De um subproduto industrial para um produto destinado à alimentação animal: produção de biomassa bacteriana

A grande diversidade das atividades industriais ocasiona durante o processo produtivo a geração de resíduos sólidos, líquidos e gasosos, que podem poluir e/ou contaminar o solo, a água e o ar. Embora existam regulamentos para o descarte desses resíduos, a inobservância às regras, a ineficiência dos tratamentos despoluentes e a inoperância de órgãos fiscalizadores permitem que, ainda hoje, sejam lançados ao ambiente grandes cargas de poluentes. Isso se aplica às indústrias de alimentos de origem animal. Felizmente, a visão dos empresários do setor vem mudando, na medida em que a imagem de uma empresa que conta com produtos e processos ambientalmente responsáveis representa parte das estratégias competitivas atuais. O objetivo deste trabalho foi demonstrar como um efluente industrial pode ser transformado em matéria prima para a obtenção de um produto com valor comercial, utilizando tecnologias reconhecidas pela indústria e a atividade microbiana. O efluente foi obtido em indústria de abate e processamento de tilápias, apresentado (valores médios) pH = 9,4, DQO = 1.127 mg/L, óleos e graxas = 1.166 mg/L, nitrogênio total = 813 mg/L, coliformes a 30–35o C = 1,0x105 NMP/mL, coliformes a 45o C = 0,41 NMP/mL, bolores e leveduras = 4,6x103 UFC/mL e, ocasionalmente, contendo Salmonella sp e Aeromonas sp. Os tratamentos físicos aplicados ao efluente incluíram gradeamento, filtração (50µm) e pasteurização (65o C/30 min). O cultivo de Rubrivivax gelatinosus foi realizado sob anaerobiose em reatores de vidro durante 7 dias, em temperatura ambiente (30±5o C) e 2.000±500 lux. A recuperação da biomassa foi feita por filtração tangencial (0,2 µm; 1,5 bar), centrifugação (3.400 g/30 min; 5o C) e liofilização (-40o C) e a pulverização foi realizada manualmente. A produção de massa celular atingiu 0,18 g/L, com produtividade de 0,0634 g/L.dia. O processo promoveu redução de 52% na DQO, 48% em óleos e graxas e 22% no nitrogênio total, gerando um resíduo com pH 7,9, livre de bactérias patogênicas e, portanto, apto ao descarte. O produto obtido apresentou cor vermelho escuro (L = 22,42; C = 14,22; h = 25,48), 4,55% de umidade, 57,39% de proteína, 11,08% de extrato etéreo, 4,05% de matéria mineral, 3,03 mg/g de oxicarotenóides, 20,27 NMP/g de coliformes a 30–35o C, <1,0 NMP/g de coliformes a 45o C, 1,2x103 UFC/g de bolores e leveduras e ausência de microrganismos patogênicos. Essas características apontam para um potencial pigmentante e nutricional positivo no produto, que pode encontrar aplicação na produção animal, com segurança microbiológica. Dessa forma, fecha-se um ciclo autosustentável que pode ser adotado na própria fonte geradora do resíduo industrial, permitindo a reversão de gastos com tratamento despoluente em receitas advindas da comercialização de um novo produto.

Vitamin A affects haematology, growth and immune response of Nile tilapia (Oreochromis niloticus, L.), but has no protective effect against bacterial challenge or cold-induced stress

Vitamin A (vitA) is an essential nutrient that acts as an endocrine regulator of several metabolic pathways, modulating normal growth and health status of animals. Although the importance of vitA for normal haematology and immune response is well documented for higher vertebrates, there is limited information on the physiological effects of vitA on fish. Therefore, we designed a 130-day feeding trial to evaluate the effect of vitA supplementation on growth, haematology, immune function and resistance to experimental infection with Aeromonas hydrophila and cold-induced stress. A group of 320 Nile tilapia fingerlings 7.49 ± 0.19 g weight (mean ± SD) were randomly stocked into 40 250 L-aquaria and fed practical diets containing graded levels of vitA (0, 0.06, 0.12, 0.24, 0.48, 0.96, 1.92, 3.84 mg retinol (ROH) kg−1 diet. Growth, haematology, plasma protein profile and immune response were significantly affected by vitA supplementation; however, no clear protective effect of vitA supplementation on disease and cold stress resistance were observed in this study. Clinical signs of vitA deficiency were: resting and abnormal swimming behaviour, exophthalmia, haemorrhages at the base of fins and on skin, serous fluids in abdominal cavity, neutropenia, reduction in red blood cell count, haematocrit and haemoglobin evolving to high mortality rates in a short period of time. A dietary level of vitA around 1.2 mg ROH kg−1 may be required to prevent gross deficiency signs and promote proper growth and health status of Nile tilapia. VitA does not seem to have a pronounced effect on leucocyte differentiation, but clearly plays an important role on maintaining normal erythropoiesis.

Immunomodulation with β-glucan in matrinxã (Brycon amazonicus)

Pós-graduação em Zootecnia - FCAV

Hemolytic activity of alternative complement pathway as an indicator of innate immunity in pacu (Piaractus mesopotamicus)

The objective of this study was to evaluate the methodology to establish the hemolytic activity of alternative complement pathway as an indicator of the innate immunity in Brazilian fish pacu (Piaractus mesopotamicus), in addition to verifying the influence of beta-glucan as an immunostimulant. Fish were fed with diets containing 0, 0.1 and 1% beta-glucan, during seven days, and then inoculated with Aeromonas hydrophila. Seven days after the challenge, they were bled for serum extraction. The methodology consisted of a kinetic assay that allows calculating the required time for serum proteins of the complement to promote 50% lysis of a rabbit red blood cell suspension. The method developed in mammals was successfully applied for pacu and determined that the hemolytic activity of the proteins of the complement system (alternative pathway) increased after the pathogen challenge, but was not influenced by the beta-glucan treatment.

Controllo adrenergico del metabolismo glucidico in Anguilla anguilla

Le conoscenze relative al controllo ormonale del metabolismo epatico dei pesci sono ancora piuttosto limitate e per molti anni sono state controverse. Per lungo tempo si è ritenuto che le catecolamine, adrenalina e noradrenalina, agissero nel fegato dei pesci soltanto attraverso i recettori adrenergici di tipo β. Quindi l’assetto recettoriale dei mammiferi, che comprende recettori α e β, era considerato frutto di un processo evolutivo che non aveva ancora avuto luogo nei pesci. Successivamente, nel fegato di vari teleostei è stata dimostrata la presenza di recettori sia α che β. Tuttavia il ruolo fisiologico dei due tipi di recettori non è ancora chiaro. Per esempio, in acciughe e sgombri non è stato fatto alcuno studio sulla risposta alle catecolamine ottenuta attraverso i recettori α e β, nel fegato di trota i recettori α non sono accoppiati alla cascata fisiologica che porta al rilascio di glucosio, e in anguilla e pesce gatto l’azione delle catecolamine attraverso recettori β è predominante rispetto a quella attraverso recettori α. L’utilizzo di ligandi farmacologici non ha portato a chiarimenti significativi, perché la loro specificità per i recettori di mammifero non trova sempre riscontro nei pesci. In questo studio, quindi, abbiamo studiato l’espressione dei geni codificanti per i recettori α e β adrenergici attraverso la tecnica della PCR real time, ottenendo i primi dati in letteratura per quanto riguarda la loro quantificazione assoluta. L’organismo modello utilizzato è stata l’anguilla, teleosteo caratterizzato da un ciclo biologico molto particolare in cui si distinguono nettamente una fase gialla ed una argentina. Le anguille argentine non sono mai state studiate a tale proposito, e date le estreme differenze nella disponibilità e nell’uso delle risorse energetiche in questi due stadi di crescita, il presente studio ha mirato a valutare la differente sensibilità alle catecolamine da parte degli epatociti isolati da anguille gialle ed argentine. I nostri dati hanno confermato quanto solo ipotizzato nei vari studi pubblicati negli ultimi due decenni, ma mai avvalorato da risultati sperimentali, cioè che i recettori α e β sono contemporaneamente espressi negli epatociti dell’anguilla, sia gialla che argentina, e la proporzione tra loro giustifica il ruolo significativamente maggiore giocato dai recettori β. Nelle anguille argentine infatti, come nelle gialle, l’effetto dell’adrenalina sul rilascio di glucosio ottenuto attraverso recettori β è chiaramente predominante. Inoltre, i nostri dati indicano che in due diverse fasi del ciclo vitale dell’anguilla, così come si osserva nell’ontogenesi dei mammiferi, i recettori adrenergici sono espressi in quantità differente.

Laribacter hongkongensis: a potential cause of infectious diarrhea

In this study, we describe the isolation of Laribacter hongkongensis, a recently described genus and species of bacterium, in pure culture on charcoal cefoperazone deoxycholate agar from the stool of six patients with diarrhea. Three patients were residents of Hong Kong, and three of Switzerland. In none of the stool samples obtained from these six patients was Salmonella, Shigella, enterohemorrhagic Escherichia coli, Vibrio, Aeromonas, Plesiomonas, or Campylobacter recovered. Rotavirus antigen detection, electron microscopic examination for viruses, and microscopic examinations for ova and cysts were all negative for the stool samples obtained from the three patients in Hong Kong. Enterotoxigenic E. coli was recovered from one of the patients in Hong Kong. Unlike L. hongkongensis type strain HKU1, all the six strains were motile with bipolar flagellae. Sequencing of the 16S ribosomal RNA genes of the six strains showed that they all had sequences with only 0-2 base differences to that of the type strain. Pulsed field gel electrophoresis of the SpeI digested genomic DNA of the six isolates and that of the type strain revealed that the seven isolates were genotypically unrelated strains. More extensive epidemiologic studies should be carried out to ascertain the causative association between L. hongkongensis and diarrhea and to define the reservoir and modes of transmission of L. hongkongensis.

Susceptibility to travelers' diarrhea and histo-blood group antigens

Histo-blood group antigens (HBGAs) have been associated with susceptibility to enteric pathogens including noroviruses (NoVs), enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni, and Vibrio cholerae. We performed a retrospective cohort study to evaluate the relationship between traveler HBGA phenotypes and susceptibility to travelers' diarrhea (TD) and post-infectious complications. 364 travelers to Guadalajara, Mexico were followed prospectively from June 1 - September 30, 2007 and from June 7–July 28, 2008 for the development of TD and at 6 months for post-infectious irritable bowel syndrome (PIIBS). Noroviruses were detected from illness stool specimens with RT-PCR. Diarrheal stool samples were also assayed for enterotoxigenic and enteroaggregative E. coli, Salmonella species, Shigella species, Vibrio species, Campylobacter jejuni, Yersinia enterocolitica, Aeromonas species, and Plesiomonas species. Diarrheal stools were evaluated for inflammation with fecal leukocytes, mucus, and occult blood. Phenotyping for ABO and Lewis antigens with an ELISA assay and FUT2 gene PCR genotyping for secretor status were performed with saliva. 171 of 364 (47%) subjects developed TD. HBGA typing for the travelers revealed O (62.9%), A (34.6%), B (1.6%), and AB (0.8%) phenotypes. There were 7% nonsecretors and 93% secretors among the travelers. AB phenotypes were more commonly associated with Cryptosporidium species (P=0.04) and ETEC ( P=0.08) as causes of TD. AB and B phenotype individuals were more likely to experience inflammatory diarrhea, particularly mucoid diarrhea ( P=0.02). However, there were relatively few individuals with AB and B phenotypes. GI and GII NoV and Cryptosporidium species infections and PI-IBS were identified only in secretors, but these differences were not statistically significant, (P=1.00), (P=1.00), and (P=0.60), respectively. Additional studies are needed to evaluate whether AB phenotype individuals may be more susceptible to developing TD associated with Cryptosporidium species or ETEC, and whether AB and B phenotype individuals may be more likely to develop inflammatory TD. Further studies are needed to investigate whether nonsecretor travelers may be at less risk for developing infections with NoVs and Cryptosporidium species and PI-IBS.^

Microbial iron transport via a siderophore shuttle: A membrane ion transport paradigm

A mechanism of ion transport across membranes is reported. Microbial transport of Fe3+ generally delivers iron, a growth-limiting nutrient, to cells via highly specific siderophore-mediated transport systems. In contrast, iron transport in the fresh water bacterium Aeromonas hydrophila is found to occur by means of an indiscriminant siderophore transport system composed of a single multifunctional receptor. It is shown that (i) the siderophore and Fe3+ enter the bacterium together, (ii) a ligand exchange step occurs in the course of the transport, and (iii) a redox process is not involved in iron exchange. To the best of our knowledge, there have been no other reports of a ligand exchange mechanism in bacterial iron transport. The ligand exchange step occurs at the cell surface and involves the exchange of iron from a ferric siderophore to an iron-free siderophore already bound to the receptor. This ligand exchange mechanism is also found in Escherichia coli and seems likely to be widely distributed among microorganisms.

Growth, survival, gene expression, microbiota and tryptic activity during feeding of Scophthalmus maximus first feeding larvae with beta-glucan (MacroGard)

Reflecting the natural biology of mass spawning fish aquaculture production of fish larvae is often hampered by high and unpredictable mortality rates. The present study aimed to enhance larval performance and immunity via the oral administration of an immunomodulator, beta-glucan (MacroGard®) in turbot (Scophthalmus maximus). Rotifers (Brachionus plicatilis) were incubated with or without yeast beta-1,3/1,6-glucan in form of MacroGard® at a concentration of 0.5 g/L. Rotifers were fed to first feeding turbot larvae once a day. From day 13 dph onwards all tanks were additionally fed untreated Artemia sp. nauplii (1 nauplius ml/L). Daily mortality was monitored and larvae were sampled at 11 and 24 dph for expression of 30 genes, trypsin activity and size measurements. Along with the feeding of beta-glucan daily mortality was significantly reduced by ca. 15% and an alteration of the larval microbiota was observed. At 11 dph gene expression of trypsin and chymotrypsin was elevated in the MacroGard® fed fish, which resulted in heightened tryptic enzyme activity. No effect on genes encoding antioxidative proteins was observed, whilst the immune response was clearly modulated by beta-glucan. At 11 dph complement component c3 was elevated whilst cytokines, antimicrobial peptides, toll like receptor 3 and heat shock protein 70 were not affected. At the later time point (24 dph) an anti-inflammatory effect in form of a down-regulation of hsp 70, tnf-alpha and il-1beta was observed. We conclude that the administration of beta-glucan induced an immunomodulatory response and could be used as an effective measure to increase survival in rearing of turbot.

Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process

Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry.

Isolation and characterization of integron-containing bacteria without antibiotic selection

The emergence of antibiotic resistance among pathogenic and commensal bacteria has become a serious problem worldwide. The use and overuse of antibiotics in a number of settings are contributing to the development of antibiotic-resistant microorganisms. The class 1 and 2 integrase genes (intI1 and intI2, respectively) were identified in mixed bacterial cultures enriched from bovine feces by growth in buffered peptone water (BPW) followed by integrase-specific PCR. Integrase-positive bacterial colonies from the enrichment cultures were then isolated by using hydrophobic grid membrane filters and integrase-specific gene probes. Bacterial clones isolated by this technique were then confirmed to carry integrons by further testing by PCR and DNA sequencing. Integron-associated antibiotic resistance genes were detected in bacteria such as Escherichia coli, Aeromonas spp., Proteus spp., Morganella morganii, Shewanella spp., and urea-positive Providencia stuartii isolates from bovine fecal samples without the use of selective enrichment media containing antibiotics. Streptomycin and trimethoprim resistance were commonly associated with integrons. The advantages conferred by this methodology are that a wide variety of integron-containing bacteria may be simultaneously cultured in BPW enrichments and culture biases due to antibiotic selection can be avoided. Rapid and efficient identification, isolation, and characterization of antibiotic resistance-associated integrons are possible by this protocol. These methods will facilitate greater understanding of the factors that contribute to the presence and transfer of integron-associated antibiotic resistance genes in bacterial isolates from red meat production animals.

Bacterial associations with salmonid eggs

Rainbow trout eggs Salmo gairdneri, Richardson, were incubated under a range of different environmental conditions. Recovery of bacteria from egg surfaces revealed that increased water temperature, slow water flow rates and high egg density all significantly increased egg surface bacterial populations. Live eggs were mainly colonized by Cytophaga sp., pseudomonas fluorescens and Aeromonas hydrophila. In contrast, dead eggs supported considerable numbers of fluorescent Pseudomonas sp. Analysis of potential nutrient sources for bacteria colonizing live egg surfaces revealed that small amounts of amino acids, phosphate and potassium may be lost by incubating eggs. Subsequently these nutrients were shown to be capable of supporting limited bacterial growth and reproduction. Dead eggs `leaked' increased amounts of the above nutrients which in turn supported higher bacterial numbers. In addition, biochemical analysis of eggs revealed amino acids and fatty acids that might be utilized by bacteria colonizing dead egg surfaces. Assessment of adhesion properties of bacteria frequently recovered from egg surfaces revealed high cell surface hydrophobicity as an important factor in successful egg colonization. Analysis of egg mortalities from groups of rainbow trout and brown trout (S.trutta L.) eggs maintained under two different incubation systems revealed that potentially a close correlation existed between egg surface bacterial numbers and mortalities in the egg during incubation. Innoculation of newly-fertilized eggs with bacteria demonstrated that groups of eggs supporting high numbers of P.fluorescens suffered significantly higher mortalities during the early part of their incubation. Exposure of incubating eggs to oxolinic acid, chlortetracycline and chloramphenicol demonstrated that numbers of bacteria on egg surfaces could be significantly reduced. However, as no corresponding increase in egg hatching success was revealed, the treatment of incubating eggs with antibiotics or antimicrobial compounds can not be recommended. In commercial hatcheries bacteria are only likely to be responsible for egg deaths during incubation when environmental conditions are unfavourable. High water temperatures, slow water flow rates and high egg density all lead to increased bacterial number of egg surfaces, reduced water circulation and low levels of dissolved oxygen. Under such circumstances sufficient amounts of dissolved oxygen may not be available to support developing embryos.

El hermafrodito: a propósito de la monstruosidad en la obra de Cristóbal de Castillejo

En la polifacética y satírica obra de Cristóbal de Castillejo es posible encontrar, entre los numerosos temas, el de la monstruosidad del hermafrodito. El tema, abordado en uno de sus poemas, permite conocer los conceptos y tratados de la época, así como la tradición desde la que se trazan una serie de elementos propios de la literatura, cultura y estética del siglo xvi.

Impact of Cryptosporidium spp. interaction with co-occurring microorganisms on moderate-to-severe diarrhea in the developing world

Diarrheal illness is responsible for over a quarter of all deaths in children under 5 years of age in sub-Saharan Africa and South Asia. Recent findings have identified the parasite Cryptosporidium as a contributor to enteric disease. We examined 9,348 cases and 13,128 controls from the Global Enteric Multicenter Study to assess whether Cryptosporidium interacted with co-occurring pathogens based on adjusted odds of moderate-to-severe diarrhea (MSD). Cryptosporidium was found to interact negatively with Shigella spp., with multiplicative interaction score of 0.16 (95% CI: 0.07 to 0.37, p-value=0.000), and an additive interaction score of -9.81 (95% CI: -13.61 to -6.01, p-value=0.000). Cryptosporidium also interacted negatively with Aeromonas spp., Adenovirus, Norovirus, and Astrovirus with marginal significance. Odds of MSD for Cryptosporidium co-infection with Shigella spp., Aeromonas spp., Adenovirus, Norovirus, or Astrovirus are lower than odds of MSD with either organism alone. This may reduce the efficacy of intervention strategies targeted at Cryptosporidium.