Bacillus thuringiensis development from 1971 to 1996: Cases of a research group in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Study of the presence of the spores of Clostridium botulinum in honey in Brazil

The isolation of Clostridium botulinum from honey samples is described. Botulism is characterized as an intoxication provoked by ingestion of contaminated foods with this toxin. Infant botulism happens by the ingestion of spores of C. botulinum together with food that in special conditions of the intestinal tract, such as those present in babies of less than 1 year old, will allow the germination and colonization of the intestine with production and absorption of botulinic toxin. The samples were subjected to dilution and to a thermal shock and cultivated in modified CMM (Difco). Cultures were subjected to Gram smears and toxicity tests in mice. The toxic cultures were purified in RFCA (Oxoid) plates and incubated in anaerobic jars. Positive samples were typed using the mouse assay neutralization test. From the 85 honey samples analyzed, six were positive for C. botulinum (7.06%), and identified as producers of type A, B, and D toxins.

Systematics and distribution of Thorea (Thoreaceae, Rhodophyta) from central Mexico and south-eastern Brazil

Thirteen populations of Thorea were analyzed from central Mexico and south-eastern Brazil. All populations were considered as belonging to a single species [Thorea hispida (Thore) Desvaux], with wide variation of morphological features. Secondary branches varying in frequency were observed in several populations with an overlapping in the range of branch density for Thorea violacea Bory and T. hispida (0-9 and 11-41 per 30 mm, respectively). As this is the most distinguishing character and on the basis of the overlapping (within a same population or even a single plant), we regarded T. violacea as a synonym of T. hispida. 'Chantransia' stage in culture, as well as gametophyte and carposporophyte were described in detail. We confirmed the coexistence of asexual monosporangia with sexual reproductive structures (carpogonia and spermatangia) and carposporangia. Size, content, arrangement and chromosome number were the most distinctive characteristics among spermatangia, carposporangia and monosporangia. Monosporangia can be promptly differentiated from spermatangia by their granulated content and larger size but they are similar to carposporangia in shape and size; however, monosporangia are not arranged in fascicles. Structures resembling bisporangia were observed in female plants of some populations. Chromosome numbers were n = 4 for spermatangia and fascicle cells, and 2n ca8 for gonimoblast filaments, carpospores and the 'Chantransia' stage cells. The populations of Thorea from central Mexico and south-eastern Brazil corroborated the known world distribution for T. hispida, consisting dominantly of tropical to subtropical rainforests, sometimes extending into warm temperate areas. Thorea hispida occurred in warm (temperature 17.6-28.0°C), neutral to alkaline (pH 7.0-8.0), high ion content (specific conductance 59-2140 μS cm-1), moderate flowing (current velocity 17-43 cm/s) and shallow waters (depth <50 cm); these data are essentially similar to previous reports.

Production of Nile tilapia Oreochromis niloticus and freshwater prawn Macrobrachium rosenbergii stocked at different densities in polyculture systems in Brazil

The effect of stocking prawns Macrobrachium rosenbergii at increasing densities in ponds with Nile tilapia Oreochromis niloticus reared at low density was evaluated. Twelve 0.01-ha earthen ponds were stocked with 1 tilapia/m2 and 0, 2, 4, or 6 post-larvae prawn/m2. Three replicates were randomly assigned to each prawn density. Postlarval prawns were stocked a week prior to tilapia juveniles and both were harvested 175 d after the beginning of the experiment. Tilapia final average weight, survival, production, and food conversion rates did not differ significantly among treatments (P > 0.05); the averages were 531 g. 67%, 3,673 kg/ha, and 1.91, respectively. Prawn survival rates did not differ for the three stocking densities (mean 90%). However, final weight and production were significantly different (P < 0.05) as follows: 34.0, 23.0, and 14.7 g and 639, 909, and 818 kg/ha, respectively for 2, 4, and 6 prawns/m2 densities. Stocking densities up to 6 prawn/m2 did not affect tilapia production and required neither additional feeding nor significant changes in management. The polyculture system allowed an increase in total production with the same amount of supplied feed, thus improving the system sustainability.

Human pythiosis, Brazil

Pythiosis, caused by Pythium insidiosum, occurs in humans and animals and is acquired from aquatic environments that harbor the emerging pathogen. Diagnosis is difficult because clinical and histopathologic features are not pathognomonic. We report the first human case of pythiosis from Brazil, diagnosed by using culture and rDNA sequencing.

Panoramic view of the occurrence of Yersinia species other than Y. pestis in Brazil

Data on the occurrence of Yersinia species, other than Y. pestis in Brazil are presented. Over the past 40 years, 767 Yersinia strains have been identified and typed by the National Reference Center on Yersinia spp. other than Y. pestis, using the classical biochemical tests for species characterization. The strains were further classified into biotypes, serotypes and phagetypes when pertinent. These tests led to the identification of Yersinia cultures belonging to the species Y. enterocolitica, Y. pseudotuberculosis, Y. intermedia, Y. frederiksenii and Y. kristensenii. Six isolates could not be classified in any of the known Yersinia species and for this reason were defined as Non-typable (NT). The bio-sero-phagetypes of these strains were diverse. The following species of Yersinia were not identified among the Brazilian strains by the classical phenotypic or biochemical tests: Y. aldovae, Y. rhodei, Y. mollaretti, Y. bercovieri and Y. ruckeri. The Yersinia strains were isolated from clinical material taken from sick and/or healthy humans and animals, from various types of food and from the environment, by investigators of various Institutions localized in different cities and regions of Brazil.

Determination of the quality index of a Paleudult under sunflower culture and different management systems

Soil is an essential resource for life and its properties are susceptible to be modified by tillage systems. The impact of management practices on soil functions can be assessed through a soil quality index. It is interesting to assess soil quality in different soil types. Therefore, the aim of this study was to determine the soil quality index of a Paleudult under different management conditions and sunflower culture. The experiment was carried out in Botucatu (SP, Brazil), in an 11-year non-tilled area used for growing soybean and maize during summer and black oat or triticale in winter. Four management systems were considered: no-tillage with a hoe planter (NTh), no-tillage with a double-disk planter (NTd), reduced tillage (RT) and conventional tillage (CT). Soil samples were taken from the planting lines at harvest time. To determine the soil quality indices, following the methodology proposed by Karlen and Stott (1994), three main soil functions were assessed: soil capacity for root development, water storage capacity of the soil and nutrient supply capacity of the soil. The studied Paleudult was considered a soil with good quality under all the observed management systems. However, the soil quality indices varied between treatments being 0.64, 0.68, 0.86 and 0.79 under NTh, NTd, RT and CT, respectively. Physical attributes such as resistance to penetration and macroporosity increased the soil quality index in RT and CT compared to NTh and NTd. The soil quality indices obtained suggested that the evaluated soil is adequate for sunflower production under our study conditions. In view of the SQI values, RT is the most suitable management for this site since it preserves soil quality and provides an acceptable sunflower yield. © 2011 Elsevier B.V.

Poult intestinal organ culture for propagation of Turkey coronavirus and assessment of host-virus interactions

Infection of young poults with turkey coronavirus (TCoV) produces a syndrome characterized by acute enteritis, diarrhea, anorexia, ruffled feathers, decreased body weight gain and uneven flock growth. The objective of this study was to standardize an intestinal organ culture (IOC) in order to assess host-virus interaction related to apoptosis. For this purpose the Brazilian strain (TCoV/Brazil/2006 with GenBank accession number FJ188401), was used for infection. Infected IOC cells had mitochondrial dysfunction and initial nuclear activation with MTT value of 90.7 (± 2.4) and apoptotic factor 2.21 (± 2.1), considered statistically different from uninfected IOC cells (p > 0.05). The kinetics of TCoV antigens and viral RNA was directly correlated to annexin-V, caspases- 2 and -3, p53, BCl-2 antigens at 24, 72 and 96 h post-infection (p.i.). Morphological and biochemical features of apoptosis, such as in situ nuclear fragmentation (TUNEL and annexin-V) and DNA ladder formation were also detected in infected cells at all assayed p.i. intervals. Moreover, different from other coronaviruses, the expression of both effective caspase-2 and - 3 and p53 antigens were considered lower. However, at all p.i., the BCl-2 antigens were expressed quantitatively and qualitatively as viral antigen measured by immunofluorescence microscopy analysis. Because the diagnosis of TCoV infection is only performed by infecting embryonated poult eggs, the pathological characteri tics related to host-virus interaction remain unclear. This is the first report on apoptosis of TCoV infected IOC, and reveals that it may be useful immunological method to assess virus pathogenesis.

Simple method for culture of peripheral blood lymphocytes of Testudinidae.

We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.

Seed and seedling surface-sterilization for in vitro culture of tillandsia gardneri (Bromeliaceae)

Tillandsia gardneri is a bromeliad with ornamental value and a wide geographical distribution over Brazil. However, due to habitat loss and illegal overcollection in the wild it is included as a vulnerable species in the official list of endangered plants of the State of Rio Grande do Sul, Brazil. The development of a protocol for T. gardneri seed propagation in vitro may be useful for reintroducing plants in their natural habitats, and for germplasm conservation. A difficult problem encountered during the establishment of an in vitro culture is explants disinfection, especially when working with endangered species, from which explant availability is restricted. Thus, the establishment of a sterilization protocol is crucial for the initiation and success of a micropropagation system for T. gardneri. The objective of this study was to evaluate the effect of sodium hypochlorite concentration and exposure time in seed and seedling surface disinfection, tissue sensitivity and development. Sodium hypochlorite solutions (10 or 20%/5, 10 or 15 min; 25%/5 or 10 min; and 50%/5 min) were effective in eliminating seed superficial contaminants. There was no significant difference among the effective sterilization treatments in relation to seed germination (%), and seedling length and number of leaves, after 120 days in vitro. Also, no damage to seed and seedling tissues were observed. Surface sterilization of seedlings, for initiation of an in vitro culture, required higher concentrations of sodium hypochlorite (25%/15 min; 20 or 50%/5, 10 or 15 min; and 40%/5 and 10 min) for controlling fungal and yeast contamination, compared to seed sterilization. No significant differences among these treatments were found in relation to seedling length and number of leaves, after 60 days in vitro.

Mume clonal rootstocks for 'Aurora-1' peach in São Paulo State, Brazil

In spite of significant results achieved with scion genetic improvement in stone fruits, the peach culture in Brazil still needs studies and new technologies regarding the use of rootstocks. A wide research project has being developed at the Faculdade de Ciências Agrárias e Veterinárias (FCAV/UNESP), Jaboticabal, Brazil, dealing with the use of mume clones (Prunus mume) as rootstocks for peach trees, which has produced promising results. In this research, two mume genotypes propagated by herbaceous cuttings were tested as rootstocks for peach cultivar Aurora-1. Three different tree spacing were used: 6 x 2 m, 6 x 3 m and 6 x 4 m. The experiment was carried out at Vista Alegre do Alto (21°10'14 S, 48°37'45 W, 700 m of altitude), São Paulo State, Brazil. Growing field conditions included Hapludalfs soil with medium sandy texture and using micro sprinkler irrigation. The region has an average chilling accumulation 17.9 hours per year. The evaluations were taken in 2005 and 2006 (2nd and 3rd year after planting, respectively). The trunk diameter was evaluated every three months, from the 24th to the 41st month after planting, totalizing seven evaluations. Plants on 'Rigitano' had higher trunk diameter on the 33rd, 39th and 41st month after planting (May/06, November/06 and February/07, respectively). No significant differences were observed in the other evaluations. The diameter at 5 cm above to the graft point was larger than below, but no incompatibility symptoms were observed between rootstocks and scion. Spacing tested did not influence trunk diameter, phenology and flower bud production in 'Aurora-1' scion. In conclusion, 'Rigitano' and 'Clone 15' are recommended for high density plantings of peach 'Aurora-1' in Brazil, and the 6 x 2 m spacing can be recommended, with productivity advantages for peach under low air relative humidity and mild winter conditions. © ISHS 2012.

Population dynamics of the seabob shrimp Xiphopenaeus kroyeri (Dendrobranchiata, Penaeidae) in south-eastern Brazil

The population dynamics, including the sex ratio, reproductive period, individual growth and longevity, and population structure of the shrimp Xiphopenaeus kroyeri, are described. The shrimps were collected monthly from July 2005 to June 2007 at four sites in Ubatuba Bay, Brazil. The salinity, temperature, depth, organic matter content and sediment grain size were measured at each site. Specimens were quantified and sexed, their carapace length measured and their gonads were classified according to the stage of maturation. A von Bertalanffy model was used to estimate the individual growth, and longevity was calculated using its inverted formula. In total, 6 470 individuals were examined and the number of males and females differed significantly in favour of females. During some periods, the segregation could be related to the reproductive period. Reproductive females were captured throughout the sampled period, and there were biannual peaks that were followed by peaks of juveniles. Carapace growth was less for males than for females, which is likely related to oocyte production and the higher fecundity of the females. The higher probability of female capture can be related to their longer foraging to sustain the enhanced food requirement during the ovarian maturation period. © 2013 Copyright NISC (Pty) Ltd.

Brucella ovis REO 198 natural and experimental infection in Santa Inês rams from Brazil

B. ovis pathogenicity was evaluated in experimentally inoculated and naturally infected rams. Ten animals were submitted to simultaneous conjunctival and intrapreputial inoculation with 2x109 CFU/ mL of B. ovis REO 198. After that, animals underwent physical examination and blood samples were collected for serology every week. Positive serology results started to be observed in the 3rd week, with fluctuations in titers. Clinical changes began in the 5th week after inoculation and were associated with positive serology in the acute phase of the disease. Presence of B. ovis in semen and urine culture was intermittent. Three non-inoculated animals showed natural infection. B. ovis was shed twice in semen of one serology-negative animal. The study underscored the pathogenic characteristics of B. ovis REO 198 in Santa Inês rams, as well as the importance of animals as potential sources of infection.

Persistent efficacy of 3.5% doramectin compared to 3.15% ivermectin against gastrointestinal nematodes in experimentally-infected cattle in Brazil

The present study aimed to evaluate the persistent efficacy of a 3.5% doramectin* (700μg/kg) formulation compared to 3.15% ivermectin** (630μg/kg) treatment, administered subcutaneously at a dose of 1mL/50kg body weight in cattle experimentally infected with gastrointestinal nematodes. Seventy-two male crossbred Holstein cattle that were negative for helminth infection were divided into nine groups. Treatments of 3.5% doramectin (Groups 2, 4, 6 and 8) and 3.15% ivermectin (Groups 3, 5, 7 and 9) were administered on days 49, 42, 35 and 28 prior to challenge with infectious nematode larvae (L3). Animals in the control group (Group 1) received saline solution on day 49 before challenge. Beginning on day zero, each animal received 50mL orally of a mixed culture containing approximately 3,000 third stage larvae (L3) of Haemonchus (60%), Oesophagostomum (20%), Cooperia (15%) and Trichostrongylus (5%) for seven consecutive days, resulting in a total challenge of 21,000 larvae/animal. Due to the large number of cattle, autopsies were performed between days 28 and 35 after the last day of inoculation. The formulation containing doramectin (700 mcg/kg) achieved persistent efficacy against H. placei and C. punctata for 49 and 35days, respectively. The persistent efficacy of ivermectin (630 mcg/kg) against H. placei lasted for 49days, but this treatment was ineffective against C. punctata. Both formulations demonstrated persistent efficacy against T. axei for 49days. The persistent efficacy of doramectin (700 mcg/kg) and ivermectin (630 mcg/kg) lasted for 49 and 42days against O. radiatum, respectively. © 2012 Elsevier Ltd.