Recombinogenic, genotoxic, and cytotoxic effects of azathioprine using in vivo assays.

Azathioprine (Aza) is a purine antimetabolite immunosuppressant that is widely employed for immunosuppressive therapy in post-transplant recipients or patients with autoimmune diseases. Chronic use of immunosuppressants might produce several side effects, including a high rate of neoplasms in these patients. Considering that genotoxic effects are associated with an increased risk of developing cancer, the aim of this study was to examine the recombinogenic, genotoxic, and cytotoxic effects of Aza using Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster, as well as comet and micronucleus assays in mouse bone marrow cells. Further, the adverse effects of Aza were determined in mouse hepatic and renal tissues using histopathological analysis. Data demonstrated that Aza induced significant increased genotoxicity in D. melanogaster and mouse bone marrow cells at all concentrations tested. Homologous recombination was the predominant genotoxic event noted for the first time to be initiated by Aza in SMART. In histopathological analysis, Aza did not show any marked toxic activity in mouse hepatic and renal tissues. Therefore, the high rate of neoplasms reported in patients with long-term use of Aza may be attributed, at least partially, to the genotoxic action of this drug.

Performance of rapid prescreening and 100% rapid review as internal quality control methods for cervical cytopathology.

BACKGROUND: An objective of quality control for cervical cytopathology is reducing high rates of false-negative results of laboratory tests. Therefore, methods to review smears such as rapid prescreening and 100% rapid review, which have shown better performance detecting false-negative results, have been widely used. The performance of rapid prescreening and the performance of 100% rapid review as internal quality control methods for cervical cytology examinations were evaluated. METHODS: For 24 months, 9318 conventional cervical cytology smears underwent rapid prescreening and routine screening. The 100% rapid review method was performed for 8244 smears classified as negative during routine screening. Any discordant results underwent detailed review to define the final diagnosis. This was considered the gold standard for evaluating the performance of rapid prescreening and 100% rapid review. RESULTS: Routine screening showed increases of 13.3% and 11.5% in the detection of abnormal smears with rapid prescreening and 100% rapid review, respectively. The relative percentage variation showed a 38.1% increase in the diagnosis of atypical squamous cells of undetermined significance with routine screening and rapid prescreening and a 12.5% increase in the diagnosis of atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion with both rapid prescreening and 100% rapid review. Sensitivity rates of rapid prescreening and routine screening were 48.2% and 83.2%, respectively. Sensitivity rates of rapid prescreening and 100% rapid review were 65.7% and 57.8%, respectively, for detecting false-negative results. CONCLUSIONS: Inclusion of rapid prescreening and/or 100% rapid review improved the diagnostic sensitivity of the cervical cytology examination and reduced false-negative results of routine screening and can provide good quality control.

Hospitalisation of ambulatory care sensitive conditions and access to primary care in Portugal.

OBJECTIVES: Our aim is to study the relationship between the incidence of hospitalisations of ambulatory care sensitive conditions (ACSCs) in small geographic areas and characteristics of the primary care delivery system that are associated with access to care such as travel time to the provider, volume of resources and patient-doctor ties. We try to assess the relative importance of access barriers and draw useful implications for healthcare planning policy. STUDY DESIGN: This work combines the approach of ACSC rates by Billings et al. (1993) and the Penchansky and Thomas (1981) framework of access, building a tool for analysing variations in small areas and assessing barriers to access for primary health care. We explicitly address and describe some of those barriers through an empirical test of the relevance on ACSC hospitalisation rates of factors that can be grouped in the access dimensions of that framework. METHODS: We examine data of hospitalisations in public hospitals and characteristics of the public primary care delivery system in small areas of Portugal in 2014, estimating two models by ordinary least squares: one with variables that capture specific aspects of access and one without such variables. We then compared the predictive power of the two models. RESULTS: Although data on specific access barriers are scarce, results show that a model with access variables explains 70% of the variation in ACSC hospitalisation rates, 11 percentage points more than a model without such variables. CONCLUSIONS: Our results suggest that increasing resources do not seem to necessarily enhance patient access to care. Other factors, such as reduced travel time and long-term patient-doctor ties, arise as more important in reducing unnecessary hospitalisations. Thus, the solution for increasing the access to primary health care and reducing disparities among small areas seems to lie more in organisational changes than in allocation of resources.

Cichlids of the Banc d'Arguin National Park, Mauritania: insight into the diversity of the genus Coptodon.

To determine the species diversity of cichlids in the Banc d'Arguin National Park (PNBA) and their phylogenetic relationships with other species in West Africa, a morphometric and meristic and molecular phylogenetic study was conducted. Both approaches not only confirm the presence of Sarotherodon melanotheron in PNBA but also demonstrate the presence of a second species from the genus Coptodon. While morphometric characteristics match the description of the Guinean tilapia Coptodon guineensis, phylogenetic reconstructions based on three mitochondrial and one nuclear DNA fragment demonstrate that C. guineensis is paraphyletic over its range. Because different lineages of C. guineensis are allopatric, the distribution of C. guineensis should be restricted to Ghana and Côte d'Ivoire. The many other lineages of this species should be considered as C. sp. aff. guineensis.

Total Mercury in Carnivorous Fish from Brazilian Southeast.

Total mercury concentrations were determined by atomic absorption spectrometry in muscle tissue of two commercially important species of carnivorous fishes croaker (Micropogonias furnieri) and cutlassfish (Trichiurus lepturus) caught in Itaipu estuary, Rio de Janeiro. In this study, whitemouth croaker presented more mercury than Atlantic cutlassfish. Bioaccumulation differences can be explained by the biological behavior of juveniles whitemouth croaker, that remain in the estuary up to maturity, which makes them good indicators of local environmental impacts (0.110 mg Kg(-1) HgT). It also can be explained by differences in nutritional requirements between the different life stages of two species. The analysis showed the presence of low levels of the metal. However, our results suggest a possible risk to human health, depending on the level of fish consumption.

The effect of carbon dioxide on the shelf life of ready-to-eat shredded chicken breast stored under refrigeration.

The objective of the present study was to determine the shelf life of ready-to-eat cooked chicken breast fillets (shredded) stored in atmospheres that were modified with different concentrations of CO2 and to establish a relationship between the concentration of this gas and bacterial growth. The samples were divided into 7 groups with different packaging conditions: aerobiosis, vacuum, and 10, 30, 50, 70, and 90% CO2 (with the remaining volume filled with N2). All of the samples were stored at 4 ± 2°C for 28 d. During this period, pH tests and counts of aerobic heterotrophic mesophyll bacteria (AHMB), aerobic heterotrophic psychotropic bacteria (AHPB), Enterobacteriaceae, and lactic acid bacteria (LAB) were performed, and the gas compositions of the packaging atmospheres were verified. The pH of the aerobic packages increased during storage. However, the other treatments resulted in the opposite trend, with the CO2 concentration decreasing over the first 24 h and then remaining constant until the end of experiment. A gradual increase in the AHMB, AHPB, Enterobacteriaceae, and LAB counts was observed during storage; this increase was faster in the meat that was packed under aerobiosis conditions than in the other treatments. The treatments with a CO2 concentration above 10% exhibited lower Enterobacteriaceae growth, whereas LAB growth was discrete in all of the treatments, independent of the CO2 concentration. The shelf life of the samples packed with 90% CO2 was 28 d. Based on the AHMB and AHPB counts, the shelf life was 3 times longer than for the samples packed under aerobiosis conditions (9 d). The increased package CO2 concentration caused a reduction in the growth rate of the examined bacteria (r = 0.99), and treatment with 90% CO2 appears promising as a method with which to increase the product's shelf life.

Microbiological quality and biogenic amines in ready-to-eat grilled chicken fillets under vacuum packing, freezing, and high-dose irradiation.

The combined effects of cooking, vacuum packing, freezing, and high-dose gamma irradiation in the microbiological conservation and in biogenic amine (BA) contents of ready-to-eat grilled breast chicken fillets are investigated in this work. After seasoning, cooking, and vacuum packing, one-third of the samples were stored at -25°C (T1). The remaining two-thirds were treated with 48 kGy, one-third being stored at -25°C (T2) and the other one-third kept at room temperature (T3). All samples were periodically analyzed to determine growth of heterotrophic aerobic mesophilic bacteria (HAMB) and levels of BA (tyramine, TYM; putrescine, PUT; cadaverine, CAD; spermidine, SPD; histamine, HYM; and spermine, SPM). Variance analysis was performed to determine significant changes in the measured data. Grilling caused HAMB counts in seasoned samples to drop from 5.3 log cfu/g to zero. In addition, no viable HAMB cells were detected in the samples throughout the 12-mo storage time. Regarding the BA analyses, the highest mean levels were measured for SPM and CAD with significantly higher levels (P < 0.05) being determined in nonirradiated samples (T1). Furthermore, significantly lower mean levels for the total content of BA were observed in the irradiated samples. Relative to T1 (7.5 ± 1.5 mg/kg), the figures were 47 ± 23% for T2 and 60 ± 25% for T3, mostly due to loss of CAD by radiolysis. Therefore, it can be concluded that the combination of grilling, vacuum packing, freezing, and high-dose gamma irradiation efficiently eliminated HAMB, while sustaining acceptable levels of BA in ready-to-eat chicken breast fillets throughout the 12 mo of storage at room temperature.

Assessment of genotoxic, cytotoxic, and protective effects of Salacia crassifolia (Mart. Ex. Schult.) G. Don. stem bark fractions in mice.

Salacia crassifolia (Mart. Ex. Schult.) G. Don., popularly known in Brazil as "bacupari", "cascudo", and "saputá", is a shrub of the Celastraceae family that is unique to the Brazilian Cerrado region. In folk medicine, this plant has been mainly used to treat skin cancer and gastric ulcers. In the present study, the genotoxic, cytotoxic, antigenotoxic, and anticytotoxic effects of S. crassifolia stem bark fractions (hexane, ethyl acetate, and hydroalcoholic extracts) were evaluated using the mouse bone marrow micronucleus test. Our results showed that none of the S. crassifolia fractions led to a significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) (P > 0.05), suggesting the absence of genotoxicity. In the antigenotoxicity assessment, a significant decrease in the MNPCE frequency was observed in all fractions of this plant (P < 0.05), demonstrating its protective action against genotoxicity induced by mitomycin C (MMC), which was used as the positive control. Only the hexane fraction of S. crassifolia significantly decreased the poly- and normochromatic erythrocyte ratio (PCE/NCE) in all doses tested (P < 0.05), demonstrating its cytotoxic activity. In association with MMC, both ethyl acetate and hydroalcoholic fractions significantly increased the PCE/NCE ratio in almost all doses tested (P < 0.05), demonstrating the protective action of S. crassifolia against the cytotoxic effect of the positive control. In contrast, the hexane fraction presented a significant decrease in the PCE/NCE ratio in all treatments (P < 0.05), demonstrating an increase in this plant's cytotoxicity in mouse bone marrow cells.

Human papillomavirus infection in Brazilian women with normal cervical cytology.

We examined the prevalence of human papillomavirus (HPV) infection in a sample of Brazilian women presenting normal cervical cytology. Possible interactions between patient characteristics and HPV infection were analyzed in order to provide background data to improve cervical cancer screening and prophylaxis. Cervical samples of 399 women, received for routine evaluation in the Health Department of Ouro Preto, MG, Brazil, were subjected to HPV-DNA testing by PCR with MY09/11 primers. HPV-positive specimens were typed by RFLP. A structured epidemiological questionnaire was administered to each woman. HPV prevalence among these cytologically normal women was 11%. Twelve viral types were detected, the most common being HPV-16, -6, -61, -83, and -66. HPV was more prevalent in younger women; high-risk viral types were detected in 61% of the infected women and 27% of the infected women had multiple HPV infections. Significant associations of HPV infection were found with age, literacy, residence, marital status, lifetime number of sexual partners, and parity. We detected a great diversity of HPV types in women with normal cytology. This kind of information about local populations is useful for HPV prevention and vaccination strategies.

In vivo wound healing effects of Symphytum officinale L. leaves extract in different topical formulations.

The present work evaluates wound healing activity of leaves extracts of Symphytum officinale L. (comfrey) incorporated in three pharmaceutical formulations. Wound healing activity of comfrey was determined by qualitative and quantitative histological analysis of open wound in rat model, using allantoin as positive control. Three topical formulations, carbomer gel, glycero-alcoholic solution and O/W emulsion (soft lotion) were compared. The histological analysis of the healing process shows significant differences in treatment, particularly on its intensity and rate. The results indicate that emulsion containing both extracts, commercial and prepared, induced the largest and furthest repair of damaged tissue. This could be evidenced from day 3 to 28 by increase in collagen deposition from 40% to 240% and reduction on cellular inflammatory infiltrate from 3% to 46%. However, 8% prepared extract in emulsion presented the best efficacy. This work clearly demonstrates that comfrey leaves have a wound healing activity. The O/W emulsion showed to be the vehicle most effective to induce healing activity, particularly with extracts obtained from comfrey leaves collected in Minas Gerais state in Brazil. It shows the best efficacy to control the inflammatory process and to induce collagen deposition at 8% concentration.

Splenectomy increases mortality in murine Trypanosoma cruzi infection.

The spleen is a secondary lymphoid organ that harbours a variety of cells such as T and B lymphocytes and antigen-presenting cells important to immune response development. In this study, we evaluated the impact of spleen removal in the immune response to experimental Trypanosoma cruzi infection. C57BL/6 mice were infected with Y strain of the parasite and infection was followed daily. Mice that underwent splenectomy had fewer parasites in peripheral blood at the peak of infection; however, mortality was increased. Histological analysis of heart and liver tissues revealed an increased number of parasites and inflammatory infiltrates at these sites. Spleen removal was associated with reduction in IFN-γ and TNF-α production during infection as well as with a decrease in specific antibody secretion. Haematological disorders were also detected. Splenectomized mice exhibited severe anaemia and decreased bone marrow cell numbers. Our results indicate that spleen integrity is critical in T. cruzi infection for the immune response against the parasite, as well as for the control of bone marrow haematological function.

Controlling bacteriological contamination of environmental surfaces at the biological isolation and containment unit of a veterinary teaching hospital.

BACKGROUND: The Biological Isolation and Containment Unit (BICU) is a subunit of the Teaching Hospital of the Faculty of Veterinary Medicine of the University of Lisbon, Portugal, for the admission of animals with confirmed infectious diseases or under clinical suspicion and waiting for a diagnosis. As a high-risk environment for the transmission of infectious agents, it is extremely important to implement programs for the surveillance of nosocomial microorganisms in these facilities. The purpose of this study was to evaluate the level of bacterial contamination of the BICU environmental surfaces and to implement corrective actions on disinfection protocols. Swab samples were collected from selected environmental surfaces in 3 different areas of the BICU (isolation, work, and preparatory rooms) to evaluate the total aerobic bacterial load and investigate the presence of 4 nosocomial microorganisms: vancomycin-resistant Enterococcus spp., methicillin-resistant Staphylococcus aureus, 3rd-generation cephalosporin-resistant Escherichia coli, and carbapenem-resistant Pseudomonas aeruginosa. Bacterial quantification was performed by using non-selective media, while specific selective media were used for the isolation of the target microorganisms. Isolates were identified based on their macro and microscopic characteristics and their biochemical profile. Subsequently, new disinfection protocols were implemented, and their effectiveness evaluated. RESULTS: The surfaces with the highest bacterial load in the isolation, preparatory, and worker's rooms were the cages, hand-held sponge, and telephone, respectively. Regarding the 4 pathogens investigated, Enterococcus spp. were the most frequently isolated (11.3%), followed by E. coli (1.5%) and P. aeruginosa (1.5%). One of the P. aeruginosa isolates obtained was resistant to imipenem. In the end, new disinfection protocols were implemented, which proved to be effective in reducing bacterial counts by 99.99% in cages and the sponge, and by 90 to 99% on the telephone. CONCLUSIONS: This study allows to conclude that the cages and the human contact surfaces were the most contaminated in the isolation rooms. Nevertheless, the new disinfection strategies seemed to be effective in reducing environmental contamination, including by some potentially nosocomial agents, although more samples must be analyzed for definitive conclusions. These results may contribute to highlight the importance of infection prevention and control measures, as fundamental tools to reduce the spread of infectious agents in the hospital environment.

Ageing down-modulates liver inflammatory immune responses to schistosome infection in mice.

Ageing is associated with several alterations in the immune system. Our aim in this study was to compare the development of immunity to Schistosoma mansoni infection in young versus aged C57Bl/6 mice using the liver as the main organ to evaluate pathological alterations and immune responses. In the acute phase, young mice had large liver granulomas with fibrosis and inflammatory cells. Chronic phase in young animals was associated with immunomodulation of granulomas that became reduced in size and cellular infiltrate. On the other hand, aged animals presented granulomas of smaller sizes already in the acute phase. Chronic infection in these mice was followed by no alteration in any of the inflammatory parameters in the liver. In concert with this finding, there was an increase in activated CD4+ T, CD19+ B and NK liver cells in young mice after infection whereas old mice had already higher frequencies of activated B, NK and CD4+ T liver cells and infection does not change these frequencies. After infection, liver production of inflammatory and regulatory cytokines such as IFN-gamma, IL-4 and IL-10 increased in young but not in old mice that had high levels of IL-4 and IL-10 regardless of their infection status. Our data suggest that the unspecific activation status of the immune system in aged mice impairs inflammatory as well as regulatory immune responses to S. mansoni infection in the liver, where major pathological alterations and immunity are at stage. This poor immune reactivity may have a beneficial impact on disease development.

Acid phosphatase activity during the interaction of the nematophagous fungus Duddingtonia flagrans with the nematode Panagrellus sp.

The use of Duddingtonia flagrans, a nematode-trapping fungus, has been investigated as a biological control method against free living larvae of gastrointestinal nematodes of livestock animals. This fungus captures and infects the nematode by cuticle penetration, immobilization and digestion of the internal contents. It has been suggested that this sequence of events occurs by a combination of physical and enzymatical activities. This report characterizes the acid phosphatase activity during the interaction of D. flagrans with the free-living nematode Panagrellus sp. The optimum pH for the hydrolysis of the acid phosphatase substrate p-nitrophenyl phosphate was 2.2, 2.8 and 5.4 from D. flagrans alone and 2.2 and 5.4 for Panagrellus sp alone, fungus-nematode interaction in liquid medium and fungus-nematode interaction in solid medium. Different acid phosphatase activity bands were detected by SDS-PAGE. Maximum acid phosphatase activity of the fungus or nematode alone and of the fungus-nematode interaction occurred within 70min of incubation in the presence of the substrate 4-methylumbelliferyl phosphate. The activity of this enzyme was significantly higher for the fungus-nematode interaction when compared to the organisms alone, indicating a synergistic response. Furthermore, structures appeared in the hyphae after 30min, nematodes were observed adhered after 40min and many were captured by the typical fungus traps after 70min of interaction. The participation of acid phosphatase activity and its importance during the interaction of the fungus with the nematode were discussed.

Chemiluminescent ELISA with multi-epitope proteins to improve the diagnosis of canine visceral leishmaniasis.

Diagnosing canine visceral leishmaniasis (CVL) is difficult because clinical signs of the disease are non-specific and a many infected animals in endemic areas, as in Brazil, are asymptomatic. Serological tests are the most common diagnostic methods employed, but most have limitations. For this reason, the implementation of a rapid, sensitive, and specific diagnostic test for CVL has become increasingly important. In this study, we adapted a chemiluminescent enzyme-linked immunosorbent assay (CL ELISA), using two multi-epitope recombinant proteins (PQ10 and PQ20) and a crude Leishmania antigen produced using promastigotes of L. infantum, as antigens to detect CVL infection in animals from Belo Horizonte. To investigate cross-reactions, samples from dogs with other infections (babesiosis, ehrlichiosis and Trypanosoma cruzi) were tested. Assay performance validations were conducted to analyse parameters such as variability, reproducibility, and stability. CL ELISA sensitivity/specificity with PQ10 antigen was 93.1%/80.0%; with the PQ20 protein 93.1%/96.6%; and with the crude antigen 75%/73.3%. Inter-assay variability and inter-operator coefficient of variation were <7% and <15%, with PQ10 and PQ20, respectively. The accuracy of the CL ELISA was classified as excellent for PQ10 (AUC = 0.95) and PQ20 (AUC = 0.98) and moderate for the crude antigen (AUC = 0.77). The kappa score for qualitative agreement between two plate lots was excellent for PQ10 (0.89) and good for PQ20 (0.65). PQ20 remained more stable than PQ10. The CL ELISA with recombinant proteins is a promising tool to diagnose CVL.

Persistence of PCR-positive tissue in benznidazole-treated mice with negative blood parasitological and serological tests in dual infections with Trypanosoma cruzi stocks from different genotypes.

OBJECTIVES: To assess different methodologies to better define an early post-therapeutic cure criterion after benznidazole treatment in BALB/c mice following mixed infection with dual Trypanosoma cruzi genotypes. METHODS: According to the classical cure criteria, animals were classified as treated not cured (TNC = 76.4%), treated cured (TC = 12.5%) and dissociated (DIS = 11.1%) using parasitological [fresh blood examination (FBE), blood culture (BC) and blood PCR] and serological methods [conventional serology (CS-ELISA) and non-conventional serology (NCS-FC-ALTA)]. Tissues were also evaluated by PCR. RESULTS: FBE was able to detect patent parasitaemia in only 18.1% of TNC and therapeutic failure was detected in 79.1% and 97.2% of TNC by BC and blood PCR, respectively. CS-ELISA should not be used before 3 months after treatment since it may lead to false-negative results. At 3 months after treatment with benznidazole, NCS-FC-ALTA was more efficient for categorizing the groups of treated mice. In the TNC group, although a decreased frequency of PCR-positive tissue was observed in several host tissues, increased positivity was also observed, despite the T. cruzi genotype combination. All TC animals presented at least two positive tissue-PCR results. CONCLUSIONS: Our results confirm that NSC-FC-ALTA and blood PCR are the most suitable methods to early detect therapeutic failure in acute murine T. cruzi infection. Additionally, our data show that BC positivity is highly dependent upon the T. cruzi genotype combination. Moreover, our findings demonstrated that PCR tests performed on tissues from animals considered cured after benznidazole treatment still detected T. cruzi DNA, most probably indicating residual infection.

Genomic characterization of Arcanobacterium pyogenes isolates recovered from the uterus of dairy cows with normal puerperium or clinical metritis.

Arcanobacterium pyogenes is considered to be the most relevant bacterium involved in the establishment of puerperal uterine infection in cattle due to its persistence in utero, resistance to treatment and synergic action with Gram negative anaerobes. Once the infection is established, A. pyogenes is responsible for the persistence of the infection. The objective of this study was to characterize A. pyogenes field isolates recovered from the uterus of cows with either normal puerperium or clinical metritis, in an attempt to identify factors that might be associated with the establishment and persistence of the disease. This characterization was based on BOX-PCR typing and on screening of eight virulence factor genes (plo, nanP, nanH, cbpA, fimA, fimC, fimE, fimG) by conventional PCR. Finally, a relationship between clonal types, virulence factors and presence of disease was investigated. A. pyogenes clonal types identified from isolates recovered from the uterus of postpartum dairy cows differed among herds. Although some clonal types were strictly associated with the development of clinical metritis, others were identified from isolates recovered from normal puerperium and clinical metritis cows. Moreover, the presence of the eight virulence factor genes was not related with the ability to induce clinical metritis, suggesting that the type of A. pyogenes may not be a determinant factor in the development of the disease. We suggest that host intrinsic factors, the synergism between A. pyogenes and other bacteria and the differential gene expression of virulence factor genes may play a more relevant role in the establishment of puerperal uterine infections.

Effects of entomopathogenic bacterium Photorhabdus temperata infection on the digestive enzymes of Diatraea saccharalis (Lepidoptera: Crambidae) larvae.

This study investigated the effects of Photorhabdus temperata infection on the activities of digestive enzymes of the sugarcane stalk borer Diatraea saccharalis. Non-infected D. saccharalis larvae present a major alpha-amylase, several proteinases, three sucrose hydrolases and two alpha-glucosidases in their midgut. Analysis of these hydrolases by electrophoresis and "in gel" assays showed that the activities of all enzymes decreased following infection, with an initial decline observed 12 h after infection. The activities of alpha-glucosidases decreased by 50% twelve hours after infection, whereas, at this time, the alpha-galactosidase activities decreased by 70%. Interestingly, the animals died 48 h after infection, but approximately 5% of all the enzymes tested remained active in the midgut following host death. At this time, most of the cultivable native intestinal bacteria had died.

Trypanosoma cruzi: immunoglobulin isotype profiles during the acute phase of canine experimental infection with metacyclic or blood trypomastigotes.

A detailed investigation has been carried out about the serological profiles of groups of dogs experimentally infected with metacyclic (MT) or blood (BT) trypomastigotes of Berenice-78 Trypanosoma cruzi strain. Peripheral blood was collected from infected dogs and uninfected controls, weekly during 35 days following the acute phase of infection, and immunoglobulin profiles were determined by ELISA. Dogs infected with BT exhibited unaltered levels of IgG2, increases in IgM, IgE, IgA, IgG and IgG1. In contrast, dogs infected with MT presented unaltered levels of IgE and IgG1 and an increase in IgM, IgA, IgG and IgG2 levels. Compared with the MT group, animals infected with BT showed significant increases in IgM on days 7, 14 and 28, in IgA on days 7, 14 and 21, in IgE on days 7 and 14, in IgG on days 14 and 28, and in IgG1 on days 7, 14 and 21. Parasitemia levels of the infected animals were measured over the same time period. No correlations were found between the immunoglobulin profiles and the parasitemia levels. The results demonstrated that the inoculum source (BT or MT) influence the immunoglobulin isotype profile that may drive distinct outcome of acute canine Chagas disease.

First assessment of microbial diversity in faecal microflora of Eurasian otter (Lutra lutra Linnaeus, 1758) in Portugal.

The Eurasian otter (Lutra lutra Linnaeus, 1758), a predator from the Order Carnivora, Family Mustelidae, evolved the ability to swim and forage in water, being an important element of biodiversity. Otters are widely spread through Portugal, and scats have been extensively used in ecology studies; however, valid information on their microbiota is scarce. This work represents a first approach to characterise the otter faecal microflora in samples collected in river stretches of the Sado river basin (Portugal) during winter 2006. Eight sampling stretches of 8 km were selected, and from each, six to eight sampling sites were visited. A total of 31 scats were analysed. The microflora studied included aerobic bacteria, spore-forming anaerobic bacteria and viruses (coronavirus, parvovirus, adenovirus, parainfluenza virus). Bacterial isolates were identified based on morphology and metabolic pathways, and virus detection was performed by polymerase chain reaction. The results revealed the high degree of bacterial diversity in the faecal microflora of L. lutra. A total of 88 Gram-negative (23 genera) and 44 Gram-positive isolates (ten genera) were identified. The identification of four isolates was inconclusive, and their identification was performed by 16S ribosomal ribonucleic acid sequencing, which confirms the need for biochemical testing optimisation regarding animal isolates. None of the scats was positive for virus detection. Identification of otter faecal microflora and of potential pathogens is an important first step towards understanding and monitoring their importance in otter population health.