Characterization of six rat strains (Rattus norvegicus) by mitochondrial DNA restriction fragment length polymorphism

Restriction fragment length polymorphism (RFLP) was used to examine the extent of mtDNA polymorphism among six strains of rats (Rattus norvegicus) - Wistar, Wistar Munich, Brown Norway, Wistar Kyoto, SHR and SHR-SP. A survey of 26 restriction enzymes has revealed a low level of genetic divergence among strains. The sites of cleavage by EcoRI, NcoI and XmnI were shown to be polymorphic. The use of these three enzymes allows the 6 strains to be classified into 4 haplotypes and identifies specific markers for each one. The percentage of sequence divergence among all pairs of haplotypes ranged from 0.035 to 0.33%, which is the result of a severe population constriction undergone by the strains. These haplotypes are easily demonstrable and therefore RFLP analysis can be employed for genetic monitoring of rats within animal facilities or among different laboratories.

Hydrolysis of xylans by enzyme systems from solid cultures of Trichoderma harzianum strains

Xylanase activity was isolated from crude extracts of Trichoderma harzianum strains C and 4 grown at 28oC in a solid medium containing wheat bran as the carbon source. Enzyme activity was demonstrable in the permeate after ultrafiltration of the crude extracts using an Amicon system. The hydrolysis patterns of different xylans and paper pulps by xylanase activity ranged from xylose, xylobiose and xylotriose to higher xylooligosaccharides. A purified ß-xylosidase from the Trichoderma harzianum strain released xylose, xylobiose and xylotriose from seaweed, deacetylated, oat spelt and birchwood xylans. The purified enzyme was not active against acetylated xylan and catalyzed the hydrolysis of xylooligosaccharides, including xylotriose, xylotetraose and xylopentaose. However, the enzyme was not able to degrade xylohexaose. Xylanase pretreatment was effective for hardwood kraft pulp bleaching. Hardwood kraft pulp bleached in the XEOP sequence had its kappa number reduced from 13.2 to 8.9 and a viscosity of 20.45 cp. The efficiency of delignification was 33%.

A comparative study of aging of the elastic fiber system of the diaphragm and the rectus abdominis muscles in rats

In the present study the age-related changes of the striated muscle elastic fiber system were investigated in the diaphragm and rectus abdominis muscles of 1-, 4-, 8- and 18-month-old rats. The activation patterns of these muscles differ in that the diaphragm is regularly mobilized tens of times every minute during the entire life of the animal whereas the rectus abdominis, although mobilized in respiration, is much less and more irregularly activated. The elastic fibers were stained by the Verhoeff technique for mature elastic fibers. Weigert stain was used to stain mature and elaunin elastic fibers, and Weigert-oxone to stain mature, elaunin and oxytalan elastic fibers. The density of mature and elaunin elastic fibers showed a progressive increase with age, whereas the amount of oxytalan elastic fibers decreased in both diaphragm and rectus abdominis muscles and their muscular fascias. These age-related quantitative and structural changes of the elastic fiber system may reduce the viscoelastic properties of the diaphragm and rectus abdominis muscles, which may compromise the transmission of tensile muscle strength to the tendons and may affect maximum total strength.

Antimicrobial resistance among invasive Haemophilus influenzae strains: results of a Brazilian study carried out from 1996 through 2000

A total of 1712 strains of Haemophilus influenzae isolated from patients with invasive diseases were obtained from ten Brazilian states from 1996 to 2000. ß-Lactamase production was assessed and the minimum inhibitory concentrations (MIC) of ampicillin, chloramphenicol, ceftriaxone and rifampin were determined using a method for broth microdilution of Haemophilus test medium. The prevalence of strains producing ß-lactamase ranged from 6.6 to 57.7%, with an overall prevalence of 18.4%. High frequency of ß-lactamase-mediated ampicillin resistance was observed in Distrito Federal (25%), São Paulo (21.7%) and Paraná (18.5%). Of the 1712 strains analyzed, none was ß-lactamase negative, ampicillin resistant. A total of 16.8% of the strains were resistant to chloramphenicol, and 13.8% of these also presented resistance to ampicillin, and only 3.0% were resistant to chloramphenicol alone. All strains were susceptible to ceftriaxone and rifampin and the MIC90 were 0.015 µg/ml and 0.25 µg/ml, respectively. Ceftriaxone is the drug of choice for empirical treatment of bacterial meningitis in pediatric patients who have not been screened for drug susceptibility. The emergence of drug resistance is a serious challenge for the management of invasive H. influenzae disease, which emphasizes the fundamental role of laboratory-based surveillance for antimicrobial resistance.

Skin morphology of the mutant hairless USP mouse

The morphology of the skin of the mutant hairless USP mouse was studied by histological, histochemical and immunohistochemical methods and compared to the skin of BALB/c mice. Representative sections of the dorsal skin from mice of both strains aged 18 days, and 1, 3, 6, and 8 months were studied. Sections stained with hematoxylin and eosin showed cystic formations called utricles and dermal cysts in the dermis that increased in size and number during growth. Skin thickness increased significantly at 8 months. Sections stained with picrosirius and examined with polarized light, displayed different colors, suggesting different thicknesses of dermal collagen fibers (probably types I and III). Weigert, Verhoeff and resorcin-fuchsin stains revealed fibers of the elastic system. The PAS and Alcian blue methods revealed neutral and acid glycosaminoglycans in the skin ground substance of both mouse strains. Immunohistochemical staining for fibronectin and laminin did not show differences between the mutant and BALB/c mice. Mast cells stained by the Gomori method and macrophages positive for HAM 56 antibodies were observed in both mouse strains. Except for the presence of enlarged cysts in the hairless strain, no qualitative differences were found during development of the skin of BALB/c and the mutant hairless mice.

Occurrence of Haemophilus influenzae strains in three Brazilian states since the introduction of a conjugate Haemophilus influenzae type b vaccine

Few vaccines in history have induced such a dramatic decline in incidence over such a short period of time as the Haemophilus influenzae type b (Hib) conjugate. This vaccine was introduced in 1988 in the United States, but only in 1999 was Hib immunization introduced by the Brazilian Ministry of Health as part of the routine infant National Immunization Program. The authors analyzed 229 H. influenzae (Hi) isolates from Public Health Laboratories in three Brazilian states: Pernambuco (Northeast, N = 54), Santa Catarina (South, N = 19), and Rio de Janeiro (Southeast, N = 156). The isolates were collected from Brazilian children 0-10 years of age with meningitis and other infections from 1990 to 2003 and were part of the research collection of the National Institute of Quality Control in Health, FIOCRUZ. Bacterial strains were characterized by serotyping and biotyping. During the pre-vaccination period the prevalence infection due to Hib was of 165 isolates and only 2 non-b Hi among all the notified meningitis infections caused by Hi. Our results showed a significant decrease in the prevalence of Hib meningitis from 165 to 33 isolates after 1999. However, during the post-vaccination period of 2001-2003 we observed an increase in the number of non-b Hi isolates: only 2 non-b strains isolated from 1990 to 1999 and 29 from 1999 to 2003. Based on the present data, the authors emphasize the need for more sensitive epidemiological and bacteriological studies aiming the improvement of the available Hib vaccine, in order to protect the susceptible population to infections due to other serological types of Hi and the reevaluation of immunization schedules used by the National Immunization Program.

Epidemiological characterization of resistance and PCR typing of Shigella flexneri and Shigella sonnei strains isolated from bacillary dysentery cases in Southeast Brazil

Shigella spp are Gram-negative, anaerobic facultative, non-motile, and non-sporulated bacilli of the Enterobacteriaceae family responsible for "Shigellosis" or bacillary dysentery, an important cause of worldwide morbidity and mortality. However, despite this, there are very few epidemiological studies about this bacterium in Brazil. We studied the antibiotic resistance profiles and the clonal structure of 60 Shigella strains (30 S. flexneri and 30 S. sonnei) isolated from shigellosis cases in different cities within the metropolitan area of Campinas, State of São Paulo, Brazil. We used the following well-characterized molecular techniques: enterobacterial repetitive intergenic consensus, repetitive extragenic palindromic, and double-repetitive element-polymerase chain reaction to characterize the bacteria. Also, the antibiotic resistance of the strains was determined by the diffusion disk method. Many strains of S. flexneri and S. sonnei were found to be multi-resistant. S. flexneri strains were resistant to ampicillin in 83.3% of cases, chloramphenicol in 70.0%, streptomycin in 86.7%, sulfamethoxazole in 80.0%, and tetracycline in 80.0%, while a smaller number of strains were resistant to cephalothin (3.3%) and sulfazotrim (10.0%). S. sonnei strains were mainly resistant to sulfamethoxazole (100.0%) and tetracycline (96.7%) and, to a lesser extent, to ampicillin (6.7%) and streptomycin (26.7%). Polymerase chain reaction-based typing supported the existence of specific clones responsible for the shigellosis cases in the different cities and there was evidence of transmission between cities. This clonal structure would probably be the result of selection for virulence and resistance phenotypes. These data indicate that the human sanitary conditions of the cities investigated should be improved.

Trailing end-point phenotype antibiotic-sensitive strains of Candida albicans produce different amounts of aspartyl peptidases

Candida albicans is an opportunistic fungal pathogen that causes severe systemic infections in immunosuppressed individuals. C. albicans resistance to antifungal drugs is a severe problem in patients receiving prolonged therapy. Moreover, trailing yeast growth, which is defined as a resistant MIC after 48 h of incubation with triazole antifungal agents but a susceptible MIC after 24 h, has been noted in tests of antifungal susceptibility against some C. albicans isolates. In this context, we recently noticed this phenomenon in our routine susceptibility tests with fluconazole/itraconazole and C. albicans clinical isolates. In the present study, we investigated the production of cell-associated and secreted aspartyl peptidases (Saps) in six trailing clinical isolates of C. albicans, since this class of hydrolytic enzymes is a well-known virulence factor expressed by this fungal pathogen. Sap2, which is the best-studied member of the Sap family, was detected by flow cytometry on the cell surface of yeasts and as a 43-kDa polypeptide in the culture supernatant, as demonstrated by Western blotting assay using an anti-Sap1-3 polyclonal antibody. Released aspartyl peptidase activity was measured with BSA hydrolysis and inhibited by pepstatin A, showing distinct amounts of proteolytic activity ranging from 5.7 (strain 44B) to 133.2 (strain 11) arbitrary units. Taken together, our results showed that trailing clinical isolates of C. albicans produced different amounts of both cellular and secreted aspartyl-type peptidases, suggesting that this phenotypic feature did not generate a regular pattern regarding the expression of Sap.

Genetic analysis of the porcine group B rotavirus NSP2 gene from wild-type Brazilian strains

Group B rotaviruses (RV-B) were first identified in piglet feces, being later associated with diarrhea in humans, cattle, lambs, and rats. In human beings, the virus was only described in China, India, and Bangladesh, especially infecting adults. Only a few studies concerning molecular analysis of the RV-B NSP2 gene have been conducted, and porcine RV-B has not been characterized. In the present study, three porcine wild-type RV-B strains from piglet stool samples collected from Brazilian pig herds were used for analysis. PAGE results were inconclusive for those samples, but specific amplicons of the RV-B NSP2 gene (segment 8) were obtained in a semi-nested PCR assay. The three porcine RV-B strains showed the highest nucleotide identity with the human WH1 strain and the alignments with other published sequences resulted in three groups of strains divided according to host species. The group of human strains showed 92.4 to 99.7% nucleotide identity while the porcine strains of the Brazilian RV-B group showed 90.4 to 91.8% identity to each other. The identity of the Brazilian porcine RV-B strains with outer sequences consisting of group A and C rotaviruses was only 35.3 to 38.8%. A dendrogram was also constructed to group the strains into clusters according to host species: human, rat, and a distinct third cluster consisting exclusively of the Brazilian porcine RV-B strains. This is the first study of the porcine RV-B NSP2 gene that contributes to the partial characterization of this virus and demonstrates the relationship among RV-B strains from different host species.

Comparative pathogenicity of Coxsackievirus A16 circulating and noncirculating strains in vitro and in a neonatal mouse model

An enterovirus 71 (EV71) vaccine for the prevention of hand, foot, and mouth disease (HMFD) is available, but it is not known whether the EV71 vaccine cross-protects against Coxsackievirus (CV) infection. Furthermore, although an inactivated circulating CVA16 Changchun 024 (CC024) strain vaccine candidate is effective in newborn mice, the CC024 strain causes severe lesions in muscle and lung tissues. Therefore, an effective CV vaccine with improved pathogenic safety is needed. The aim of this study was to evaluate the in vivo safety and in vitro replication capability of a noncirculating CVA16 SHZH05 strain. The replication capacity of circulating CVA16 strains CC024, CC045, CC090 and CC163 and the noncirculating SHZH05 strain was evaluated by cytopathic effect in different cell lines. The replication capacity and pathogenicity of the CC024 and SHZH05 strains were also evaluated in a neonatal mouse model. Histopathological and viral load analyses demonstrated that the SHZH05 strain had an in vitro replication capacity comparable to the four CC strains. The CC024, but not the SHZH05 strain, became distributed in a variety of tissues and caused severe lesions and mortality in neonatal mice. The differences in replication capacity and in vivo pathogenicity of the CC024 and SHZH05 strains may result from differences in the nucleotide and amino acid sequences of viral functional polyproteins P1, P2 and P3. Our findings suggest that the noncirculating SHZH05 strain may be a safer CV vaccine candidate than the CC024 strain.

Lactobacillus plantarum strains isolated from naturally fermented sausages and their technological properties for application as starter cultures

In the present study, technological properties of L. plantarum strains isolated from naturally fermented sausages manufactured in the South region of Brazil were investigated in order to obtain a starter culture. The technological properties evaluated were the following: ability to growth at different pH values, at different temperatures, in different salt concentrations and in the presence of commercial curing salt, fast production of acid, determination of D - and L - lactic acid; nitrate reductase activity; antagonistic activity and stability of the isolated cultures after fermentation, concentration, and freeze-drying process. The isolated strains showed effectiveness to improve technological properties as starter cultures.

Viscosity of egg white from hens of different strains fed with commercial and natural additives

Yolk color and egg white (albumen) cleanliness and viscosity are important parameters by which consumers judge the quality of eggs. This study aimed to investigate changes in albumen viscosity during storage of eggs for up to 36 days from two different commercial laying hen strains (Carijo Barbada and Isa Brown) fed a diet containing annatto (1.5 and 2.0%) or a synthetic additive without synthetic colorants (control). Analyses of humidity, albumen height, pH, viscosity, foam formation, and stability were carried out on eggs. Carijo Barbada strain had smaller albumen, lower humidity and higher egg white viscosity than Isa Brown strain; however, with storage, viscosity lowered significantly on both strains. Initially, the addition of 2.0% of annatto or a synthetic additive increased viscosity in both strains, but with storage only the control maintained longer viscosity. Lower viscosity did not change foam density and stability.

Isolation and characterization of bacterial strains with a hydrolytic profile with potential use in bioconversion of agroindustial by-products and waste

There is a trend towards the use of novel technologies nowadays, mainly focused on biological processes, for recycling and the efficient utilization of organic residues that can be metabolized by different microorganisms as a source of energy. In the present study the isolation of bacterial strains from six different agro-industrial by-products and waste was performed with the objective of evaluating their hydrolytic capacities and suitability for use in bioconversion of specific substrates. The 34 isolated strains were screened in specific culture media for the production of various hydrolytic enzymes (lipase, protease, cellulase, and amylase). It was found that 28 strains exhibited proteolytic activity, 18 had lipolytic activity, 13 had caseinolytic activity, 15 had amylolytic activity, and 11 strains exhibited cellulolytic activity. The strains that showed the highest hydrolytic capacities with biotechnological potential were selected, characterized genotipically, and identified as Bacillus, Serratia, Enterococcus, Klebsiella, Stenotrophomonas, Lactococcus, and Escherichia genera. It was concluded that the strain isolates have a high potential for use in the bioconversion of agro-industrial waste, both as a pure culture and as a microbial consortium.

Survival of Lactobacillus rhamnosus probiotic strains in peach jam during storage at different temperatures

The survival of six probiotic wild strains of Lactobacillus rhamnosus was compared with that of a type strain during 78 days of storage at 25 and 5 ºC in peach synthetic medium (PSM) and commercial peach jam (PJ). Changes in viable cell counts, pH values, sugar content, and colour parameters were monitored. All strains exhibited better performances in PJ than in PSM, showing count values higher than 7 Log cfu g-1 up to 78 days of storage at 5 ºC. Almost all wild strains remained above the critical value of 6 Log cfu g-1 in samples stored at 25 ºC up to 45 days, while the Lb. rhamnosus GG type strain, used as control, was not able to survive later than 15 days. In the synthetic medium used, the strains showed better survival in the samples incubated at 25 ºC, remaining viable above the critical level up to 45 days of storage, except for the strain H12. The probiotic cultures added to jam did not significantly change the colour parameters of the product; however the metabolism of lactobacilli did cause changes in the pH and in the composition of sugars.

Characterization of the chitinolytic system during the mycoparasitic interaction between Trichoderma aggressivum f. aggressivum and different host strains of Agaricus bisporus /

Green mould is a serious disease of commercially grown mushrooms, the causal agent being attributed to the filamentous soil fungus Triclzodenna aggressivum f. aggressivu11l and T. aggressivum f. ellropaellm. Found worldwide, and capable of devastating crops, this disease has caused millions of dollars in lost revenue within the mushroom industry. One mechanism used by TricllOdenlla spp. in the antagonism of other fungi, is the secretion of lytic enzymes such as chitinases, which actively degrade a host's cell wall. Therefore, the intent of this study was to examine the production of chitinase enzymes during the host-parasite interaction of Agaricus bisporus (commercial mushroom) and Triclzodemza aggressivum, focusing specifically on chitinase involvement in the differential resistance of white, off-white, and brown commercial mushroom strains. Chitinases isolated from cultures of A. bisporus and T. aggressivu11l grown together and separately, were identified following native PAGE, and analysis of fluorescence based on specific enzymatic cleavage of 4-methylumbelliferyl glucoside substrates. Results indicate that the interaction between T. aggressivulll and A. bisporus involves a complex enzyme battle. It was determined that T. aggressivum produces a number of chitinases that appear to correlate to those isolated in previous studies using biocontrol strains of T. Izarziallilm. A 122 kDa N-acetylglucosaminidase of T. aggressivu11l revealed the highest and most variable activity, and is therefore believed to be an important predictor of antifungal activity. Furthermore, results indicate that brown strain resistance of mushrooms may be related to high levels of a 96 kDa N-acetylglucosaminidase, which showed elevated activity in both solitary and dual cultures with T. aggressivum. Overall, each host-parasite combination produced unique enzyme profiles, with the majority of the differences seen between day 0 and day 6 for the extracellular chitinases. Therefore, it was concluded that the antagonistic behaviour of T. aggressivli1ll does not involve a typical response, always producing the same types and levels of enzymes, but that mycoparasitism, specifically in the form of chitinase production, may be induced and regulated based on the host presented.