Paulo Fernandes Marcusso

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.

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

Cats 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.

Humoral immune response in Oreochromis niloticus: modified direct agglutination test

Understanding the functional mechanisms of the humoral and cellular immune responses among teleost broadens the possibilities for intervention and improvement of vaccination methods. The aim of this study was to evaluate the dynamics of the humoral immune response, hematological biochemical changes, and electrophoretic profile of proteins in Oreochromis niloticus, when submitted to antigen stimulation using sheep erythrocytes in a single dose and with booster doses. Seventy O. niloticus were distributed in two groups (n = 35): one received a single dose and the other received two doses of antigen. Evaluations were made at the following predetermined times: zero (pre-immune, i.e., before inoculation with the first injection of sheep erythrocytes, negative control) and on 7th, 14th, 21st, and 28th days after the first immunization to evaluate the hemogram, biochemistry, and electrophoresis of proteins and direct hemagglutination. Twenty-eight days after antigen inoculation, it was observed that there had been an increase in the serum antibody titer and fractions gamma globulins after second antigen stimulation. The results suggest that teleost fish have the capacity to produce antibodies from a single stimulation, and a second antibody production wave after second stimulation.

Parasitic infections in ornamental cichlid fish in the Peruvian Amazon

The aim of this study was to evaluate the prevalence and seasonal distribution of the main parasite species in Amazonian ornamental cichlids that affect their trade. The study was conducted from August 2007 to September 2009. We sampled 3042 specimens from 9 different species, of which 9.47% had at least one type of external parasite. 81.25% of the cases occurred in the dry season. Crenicichla anthurus (28.57%) was the most parasitized, followed by Aequidens diadema (26.32%), Pterophyllum scalare (22.69%), Cichlasoma sp. (9.52%), Apistogramma sp. (3.88%) and Symphysodon aequifasciatus (3.66%). Monogenea was the most abundant group of parasites, occurring in 66.67% of the cases, of which 96.88% occurred in the dry season. This parasite infested 95.68% of Pterophyllum scalare, 76.67% of Apistogramma sp, 33.33% of Cichlasoma sp. and 23.81% of Symphysodon aequifasciatus cases. Ichthyophthirius multifiliis infested 100% of Aequidens diadema, 76.19% of Symphysodon aequifasciatus, 66.67% of Cichlasoma sp, 41.67% of Crenicichla anthurus and 23.33% of Apistogramma sp cases. Myxosporidia infested 58.33% of Crenicichla anthurus. Trichodina infested 4.32% of Pterophyllum scalare. The prevalence of these parasites is related to the season, preferred habitat, fish behavior, individual susceptibility and handling of animals during transportation by fishermen.

Clinical features and corneal optical anisotropies in a rabbit model of limbal stem cell deficiency

PURPOSES To investigate the intra-laboratory reproducibility of clinical features and to evaluate corneal optical anisotropies in a rabbit model of limbal stem cell deficiency. METHODS Limbal injury was induced in the right eye of 23 adult New Zealand White rabbits using a highly aggressive protocol that combined 360 degrees limbal peritomy, keratolimbectomy, alkaline chemical burn, and mechanical removal of the epithelium. Clinical evaluation of the injured eyes was performed for 28 days and included corneal impression cytology. Corneas with a severe clinical outcome set typical of limbal stem cell deficiency were then collected, subjected to a histopathological examination, and examined for optical anisotropies. Corneas from healthy rabbit eyes were used as controls. Differences in optical path due to stromal collagen birefringence, as well as linear dichroism related to the expression and spatial orientation of glycosaminoglycan chains from proteoglycans, were measured from cross-sections under a quantitative polarized light microscope. RESULTS One eye showed signs of hypopyon and was excluded. Signs of ocular inflammation were observed in all eyes studied (n=22). Corneal impression cytology did not detect goblet cells. Twelve of the 22 corneas presented a clinical outcome set typical of limbal stem cell deficiency, which is characterized by the presence of epithelial defects, inflammatory cells, moderate-to-severe opacity, and neovascularization. Microscopic studies under polarized light revealed that relative to controls, limbal stem cell deficiency caused a 24.4% increase in corneal optical path differences. Further, corneas with limbal stem cell deficiency were less dichroic than controls. CONCLUSIONS These results suggest that rabbit models of limbal stem cell deficiency must be rigorously screened for use in preclinical studies to ensure experimental homogeneity because protocols used to create limbal stem cell deficiency could be not associated with good intra-laboratory reproducibility of clinical features. Limbal stem cell deficiency, as induced herein, altered the optical anisotropic properties of the corneal stroma. Such alterations are indicative of changes in collagen packing and the spatial orientation of glycosaminoglycan chains from proteoglycans. Knowledge of these changes is important to potentiate strategies aimed at restoring the morphofunctional integrity of the corneal stroma affected by limbal stem cell deficiency.

UTILIZAÇÃO DE RETALHO DE AVANÇO DE PADRÃO SUBDÉRMICO PARA CORREÇÃO DE LESÃO NECRÓTICA NA PORÇÃO ROSTRAL DO LÁBIO SUPERIOR - RELATO DE CASO

Objetivou-se descrever o caso de um cao com extensa ferida traumatica necrosada no labio superior que obteve bons resultados apos cirurgia reconstrutiva. O dono referiu que animal evadiu-se de sua residencia e retornou apos 15 dias com a lesao. Na avaliacao clinica pode-se observar uma ferida extensa com odor fetido acometendo grande parte do labio superior esquerdo com evolucao estimada de sete dias. No centro da lesao, observaram-se areas de necrose evidenciada pela coloracao escurecida com regioes avasculares e desvitalizadas. O paciente foi submetido a reconstrucao da face com retalho rotacional e apos 15 dias do procedimento cirurgico observou-se boa cicatrizacao da ferida. Concluiu-se entao com este relato que feridas necrosadas podem ser suturadas apos amplo desbridamento e limpeza local.

Transferência passiva de soro hiperimune anti-Streptococcus agalactiae e seu efeito profilático em tilápias-do-nilo infectadas experimentalmente: sobrevivência e títulos de anticorpos

A bacteria Streptococcus agalactiae e um potente agente causador de surtos por doencas bacterianas em peixes. O estresse provocado pelo manejo zootecnico e pela ma qualidade ambiental torna a tilapia susceptivel as infeccoes por essa bacteria. O objetivo do presente trabalho foi avaliar a resistencia de tilapias-do-nilo imunizadas com soro hiperimune anti-S. agalactiae, posteriormente desafiadas com cepa homologa da mesma bacteria. Apos determinacao da DL 50 de S. agalactiae, 36 tilapias foram distribuidas em quatro aquarios, dois para o grupo controle e dois para inoculacao celomatica para producao de anticorpos anti-S. agalactiae. No 21° e 28° dias, foi coletado sangue para obtencao de soro hiperimune utilizado na transferencia passiva. Em seguida, 30 tilapias foram distribuidas em tres aquarios e submetidas a tres tratamentos: GI: controle; GII: imunizadas com o soro inativado; GIII: imunizadas com soro ativo. Apos 48 horas e sete, 14, 21, 28 e 35 dias, foram realizadas coletas de sangue para titulacao de anticorpos anti-S. agalactiae utilizando-se o teste de aglutinacao direta. Para avaliar a taxa de sobrevivencia, outras 30 tilapias foram distribuidas em tres aquarios e submetidas a tres tratamentos (GI: controle; GII: imunizadas com soro inativado; GIII: imunizadas com soro ativo). Apos 48 horas da imunizacao, as tilapias foram desafiadas via celomatica com 100µL de S. agalactiae e avaliadas duas vezes ao dia, pelo periodo de 35 dias. Os resultados dos titulos sericos de anticorpos foram detectados pela aglutinacao direta ate o 21° dia pos-transferencia passiva, e, no mesmo periodo, houve protecao de 80% entre os grupos imunizados com soro inativado e soro ativo contendo anticorpos anti-S. agalactiae. Ao final, os grupos soro inativado e soro ativo apresentaram 60 e 80% de protecao, respectivamente, enquanto no grupo controle 100% dos peixes adoeceram, apresentando sinais graves da infeccao, e foram eutanasiados. Nao houve diferenca estatistica significativa na taxa de protecao entre os grupos imunizados.

Desempenho e hematologia de tilápias‑do‑nilo alimentadas com Saccharomyces cerevisiae e vacinadas contra Streptococcus agalactiae

The objective of this work was to evaluate the inter-relationship between dietary supplementation with Saccharomyces cerevisiae cell wall and vaccination against Streptococcus agalactiae, and its effect on the productive performance and hematological variables of Nile tilapia (Oreochromis niloticus). Eighty-four Nile tilapia were distributed in 12 fiber boxes (n=7), in a 2x2x3 factorial arrangement, corresponding to two levels of supplementation with yeast cell wall, two types of inoculation, and three evaluation times. Fish were fed during 77 days. Vaccination of fish was done 60 days after feeding started. Fifteen days after vaccination, all fish were subjected to challenge with live strain of S. agalactiae, and 6, 24, and 48 hours after the challenge, blood was collected from the caudal vein for evaluations. Fish fed with supplemented diets show greater weight gain and specific growth rate, and the interaction between the diet and vaccination effects results in higher hematocrit, hemoglobin, and leukocyte rates.