexpectedly on growth parameters. However, dietary supplementation of this herb did not affect red blood cells count (RBC), hemoglobin, and there were no observed histological lesions in gills, intestine, spleen, and liver. The current results demonstrate for the first time that U. tomentosa can stimulate fish immunity and improve growth performance in Nile tilapia.

Acute Toxicity and Determination of the Active Constituents of Aqueous Extract of Uncaria tomentosa Bark in Hyphessobrycon eques.

Uncaria tomentosa is a medicinal plant used in folk medicine by Amazon tribes. In this study the constituents of aqueous extract of U. tomentosa bark were quantified by chromatographic technique and its lethal concentration 50 (48 h) in Hyphessobrycon eques was determined. The chromatography showed high levels of oxindole alkaloids, quinovic acid glycosides, and low molecular weight polyphenols. The CL50 48 h was 1816 mg/L. Fish showed behavior changes at concentrations above 2000 mg/L, accompanied by a significant decrease of dissolved oxygen. At the highest concentration 100% mortality was observed attributed to oxygen reduction by the amount of oxindole alkaloids, polyphenols accumulation of the extract in the gills, and the interaction of these compounds with dopamine. In conclusion, the aqueous extract of U. tomentosa did not alter the chemical components and it was shown that U. tomentosa has low toxicity to H. eques; therefore, it can be used safely in this species.