Bacterial septicemia and herpesvirus infection in Antarctic fur seals (Arctocephalus gazella) stranded in the São Paulo coast, Brazil.

In August 2021, two juvenile male Antarctic fur seals (Arctocephalus gazella) stranded in the southeastern Brazilian coast and were referred to rehabilitation centers. The animals presented increased body temperature, prostration, respiratory distress and despite treatment died. A necropsy following a standardized protocol was performed, and formalin-fixed tissues were processed for microscopic examination. Samples were screened for morbillivirus, herpesvirus, and Brucella spp. by molecular analyses (PCR, RT-PCR). Bacteriological culture was performed in samples collected from the lungs, trachea, and lymph nodes of both cases. The main histopathologic findings were of infectious nature, including multifocal necrotizing and fibrinous mixed interstitial pneumonia, bronchiolitis, and bronchitis, with intralesional myriad bacteria associated with vascular fibrinoid necrosis. Pseudomonas aeruginosa was isolated from tracheal and lung swabs of Case 1, and Klebsiella oxytoca was found in nostril swabs, tracheobronchial lymph nodes, and lung of Case 2. Gammaherpesvirus infection was detected in both cases, and the sequences retrieved were classified into the genus Percavirus. All tested samples were PCR-negative for Brucella spp. and morbillivirus. We hypothesize that the deficient immunological status in association with starvation predisposed the reactivation of herpesvirus and secondary bacterial co-infections. To the authors' knowledge, this is the first molecular detection of herpesvirus in an Antarctic pinniped. These findings reinforce that Otariid gammaherpesvirus circulating in the Southern Hemisphere are likely endemic in the Arctocephalus genus. This report contributes to the current knowledge of health aspects affecting wild pinnipeds, especially in the poorly studied Antarctic species.

Effect of a multifaceted quality improvement intervention on the prescription of evidence based treatment in patients at high cardiovascular risk in Brazil The BRIDGE Cardiovascular Prevention Cluster Randomized Clinical Trial

RESULTS Of the 1619 included patients,1029 (63.6%) weremale,1327(82.0%) had coronary artery disease (843[52.1%] with prioracutemyo cardial infarction),355(21.9%)had priorischemicstroke ortransientischemicattack,and197 (12.2%) had peripheral vascular disease,andthemean( SD) age was 65.6 (10.5) years. Among randomized clusters, 30 (75%) were cardiology sites, 6 (15%) were primary careunits,and 26 (65%) were teaching institutions.Amonge ligible patients,thosein intervention clusters were more like ly to receive aprescription of evidence-based therapies thant hose in control clusters (73.5%[515of701] vs58.7% [493of840];oddsratio,2.30;95%CI,1.14-4.65). There were no differences between the intervention and control group swithregard storisk factor control(ie,hyperlipidemia,hypertension,ordiabetes).Ratesofeducationforsmokingcessationwere higher among current smokers in the intervention group thanin the control group (51.9%[364of701] vs18.2%[153of840];oddsratio,11.24;95%CI,2.20-57.43).Therateofcardiovascularmortality,acute myocardial infarction,andstrokewas2.6%for patients from intervention cluster sand 3.4%forthose in the control group (hazardratio, 0.76;95%CI,0.43-1.34). (AU)

ß-lapachone and α-nor-lapachone modulate Candida albicans viability and virulence factors.

BACKGROUND: Candida albicans is the most important fungal pathogen that causes infections in humans, and the search for new therapeutic strategies for its treatment is essential. OBJECTIVE: The aim of this study was to evaluate the activity of seven naphthoquinones (ß-lapachone, ß-nor-lapachone, bromide-ß-lapachone, hydroxy-ß-lapachone, α-lapachone, α-nor-lapachone and α-xyloidone) on the growth of a fluconazole-resistant C. albicans oral clinical isolate and the effects of these compounds on the viability of mammalian cells, on yeast's morphogenesis, biofilm formation and cell wall mannoproteins availability. RESULTS: All the compounds were able to completely inhibit the yeast growth. ß-lapachone and α-nor-lapachone were the less cytotoxic compounds against L929 and RAW 264.7 cells. At IC50, ß-lapachone inhibited morphogenesis in 92%, while the treatment of yeast cells with α-nor-lapachone decreased yeast-to-hyphae transition in 42%. At 50µg/ml, ß-lapachone inhibited biofilm formation by 84%, whereas α-nor-lapachone reduced biofilm formation by 64%. The treatment of yeast cells with ß-lapachone decreased cell wall mannoproteins availability in 28.5%, while α-nor-lapachone was not able to interfere on this virulence factor. Taken together, data show that ß-lapachone and α-nor-lapachone exhibited in vitro cytotoxicity against a fluconazole-resistant C. albicans strain, thus demonstrating to be promising candidates to be used in the treatment of infections caused by this fungus.

Vaccine effectiveness and use of collar impregnated with insecticide for reducing incidence of Leishmania infection in dogs in an endemic region for visceral leishmaniasis, in Brazil.

Although a national programme for control of visceral leishmaniosis (VL) is being run in Brazil, the disease continues to spread. This programme is essentially based on culling infected dogs from endemic regions. Thus, there is an urgent need to develop other control measures against VL to deter its advance. Here, a subunit vaccine, a recombinant vaccine, an insecticide-impregnated collar and the associations between these measures were evaluated for reducing the incidence of Leishmania infection in dogs. This was through a cohort study conducted in an endemic region of Brazil, considering the incidence and time of total exposure over a period of 1 year. The incidence of VL was estimated by means of serological and molecular diagnostic tests, 180 and 360 days after the application of the control measures. The estimates of the effectiveness (EF) were not significant in any cohort. The EF of the subunit vaccine, the recombinant vaccine and the collar were 26.4%, 32.8% and 57.7% and the upper limit of the 95% confidence interval for EF were 63.7%, 67.9% and 82.5%, respectively. In conclusion, under the conditions of this study, none of the immunogens for VL control was sufficiently effective to protect dogs against infection. On the other hand, use of collars impregnated with insecticide seems to constitute a method with better prognosis, corroborating other studies in this field.

Histatin-5 induces the reversal of Pdr5p mediated fluconazole resistance in Saccharomyces cerevisae.

BACKGROUND: Candidiasis is a major opportunistic fungal infection in humans. The low number of antifungal drugs available to treat Candida infections and the increasing incidence of multidrug resistant (MDR) strains point to an urgent need of identifying new therapeutic options. The role of salivary components can provide insights for the development of new methodologies of control. OBJECTIVE: The aim of this study was to evaluate the ability of histatin-5, a constitutive immunological peptide present in saliva, in reversing fungal MDR phenotype, using a resistant Saccharomyces cerevisiae strain as model of study. RESULTS: A total of 2.5µg and 5µg of histatin-5 revealed to be able to chemosensitize (to revert antifungal resistance) a MDR strain to fluconazole impairing its intrinsic resistance. The presence of histatin-5 decreased the strain growth when associated to fluconazole, and also assisted in the retention of rhodamine 6G within cell cytoplasm. The ATPase activity of Pdr5p, an ABC efflux transporter, was significantly reduced up to 65% within physiological concentration of the peptide. CONCLUSION: Results revealed that histatin-5 is able to revert MDR phenotype and may be considered a potential alternative MDR inhibitor. Since Pdr5p is homologous to Candida albicans CaCdr1p and CaCdr2p, data obtained might be extrapolated to these transporters, inferring that associating fluconazole and histatin-5 may be a useful tool to circumvent failure treatments of infections caused by Candida MDR strains.

Serological and molecular diagnostic tests for canine visceral leishmaniasis in Brazilian endemic area: one out of five seronegative dogs are infected.

Euthanasia of infected dogs is one of the measures adopted in Brazil to control visceral leishmaniasis (VL) in endemic areas. To detect infected dogs, animals are screened with the rapid test DPP® Visceral Canine Leishmaniasis for detection of antibodies against K26/K39 fusion antigens of amastigotes (DPP). DPP-positives are confirmed with an immunoenzymatic assay probing soluble antigens of promastigotes (ELISA), while DPP-negatives are considered free of infection. Here, 975 dogs from an endemic region were surveyed by using DPP, ELISA and real-time PCR (qPCR) for the diagnosis of VL. When DPP-negative dogs were tested by qPCR applied in blood and lymph node aspirates, 174/887 (19·6%) were positive in at least one sample. In a second sampling using 115 cases, the DPP-negative dogs were tested by qPCR in blood, lymph node and conjunctival swab samples, and 36/79 (45·6%) were positive in at least one sample. Low-to-moderate pairwise agreement was observed between all possible pair of tests. In conclusion, the official diagnosis of VL in dogs in Brazilian endemic areas failed to accuse an expressive number of infected animals and the impact of the low accuracy of serological tests in the success of euthanasia-based measure for VL control need to be assessed.

Low toxicity superparamagnetic magnetite nanoparticles: One-pot facile green synthesis for biological applications.

Superparamagnetic magnetite nanoparticles have been synthesized by a highly reproducible polyvinyl alcohol (PVA)-based modified sol-gel process using water as the only solvent. The synthesis method has proven to be effective, time and cost saving and environmental friendly, resulting in PVA-coated magnetite nanoparticles as direct product from the synthesis, without any special atmosphere or further thermal treatment. X-ray diffraction and transmission electron microscopy revealed that the biocompatible PVA-coating prevents the nanoparticle agglomeration, giving rise to spherical crystals with sizes of 6.8nm (as-cast) and 9.5nm (heat treated) with great control over size and shape with narrow size distribution. Complementary compositional and magnetic characterizations were employed in order to study the surface chemistry and magnetic behavior of the samples, respectively. Cytotoxicity endpoints including no observed adverse effect concentration (NOAEC), 50% lethal concentration (LC50) and total lethal concentration (TLC) of the tested materials on cell viability were determined after 3, 24 and 48h of exposure. The PVA coating improved the biocompatibility of the synthesized magnetite nanoparticles showing good cell viability and low cytotoxicity effects on the MTT assay performed on BHK cells. Preliminary assessment of nanoparticles in vivo effects, performed after 48h on Balb/c mice, exposed to a range of different sub-lethal doses, showed their capacity to penetrate in liver and kidneys with no significant morphological alterations in both organs.

On-line identification of fermentation processes for ethanol production.

A strategy for monitoring fermentation processes, specifically, simultaneous saccharification and fermentation (SSF) of corn mash, was developed. The strategy covered the development and use of first principles, semimechanistic and unstructured process model based on major kinetic phenomena, along with mass and energy balances. The model was then used as a reference model within an identification procedure capable of running on-line. The on-line identification procedure consists on updating the reference model through the estimation of corrective parameters for certain reaction rates using the most recent process measurements. The strategy makes use of standard laboratory measurements for sugars quantification and in situ temperature and liquid level data. The model, along with the on-line identification procedure, has been tested against real industrial data and have been able to accurately predict the main variables of operational interest, i.e., state variables and its dynamics, and key process indicators. The results demonstrate that the strategy is capable of monitoring, in real time, this complex industrial biomass fermentation. This new tool provides a great support for decision-making and opens a new range of opportunities for industrial optimization.

Brucella canis infection in dogs from commercial breeding kennels in Brazil.

Canine brucellosis caused by Brucella canis is a neglected zoonosis worldwide and is a leading cause of reproductive failure in dogs, often causing substantial economic losses in breeding kennels. This study aimed to investigate the occurrence of B. canis infection in dogs of commercial breeding kennels located in São Paulo State, Brazil. A total of 753 dogs (183 males and 570 females) from 38 commercial kennels were clinically examined, and blood samples were collected for brucellosis diagnosis through blood culture. The association between clinical manifestations suggestive of brucellosis and positive results through blood culture was determined. Of the 753 dogs tested, 166 (22.0%) had at least one clinical sign suggestive of brucellosis and 158 (20.9%) had positive blood cultures. Seventy-two dogs had positive blood culture and had at least one clinical sign suggestive of brucellosis, while 91 dogs showed at least one clinical manifestation suggestive of brucellosis although blood culture was negative. Of the 38 kennels, 16 (42.1%) had at least one positive dog. The prevalence of infection in each kennel varied from 3.8% to 62.6%. Abortion/stillbirth, failure to conceive and enlargement of lymph nodes were significantly associated with brucellosis in female. No association of clinical signs and positive results in blood culture was observed in males. None of the kennels has been carrying out programmes to control brucellosis, and the sale of infected dogs was considered a common practice yielding risks to the public health, in view of the zoonotic potential of the infection.

Phenotypic and genotypic characterization of two Toxoplasma gondii isolates in free-range chickens from Uberlândia, Brazil.

The aim of this study was to determine the seroprevalence of Toxoplasma gondii infection in free-range chickens from Uberlândia, Minas Gerais state, Brazil, and characterize the genotypic and phenotypic features of two isolates of this parasite, considering the importance of these hosts in the epidemiology of toxoplasmosis. Serum samples from 108 free-range chickens were obtained from ten different districts, and submitted to the modified agglutination test (MAT) for the presence of anti-T. gondii antibodies, and brain and heart tissue samples from infected chickens were processed for mouse bioassay. An overall seroprevalence of 71·3% was found and antibody titres ranged from 16 to 4096. After confirmation of seropositivity by mouse bioassay, the determination of the T. gondii genotypes of two isolates was performed by PCR-RFLP, using primers for the following markers: SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, new SAG2, Apico and CS3. These T. gondii isolates, designated TgChBrUD1and TgChBrUD2, were obtained from heart samples of free-range chickens. The TgChBrUD1 isolate belonged to ToxoDB PCR-RFLP genotype 11 and the TgChBrUD2 isolate belonged to ToxoDB PCR-RFLP genotype 6. Both isolates demonstrated high virulence in a rodent model, with the TgChBrUD1 isolate able to induce brain cysts, in accord with its pattern of multiplication rates in human fibroblast culture. Taken together, these results reveal high prevalence of T. gondii infection in free-range chickens throughout Uberlândia, indicating an important degree of oocyst environmental contamination and the existence of considerable risk for T. gondii transmission to humans by consumption of free-range chicken as a food source.

Evaluation of an Immunochromatographic Test to the Diagnosis of Canine Brucellosis Caused by Brucella canis.

This study evaluated the performance of an immunochromatographic test (ICT) for the diagnosis of canine brucellosis caused by Brucella canis, comparing its results with that of the rapid slide agglutination test with and without the use of 2-mercaptoethanol and the agar gel immunodiffusion test (AGID). The microbiological culture, PCR and clinical examination were used as reference. According to the results obtained in clinical examination, blood culture, culture of semen and vaginal swab and PCR in blood, semen and vaginal swab, a total of 102 dogs were divided into three groups: B. canis-infected dogs (Group 1), B. canis-non-infected dogs (Group 2) and dogs with suspected brucellosis (Group 3). The diagnostic sensitivity of RSAT, 2ME-RSAT, AGID and ICT in Group 1 was, respectively, 75%, 37.5%, 27.8% and 89.58%. The diagnostic specificity of RSAT, 2ME-RSAT, AGID and ICT in Group 2 was, respectively, 91%, 100%, 100%, and 100%. In dogs with suspected brucellosis, 9.67% were RSAT positive, none was positive by 2ME-RSAT, 3.22% were AGID positive and 6.45% were ICT positive. The main drawback concerning canine brucellosis diagnosis is the lack of a highly sensitive serological assay to be used as a screening test to the rapid identification of infected animals. The ICT showed a high diagnostic specificity and a diagnostic sensitivity value greater than that observed in the RSAT, 2ME-RSAT and AGID. However, 10.41% of infected dogs had negative results by ICT. These dogs were positive by microbiological culture and/or PCR, indicating active infection and consequently a higher potential of spreading Brucella. Although rapid and simple to perform, the ICT lacked sensitivity to be used as a screening test.

Variables Associated with Infections of Cattle by Brucella abortus., Leptospira spp. and Neospora spp. in Amazon Region in Brazil.

The frequency of Neospora spp., Leptospira spp. and Brucella abortus infections in adult cattle was determined in herds of the State of Pará, Brazil, which is an important region for cattle production located in the Amazon region. A total of 3466 adult female cattle from 176 herds were tested, leading to a frequency of seropositive animals of 14.7%, 3.7% and 65.5% and a herd positivity of 87.4%, 41.3% and 98.8% for infections caused by Neospora spp., B. abortus and Leptospira spp., respectively. The five most frequently diagnosed serologic responses to Leptospira spp. were those against serovars hardjo, wolfii, grippotyphosa, hebdomadis and shermani. The following associations were found: practice of artificial insemination, large farm size, large herd size, large number of dogs and high number of total abortions per year with the presence of antibodies against serovar hardjo; positive results to serovar grippotyphosa with the presence of dogs; inappropriate disposal of aborted foetuses with positivity to serovar hebdomadis. Serovar grippotyphosa was also associated with number of episodes of abortions. Neospora spp. positive herds were associated with episodes of abortion and B. abortus infection with the disposal of dead animals and aborted foetuses on pastures and with the use of artificial insemination. In conclusion, the high frequency of brucellosis, leptospirosis and neosporosis in the region may be a consequence of social, natural and raising conditions as: (i) climate conditions that favour the survival and spread of pathogens in the environment; (ii) farms located in regions bordering forest areas; (iii) farms in areas of difficult access to the veterinary service; (iv) extensive beef herds raised at pastures with different age and productive groups inter-mingled; and (v) minimal concerns regarding hygiene practices and disease prevention measures.

Betacoronavirus 1 in alpacas (Vicugna pacos) in the High Peruvian Andes.

Genetic sequences highly related to Bovine coronavirus (BCoV) were detected in fecal samples from Peruvian 1-3 week old alpaca crias located on six farms in Puno department, some of which shared pastures with cattle. A total of 60 samples were screened for coronavirus using a nested PCR amplification of a fragment of the RNA-dependent RNA polymerase (RdRp) gene. Sequences from 11 positive samples were highly similar to the Kakegawa, Quebec and Mebus BCoV strains (99.5-100.0%) and 99.2% identical to an alpaca Coronavirus (CoV) previously detected in the USA. The detection of genetic sequences related to BCoV from Peruvian alpaca crias suggests possible role of this virus on enteric disorders etiology in the High Andes.

Genetic characterization of Toxoplasma gondii from Brazilian wildlife revealed abundant new genotypes.

This study aimed to isolate and genotype T. gondii from Brazilian wildlife. For this purpose, 226 samples were submitted to mice bioassay and screened by PCR based on 18S rRNA sequences. A total of 15 T. gondii isolates were obtained, including samples from four armadillos (three Dasypus novemcinctus, one Euphractus sexcinctus), three collared anteaters (Tamandua tetradactyla), three whited-lipped peccaries (Tayassu pecari), one spotted paca (Cuniculus paca), one oncilla (Leopardus tigrinus), one hoary fox (Pseudalopex vetulus), one lineated woodpecker (Dryocopus lineatus) and one maned wolf (Chrysocyon brachyurus). DNA from the isolates, originated from mice bioassay, and from the tissues of the wild animal, designated as "primary samples", were genotyped by PCR-restriction fragment length polymorphism (PCR/RFLP), using 12 genetic markers (SAG1, SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L258, PK1, CS3 and Apico). A total of 17 genotypes were identified, with 13 identified for the first time and four already reported in published literature. Results herein obtained corroborate previous studies in Brazil, confirming high diversity and revealing unique genotypes in this region. Given most of genotypes here identified are different from previous studies in domestic animals, future studies on T. gondii from wildlife is of interest to understand population genetics and structure of this parasite.

In vivo genotoxicity of treated water containing the cylindrospermopsin-producer Cylindrospermopsis raciborskii.

Cylindrospermopsin (CYN) is an alkaloid commonly produced by some cyanobacteria that has been implicated in outbreaks of human illness. The aim of this study was to investigate the genotoxicity of Cylindrospermopsis raciborskii cellular content (including CYN) and its byproducts resulting from chlorination during water treatment. DNA damage in blood and liver cells was analysed by the comet assay and micronucleus test (MN). Mice were injected intraperitoneally with the following treatments: (a) physiological saline, (b) treated water, (c) treated water plus C. raciborskii extract (CYN producer strain, CYPO-011 K), (d) C. raciborskii extract (CYN producer strain, CYPO-011 K), (e) C. raciborskii extract (CYN non producer strain), and (f) treated water plus C. raciborskii extract (CYN non producer strain) extract. After 48 h, samples were taken to perform tests (blood and liver cells to the comet assay and bone marrow to MN test). The CYPO-011 K had a genotoxic and mutagenic effects on liver and bone marrow cells. The group that received chlorine-treated water plus CYPO-011 K also exhibited genotoxic effects in the liver, as well as in the blood, and a mutagenic effect in blood marrow cells. The results emphasise the need of improving CYN monitoring in waters bodies in order to reduce the risk of human exposure.

Dualistic evolution of liver damage in mice triggered by a single sublethal exposure to Microcystin-LR.

Microcystins (MCYST) are the most frequently reported cyanotoxins in human poisoning incidents. Despite the well-described mechanism of acute and lethal injury, the sublethal effects of this toxin require further investigation. The aim of this study was to contribute to the knowledge of the variant MCYST-LR effects at sublethal doses by investigating biochemical changes and tissue damage in a murine model. For this purpose, mice were intraperitoneally injected with 45 µg of MCYST-LR/kg body weight. Their organs were collected at 2, 8, 24, 48 or 96 h after injection. Control animals received saline solution. We detected oxidative imbalance in the liver, particularly at 8 h after exposure. Furthermore, biomarkers of liver injury were detected in high concentration in the serum of the exposed animals. Stereological analyses of the liver indicated two different phases in the intoxication process: an initial phase characterized by an increase in steatosis was followed by a second, later phase characterized by increased inflammation and hepatocyte binucleation. Formation of areas of necrosis and increased blood vessel diameter were observed throughout the experimental period. The number of hepatocytes per area unit also decreased. However, these parameters recovered over the period of exposure. MCYST accumulated in liver and was detectable until the end of the monitoring period. These results confirm the necessity for further studies of processes involved in sublethal exposure to MCYST.

A novel nerolidol-rich essential oil from Piper claussenianum modulates Candida albicans biofilm.

Candidiasis is a major opportunistic fungal infection in humans, and its incidence has increased steadily over the last two decades. Candida albicans, the main species of the genus, has a large arsenal of virulence attributes that contribute to successful infections, such as dimorphism and biofilm formation. The adverse effects of eukaryotic antimicrobial therapies associated with an increase in resistance to the compounds presently available have boosted efforts to improve the therapeutic arsenal against candidiasis with a newer and cheaper range of drugs. In this study, a novel nerolidol-rich essential oil (EO) derived from Piper claussenianum (Miq.) C. DC., Piperaceae, was tested on the growth, transition (yeast to hyphae), formation and stability of biofilms produced by C. albicans. Both inflorescence and leaf EOs were evaluated and revealed MIC values ranging from 0.04 to 0.1 % and 0.2 to 1.26 %, respectively. Furthermore, leaf EO managed to downregulate the yeast-to-hyphae transition by 81 %, as well as reducing biofilm formation by about 30 and 50 % after incubation for 24 and 48 h, respectively. The EO was also able to reduce the viability of pre-formed biofilm by 63.9 %. Finally, the association between the leaf EO and fluconazole was evaluated and revealed an interesting synergistic effect. Taken together, these results demonstrate that this novel compound could be a promising agent and could reinforce the arsenal of therapeutic alternatives for the treatment of candidiasis. Furthermore, it may represent a novel and natural source of nerolidol, which could be of interest pharmaceutically.

Evaluation of the Joypex 5 and Root ZX II: an in vivo and ex vivo study.

AIM: To evaluate in vivo and ex vivo the accuracy of the multifrequency electronic root canal length measurement device (ERCLMD) Joypex 5 and compare it with the Root ZX II. The ex vivo methodology for evaluation of the accuracy of ERCLMDs was also validated by comparison with in vivo results. METHODOLOGY: Twenty single-rooted human teeth that were scheduled for extraction were selected. Working length measurements were performed in vivo with the Joypex 5 and Root ZX II. After the teeth were extracted, each canal was measured with the ERCLMDs ex vivo. Then, a size 15 K-file was used to determine the reference working length (RWL), which was established 0.5 mm from the major foramen by direct observation. The measurements of working length obtained with the different methods and ERCLMDs were compared by analysis of variance and Tukey's test at P < .05. Statistical correlations were also performed. RESULTS: No significant difference was observed between in vivo and ex vivo measurements or the different types of ERCLMDs. The Bland-Altman plot confirmed the agreement of different methods and ERCLMDs to measure canal length. The R(2) coefficient obtained in both situations, comparing the ERCLMDs, was close to 1, denoting a strong agreement between measurements obtained with the Joypex 5 and Root ZX II and between in vivo and ex vivo methodologies. CONCLUSION: The Joypex 5 and Root ZX II had similar accuracy in determining working length of root canals. The ex vivo methodology for evaluation of the accuracy of ERCLMDs was validated when compared with the in vivo results.

Extensively variable surface antigens of Sarcocystis spp. infecting Brazilian marsupials in the genus Didelphis occur in myriad allelic combinations, suggesting sexual recombination has aided their diversification.

Sarcocystis neurona and Sarcocystis falcatula are very similar species of Apicomplexan protozoa that use marsupials of the genus Didelphis as definitive hosts. These mammals can serve as definitive hosts not only for these two parasites, but for other Sarcocystis such as Sarcocystis speeri and Sarcocystis lindsayi. Sarcocystis shed by opossums (with the exception of S. neurona) can cause disease in a great variety of birds, being commonly associated with acute pulmonary sarcocystosis in zoos. S. neurona is the most commonly associated parasite with the equine protozoal myeloencephalitis in horses. Herein we assessed the variability of Sarcocystis spp. isolated from opossums of the state of Rio Grande do Sul, Brazil, by sequencing fragments of genes coding for glycosylphosphatidylinositol-anchored surface antigens (termed surface antigen or SAG), SAG2, SAG3 and SAG4. Two genetic groups were identified, one of them related to S. falcatula and the other related to S. neurona. Various allelic combinations of SAG2, SAG3 and SAG4 occur among S. falcatula related isolates and strong evidences suggest that such isolates may exchange high divergent alleles in possible sexual recombination processes. Regarding the group S. neurona-like (isolates G37 and G38), none of the individuals in this group share alleles with individuals of the other group. Comparing G37 and G38 strains and North American strains of S. neurona, four polymorphisms were identified at SAG-3, five at SAG-2 and three at SAG-4. Gene sequences of locus SAG-3 from isolates G37 and G38 differed from the other sequences by an insertion 81bp long. This insertion contains several dinucleotide repeats of AT, resembling a microsatellite locus and has already been detected in SAG3 sequences of S. neurona from North America. When aligned against North American strains of S. neurona, G37 and G38 isolates have a deletion of 8 nucleotides within this intron which indicate that S. neurona strains of South America are divergent from that of North America. From the results obtained so far, we have shown extensive variability in surface antigens coding sequences among Sarcocystis eliminated by mammals of the genus Didelphis spp. In addition, such divergent alleles may be exchanged in possible sexual recombination processes between different isolates of S. falcatula related isolate. The evolutionary relationships within S. falcatula related isolates will be best clarified after markers less subjected to selection pressures are analyzed in conjunction with surface antigen genes. These results may have a striking impact on the knowledge of the Sarcocystis species that infect opossums in Brazil and also in the epidemiology of the infections caused by these protozoans.

PCR-RFLP genotyping of Toxoplasma gondii from chickens from Espírito Santo state, Southeast region, Brazil: new genotypes and a new SAG3 marker allele.

Brazil is one of the regions with the highest prevalences of Toxoplasma gondii in humans and animals. Because free-range chickens become infected by feeding from ground contaminated with oocysts, the prevalence of T. gondii in this host has been widely used as an indicator of the strains prevalent in the environment. The genetic variability among T. gondii isolates from different healthy and sick hosts all over the world has been recently studied. Three clonal genetic lineages (Types I, II and III) were initially recognised as predominant in Western Europe and the United States. T. gondii strains are genetically diverse in South America. In Brazil, recombination plays an important role in strain diversification. The objective of this study was to genetically characterise T. gondii isolates from free-range chickens from Espírito Santo state, Southeast region, Brazil, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). A total of 44 isolates among 47 previously described isolates (TgCkBr234-281) from free-range chickens were included in this study. Strain typing was performed using 12 PCR-RFLP markers: SAG1, SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3. Eleven genotypes were identified. Ten isolates (23%) were grouped into four novel genotypes. Four isolates, distributed in four counties, corresponded to the Type BrI lineage, the genotype found most frequently in Brazil. No clonal Types I, II or III lineages were found. Two novel genotypes were represented by single isolates. Unique alleles were identified for the markers SAG1, c22-8 and CS3, and for the first time, a unique allele was found for the marker SAG3. Although a large number of T. gondii genotypes have already been identified from a variety of animal hosts in Brazil, new genotypes are continuously identified from different animal species. This study confirmed the diversity of T. gondii in Brazil and demonstrates clonal Type I, II and III lineages are rare in this country.