Genomic analysis of multidrug-resistant Escherichia coli strains carrying the mcr-1 gene recovered from pigs in Lima-Peru.

Antibiotic resistance is a current problem that significantly impacts overall health. The dissemination of antibiotic resistance genes (ARGs) to urban areas primarily occurs through ARG-carrying bacteria present in the gut microbiota of animals raised in intensive farming settings, such as pig production. Hence, this study aimed to isolate and analyzed 87 Escherichia coli strains from pig fecal samples obtained from intensive farms in Lima Department. The isolates were subjected to Kirby-Bauer-Disk Diffusion Test and PCR for mcr-1 gene identification. Disk-diffusion assay revealed a high level of resistance among these isolates to oxytetracycline, ampicillin, cephalothin, chloramphenicol, ciprofloxacin, and doxycycline. PCR analysis identified the mcr-1 gene in 8% (7/87) E. coli isolates. Further, whole genome sequencing was conducted on 17 isolates, including multidrug resistance (MDR) E. coli and/or mcr-1 gene carriers. This analysis unveiled a diverse array of ARGs. Alongside the mcr-1 gene, the blaCTX-M55 gene was particularly noteworthy as it confers resistance to third generation cephalosporins, including ceftriaxone. MDR E. coli genomes exhibited other ARGs encoding resistance to fosfomycin (fosA3), quinolones (qnrB19, qnrS1, qnrE1), tetracyclines (tetA, tetB, tetD, tetM), sulfonamides (sul1, sul2, sul3), amphenicols (cmlA1, floR), lincosamides (inuE), as well as various aminoglycoside resistance genes. Additionally, Multi Locus Sequence Typing (MLST) revealed a high diversity of E. coli strains, including ST10, a pandemic clone. This information provides evidence of the dissemination of highly significant ARGs in public health. Therefore, it is imperative to implement measures aimed at mitigating and preventing the transmission of MDR bacteria carrying ARGs to urban environments.

Bioactivity analysis of calcium silicate-based sealers and repair cements on the phenotype and cytokine secretion profile of CD14+ monocytes: An ex vivo study.

AIM: This study evaluated the immune bioactivity of testing media (TM) obtained from different calcium silicate-based sealers and cements on monocyte morphology, activation, differentiation and cytokine secretion. METHODS: Blood-derived CD14+ monocytes were isolated and cultured for 5 days with 25% TM from the following calcium silicate-based materials: TotalFill BC RRM Fast-Set Putty, Biodentine, TotalFill BC Sealer and BioRoot-Root-Canal-Sealer (RCS). A resin-based endodontic cement was used as a control. The expression of surface markers such as CD86, HLA-DR, CD16, CD309 and CD209, and cytokine secretion were analysed by flow cytometry. Data were analysed using the one-way repeated measures analysis of variance (anova) multiple comparison test and a Holm-Sidak multiple comparison post-hoc test (p < .05). RESULTS: This comparative analysis revealed that monocytes co-cultured with calcium silicate-based materials showed a spindle-shaped morphology compared with the round shape observed in the control. Regarding activation markers, BioRoot-RCS and Biodentine significantly increased CD86 expression compared with the control sample, whereas no significant differences (p > .05) were observed in HLA-DR expression. In addition, no differences were observed among the differentiation markers. When the inflammatory cytokines were analysed, BioRoot-RCS increased the secretion of IL-1ß, IL-6, IL-10 and TNF-α, whereas BioRoot-RCS and Biodentine significantly decreased IL-8 production (p < .05). CONCLUSIONS: These data showed that the calcium silicate-based materials tested changed the morphology of CD14+ monocytes; however, only BioRoot-RCS and Biodentine significantly upregulated CD86. In addition, BioRoot-RCS was the sealer with the highest immunomodulatory properties for cytokine production which means that it can contribute with the in vivo healing process and regeneration of periapical lesions.

Genomic insights into ESBL-producing Escherichia coli isolated from non-human primates in the Peruvian Amazon.

Introduction: Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae are on the WHO priority pathogens list because they are associated with high mortality, health-care burden, and antimicrobial resistance (AMR), a serious problem that threatens global public health and should be addressed through the One Health approach. Non-human primates (NHP) have a high risk of acquiring these antibiotic-resistant bacteria due to their close phylogenetic relationship with humans and increased anthropogenic activities in their natural environments. This study aimed to detect and analyze the genomes of ESBL-producing Escherichia coli (ESBL-producing E. coli) in NHP from the Peruvian Amazon. Materials and methods: We collected a total of 119 fecal samples from semi-captive Saguinus labiatus, Saguinus mystax, and Saimiri boliviensis, and captive Ateles chamek, Cebus unicolor, Lagothrix lagothricha, and Sapajus apella in the Loreto and Ucayali regions, respectively. Subsequently, we isolated and identified E. coli strains by microbiological methods, detected ESBL-producing E. coli through antimicrobial susceptibility tests following CLSI guidelines, and analyzed their genomes using previously described genomic methods. Results: We detected that 7.07% (7/99) of E. coli strains: 5.45% (3/55) from Loreto and 9.09% (4/44) from Ucayali, expressed ESBL phenotype. Genomic analysis revealed the presence of high-risk pandemic clones, such as ST10 and ST117, carrying a broad resistome to relevant antibiotics, including three blaCTX-M variants: blaCTX-M-15, blaCTX-M-55, and blaCTX-M-65. Phylogenomic analysis confirmed the clonal relatedness of high-risk lineages circulating at the human-NHP interface. Additionally, two ESBL-producing E. coli strains were identified as EPEC (eae) and ExPEC according to their virulence profiles, and one more presented a hypermucoviscous phenotype. Discussion: We report the detection and genomic analysis of seven ESBL-producing E. coli strains carrying broad resistome and virulence factors in NHP from two regions of the Peruvian Amazon. Some of these strains are closely related to high-risk pandemic lineages previously reported in humans and domestic animals, highlighting the negative impact of anthropogenic activities on Amazonian wildlife. To our knowledge, this is the first documentation of ESBL-producing E. coli in NHP from the Amazon, underscoring the importance of adopting the One Health approach to AMR surveillance and minimizing the potential transmission risk of antibiotic-resistant bacteria at the human-NHP interface.

Occurrence and Genomic Characterization of mcr-1-Harboring Escherichia coli Isolates from Chicken and Pig Farms in Lima, Peru.

Resistance to colistin generated by the mcr-1 gene in Enterobacteriaceae is of great concern due to its efficient worldwide spread. Despite the fact that the Lima region has a third of the Peruvian population and more than half of the national pig and poultry production, there are no reports of the occurrence of the mcr-1 gene in Escherichia coli isolated from livestock. In the present work, we studied the occurrence of E. coli carrying the mcr-1 gene in chicken and pig farms in Lima between 2019 and 2020 and described the genomic context of the mcr-1 gene. We collected fecal samples from 15 farms in 4 provinces of Lima including the capital Lima Metropolitana and recovered 341 E. coli isolates. We found that 21.3% (42/197) and 12.5% (18/144) of the chicken and pig strains were mcr-1-positive by PCR, respectively. The whole genome sequencing of 14 mcr-1-positive isolates revealed diverse sequence types (e.g., ST48 and ST602) and the presence of other 38 genes that confer resistance to 10 different classes of antibiotics, including beta-lactamase blaCTX-M-55. The mcr-1 gene was located on diverse plasmids belonging to the IncI2 and IncHI1A:IncHI1B replicon types. A comparative analysis of the plasmids showed that they contained the mcr-1 gene within varied structures (mikB-mcr1-pap2, ISApl1-mcr1-pap2, and Tn6330). To the best of our knowledge, this is the first attempt to study the prevalence of the mcr-1 gene in livestock in Peru, revealing its high occurrence in pig and chicken farms. The genetic diversity of mcr-1-positive strains suggests a complex local epidemiology calling for a coordinated surveillance under the One-Health approach that includes animals, retail meat, farmers, hospitals and the environment to effectively detect and limit the spread of colistin-resistant bacteria.

Genomic Characterization of Salmonella Typhimurium Isolated from Guinea Pigs with Salmonellosis in Lima, Peru.

Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) is one of the most important foodborne pathogens that infect humans globally. The gastrointestinal tracts of animals like pigs, poultry or cattle are the main reservoirs of Salmonella serotypes. Guinea pig meat is an important protein source for Andean countries, but this animal is commonly infected by S. Typhimurium, producing high mortality rates and generating economic losses. Despite its impact on human health, food security, and economy, there is no genomic information about the S. Typhimurium responsible for the guinea pig infections in Peru. Here, we sequence and characterize 11 S. Typhimurium genomes isolated from guinea pigs from four farms in Lima-Peru. We were able to identify two genetic clusters (HC100_9460 and HC100_9757) distinguishable at the H100 level of the Hierarchical Clustering of Core Genome Multi-Locus Sequence Typing (HierCC-cgMLST) scheme with an average of 608 SNPs of distance. All sequences belonged to sequence type 19 (ST19) and HC100_9460 isolates were typed in silico as monophasic variants (1,4,[5],12:i:-) lacking the fljA and fljB genes. Phylogenomic analysis showed that human isolates from Peru were located within the same genetic clusters as guinea pig isolates, suggesting that these lineages can infect both hosts. We identified a genetic antimicrobial resistance cassette carrying the ant(3)-Ia, dfrA15, qacE, and sul1 genes associated with transposons TnAs3 and IS21 within an IncI1 plasmid in one guinea pig isolate, while antimicrobial resistance genes (ARGs) for ß-lactam (blaCTX-M-65) and colistin (mcr-1) resistance were detected in Peruvian human-derived isolates. The presence of a virulence plasmid highly similar to the pSLT plasmid (LT2 reference strain) containing the spvRABCD operon was found in all guinea pig isolates. Finally, seven phage sequences (STGP_Φ1 to STGP_Φ7) were identified in guinea pig isolates, distributed according to the genetic lineage (H50 clusters level) and forming part of the specific gene content of each cluster. This study presents, for the first time, the genomic characteristics of S. Typhimurium isolated from guinea pigs in South America, showing particular diversity and genetic elements (plasmids and prophages) that require special attention and also broader studies in different periods of time and locations to determine their impact on human health.

Somatic mutation rates scale with lifespan across mammals.

The rates and patterns of somatic mutation in normal tissues are largely unknown outside of humans1-7. Comparative analyses can shed light on the diversity of mutagenesis across species, and on long-standing hypotheses about the evolution of somatic mutation rates and their role in cancer and ageing. Here we performed whole-genome sequencing of 208 intestinal crypts from 56 individuals to study the landscape of somatic mutation across 16 mammalian species. We found that somatic mutagenesis was dominated by seemingly endogenous mutational processes in all species, including 5-methylcytosine deamination and oxidative damage. With some differences, mutational signatures in other species resembled those described in humans8, although the relative contribution of each signature varied across species. Notably, the somatic mutation rate per year varied greatly across species and exhibited a strong inverse relationship with species lifespan, with no other life-history trait studied showing a comparable association. Despite widely different life histories among the species we examined-including variation of around 30-fold in lifespan and around 40,000-fold in body mass-the somatic mutation burden at the end of lifespan varied only by a factor of around 3. These data unveil common mutational processes across mammals, and suggest that somatic mutation rates are evolutionarily constrained and may be a contributing factor in ageing.

Carnivore protoparvovirus 1 in Peruvian dogs: Temporal/geographical and evolutionary dynamics of virus.

Canine parvovirus (CPV) has been recognized all around the world as the causal agent of a contagious and highly mortal disease in domestic dogs. In Peru, the infection is endemic and unvaccinated animals and puppies are the most at risk. In order to analyze viral diversity and determine the evolutionary genetic relationships and transmission dynamic of Peruvian CPV-2, were collected during the period of 2016-2017 rectal swabs from puppies with parvovirosis compatible symptoms. Viral DNA was amplified by PCR using primers that flanked the ends of the viral genome and sequenced by Illumina Miseq platform. Twenty-six genomic sequences (NSP1-VP1) of CPV from several districts in Lima Metropolitan area were obtained. The VP2 gene analysis demonstrated the presence of the New CPV-2a, New CPV-2b and 2c variants. The phylodynamic analysis of the viral genomes determined that all Peruvian sequences were clustered into a big clade named South American clade that emerged from the west region of Europe (Italy). The Time to the Most Recent Common Ancestor (TMRCA) of the South American clade was dated to 1993. Peruvian sequences were distributed into three subclades, and the 92% of these sequences were related to Ecuadorian CPV-2. The results suggests that three independent introduction events of virus from other countries could have occurred, in two of these events, CPV-2 from Ecuador were introduced in Peru in 2003 and 2009, and another introduction event, in 2000, from Europe. Overall, these results indicate a viral genetic relationship between Peruvian with Ecuadorian and European virus, and the circulation of several viral subpopulations in Lima Metropolitan.

In vitro antibacterial activity of endodontic bioceramic materials against dual and multispecies aerobic-anaerobic biofilm models.

The aim of this in vitro study was to evaluate the antibacterial activity of calcium silicate repair cements and sealers against a dual-species planktonic aerobic model with different aging times and the ability to inhibit the formation of a mature 21-day-old multispecies anaerobic biofilm. The antibacterial activity of ProRoot MTA, MTA Angelus, Biodentine, BioRoot RCS and TotalFill BC sealer against a dual-species aerobic planktonic model, as well as measuring how materials were affected by aging, was evaluated using the Modified Direct Contact Test. Subsequently, the ability to inhibit the formation of a mature multispecies anaerobic biofilm was evaluated using scanning electron microscopy complemented with confocal laser scanning microscopy. Biodentine and BioRoot RCS had higher antibacterial action, and Biodentine was able to maintain its antibacterial action after a prolonged aging period in vitro. Calcium silicate repair cement MTA ProRoot and Biodentine had higher antibiofilm action.

Somatic mutation landscapes at single-molecule resolution.

Somatic mutations drive the development of cancer and may contribute to ageing and other diseases1,2. Despite their importance, the difficulty of detecting mutations that are only present in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. Here, to overcome these limitations, we developed nanorate sequencing (NanoSeq), a duplex sequencing protocol with error rates of less than five errors per billion base pairs in single DNA molecules from cell populations. This rate is two orders of magnitude lower than typical somatic mutation loads, enabling the study of somatic mutations in any tissue independently of clonality. We used this single-molecule sensitivity to study somatic mutations in non-dividing cells across several tissues, comparing stem cells to differentiated cells and studying mutagenesis in the absence of cell division. Differentiated cells in blood and colon displayed remarkably similar mutation loads and signatures to their corresponding stem cells, despite mature blood cells having undergone considerably more divisions. We then characterized the mutational landscape of post-mitotic neurons and polyclonal smooth muscle, confirming that neurons accumulate somatic mutations at a constant rate throughout life without cell division, with similar rates to mitotically active tissues. Together, our results suggest that mutational processes that are independent of cell division are important contributors to somatic mutagenesis. We anticipate that the ability to reliably detect mutations in single DNA molecules could transform our understanding of somatic mutagenesis and enable non-invasive studies on large-scale cohorts.

Percepción Intraoperatoria del Paciente sobre la Preparación de la Cavidad de Acceso Endodóntico / Patient Intraoperatory Perception About Endodontic Access Cavity Preparation

RESUMEN: La creencia de que el tratamiento de endodoncia es el tratamiento dental más doloroso es algo habitual. A pesar de ello, la percepción intraoperatoria durante el tratamiento de endodoncia ha sido poco estudiada. Por ello, el objetivo de esta investigación fue describir la percepción de dolor y la experiencia intraoperatoria del paciente tratado en endodoncia, durante la etapa de acceso endodóntico. Se analizaron las valoraciones sobre dolor intraoperatorio realizadas por 20 pacientes, tratados en la clínica de especialidad de una universidad tradicional chilena, durante la etapa de acceso endodóntico en molares. Para ello se utilizaron escalas cuantitativas de valoración de la intensidad del dolor y el análisis de contenido cualitativo de una entrevista semi-estructurada. Se observó que los participantes de sexo femenino, los tratamientos en molares mandibulares y en dientes con diagnóstico de pulpitis irreversible sintomática obtuvieron medias mayores en cuanto a la valoración de la intensidad del dolor. Sin embargo, en la mayoría de los casos el dolor fue descrito como leve. En el análisis cualitativo lo descrito por los participantes se agrupó en 10 categorías. Se observó un mayor porcentaje de referencias a la categoría "Ansiedad ante el tratamiento" (16 %) seguido de "Percepción de la atención profesional" (14 %). Sin embargo, también destacaron las referencias al dolor en su totalidad (25 %) ya sea a "Ausencia de dolor" (13 %) o a "Presencia de dolor en algún grado" (12 %). Se concluyó que entre los participantes de esta investigación existió percepción de dolor intraoperatorio durante la etapa de acceso endodóntico, sin embargo, este fue de carácter leve en la mayoría de los casos.

ABSTRACT: There is a common belief that endodontic treatment is the most painful dental treatment of all. Despite this idea, intraoperative perception during endodontic treatment has not been fully studied. Therefore, the aim of this research was to describe the perception of pain and the intraoperative experience of endodontic treated patients, during the endodontic access cavity preparation. We analyzed the valuations on intraoperative pain completed by 20 patients, treated at the endodontic clinic of a traditional Chilean university, during the endodontic access cavity preparation in molars. For this purpose, quantitative scales of pain intensity assessment and qualitative content analysis of a semi-structured interview were employed. Female participants, treatments in mandibular molars and in teeth diagnosed with symptomatic irreversible pulpitis obtained higher means in terms of assessment of pain intensity. However, in most cases the pain was described as mild. In the qualitative analysis the participant's descriptions were grouped into 11 categories. There was a higher percentage of references to the category "Anxiety before treatment" (16 %) followed by "Perception of professional care" (14 %). However, references to pain in its totality (25 %) either to "Absence of pain" (13 %) or to "Presence of pain to some degree" (12 %) also stood out. It was concluded that among the participants of this investigation there was perception of intraoperative pain during the endodontic access stage, however, this was mild in most cases.

Canine parvovirus types 2a and 2c detection from dogs with suspected parvoviral enteritis in Peru.

Canine parvovirus type 2 (CPV-2) has been reported worldwide as the main agent related to acute hemorrhagic enteritis of high morbidity and variable mortality in puppies. The detection and characterization of this virus is essential to understand the etiology of the disease and to develop control measures. To characterize the virus circulating in Peruvian dogs and to provide new insights into the local diversity of CPV-2, rectal swabs from 39 puppies with clinical symptoms and with no history of previous vaccinations were analyzed. Total DNA was extracted by fast boiling method, and PCR and sequencing were performed using specific primers that amplify a 1316 bp fragment corresponding to the VP2 gene of CPV-2. CPV-2 was detected in 62% of the analyzed samples. The sequencing of PCR product was possible in 9 samples, which were identified as type 2a (4 samples) and type 2c (5 samples). A phylogenetic analysis of both variants circulating in Peruvian dogs showed similarities to Equatorian and Uruguayan strains. This work constitutes the first report about genetic characterization of CPV-2 in Peru.

Permeabilización del Canal Radicular y Transporte Apical: Comparación de Tres Sistemas / Root Canal Glide Path and Apical Transportation: Three System Comparison

La mantención de la anatomía original del canal radicular incide directamente en el éxito del tratamiento endodóntico. Para ello, los sistemas rotatorios de instrumentación requieren de canales radiculares permeables. Existen distintas limas y sistemas para la permeabilización o glide path como las limas tipo K manuales de acero inoxidable y los sistemas de NiTi rotatorios PathFile y ProGlider (Dentsply, Maillefer). Así, el objetivo de esta investigación fue comparar la capacidad de distintos sistemas de permeabilización para mantener la anatomía radicular sin producir transporte apical en canales mesiales de molares inferiores humanos extraídos. Se realizó un estudio cuantitativo experimental cuya muestra estuvo conformada por 36 canales mesiales de molares mandibulares humanos extraídos. Las muestras fueron divididas aleatoriamente en tres grupos conformados por 12 canales, cada uno de los cuales fueron sometidos a distintos sistemas de permeabilización (Lima K, PathFile y ProGlider). Las muestras fueron fotografiadas antes y después de la permeabilización utilizando un microscopio con magnificación 30X. Se cuantificó el desplazamiento del canal radicular en sentido mesio - distal y buco - lingual posterior a la permeabilización. Para el análisis de los datos se utilizó el paquete estadístico InfoStat/L y se aplicó la prueba de ANOVA / Tukey. Se observó que el sistema PathFile produce mayor transporte del canal radicular en su tercio apical en la dirección mesio-distal (p=0,77) y el sistema ProGlider en la dirección buco-lingual (p=0,57). Sin embargo, estas diferencias no fueron significativas. En conclusión, los sistemas de permeabilización analizados no presentaron diferencias en cuanto a su capacidad para mantener la anatomía sin producir transporte apical.

Preservation of the original root canal anatomy has a direct influence on the success of an endodontic treatment. In order to achieve this, rotary instrumentation systems require permeable root canals. Different files and systems are used for the establishment of a glide path such as manual stainless steel K files and NiTi rotatory systems like PathFile and ProGlider (Dentsply, Maillefer). Thus, the objective of this research was to compare the ability of different systems to create a glide path and maintain root canal anatomy without producing apical transportation in mesial root canals of extracted human lower molars. A quantitative experimental study was performed with a sample of 36 mesial root canals of extracted human mandibular molars. The samples were randomly divided into three groups consisting of 12 root canals each, which were subjected to different glide path systems (K-Files, PathFile and ProGlider). Samples were photographed before and after creating glide path using a microscope with 30X magnification. The displacement of the root canal in a mesio - distal and bucco - lingual direction was quantified after creating glide path. Data was analyzed using the statistical package InfoStat / L and the ANOVA / Tukey test was applied. The PathFile system produced greater transport of the root canal in its apical third in the mesio-distal direction (p = 0.77) and the ProGlider system in the bucco-lingual direction (p = 0.57). However, these differences were not significant. In conclusion, the glide path systems analyzed do not present any differences in their ability to maintain the anatomy without producing apical transportation.

Antimicrobial evaluation of copper sulfate (II) on strains of Enterococcus faecalis: in vitro study / Evaluación antimicrobiana del sulfato de cobre (II) sobre cepas de Enterococcus faecalis: estudio in vitro

Introduction: Controlling Enterococcus faecalis is of vital importance in endodontics, as this pathogen is associated with endodontic failure. Experimental evidence has shown that copper has antibacterial activity against other pathogens with similar characteristics. The objective of this study was to determine the antimicrobial activity of copper (II) or cupric (SC-II) sulfate on strains of Enterococcus faecalis and to compare it with the most commonly used antimicrobials. Methodology: We used 33 strains of Enterococcus faecalis isolated from different clinical pictures in different Chilean hospitals. The minimum inhibitory concentration (MIC) of SC-II, chlorhexidine and calcium hydroxide was determined by the broth microdilution technique, following the recommendations given by the Clinical and Laboratory Standards Institute. Results: The MIC for CHX varied in the range of 5-10 ug/ml; SC-II from 1.5 to 12 ug/ml, and HC was >32 mg/ml. The geometric mean of SCII was 6 ug/ml, lower than that of CHX, which was 7.29 ug/ml. Conclusions: SCII showed antimicrobial activity at lower concentrations than CHX. HC (which could have been affected by the buffer effect of the broth microdilution technique) showed high values, not comparable to other compounds. We suggest carrying out further studies on the properties of SC-II, such assessing its biocompatibility and reaction with other materials to be used clinically in endodontic therapy.

Introducción: El control de Enterococcus faecalis es de vital importancia en endodoncia, ya que este patógeno está asociado al fracaso endodóntico. Evidencias experimentales que han demostrado que el cobre presenta actividad antibacteriana en otros patógenos de similares características. El objetivo de este estudio es determinarla actividad antimicrobiana del sulfato de cobre (II) o cúprico (SC-II) sobre cepas de Enterococcus faecalis y compararla con los antimicrobianos más usados en la actualidad. Metodología: Estudio in vitro. Se utilizó la técnica de microdilución en caldo según lineamientos del Clinical and Laboratory Standards Institute, incluyendo 33 cepas de Enterococcus faecalis obtenidas desde hospitales chilenos, para cada una de las cuales se determinó las concentraciones mínimas inhibitorias (CMI) de: SC-II, Clorhexidina (CHX) e Hidróxido de calcio (HC). Resultados: La CMI para CHX tuvo un rango de 5-10 ug/ml, el SC-II de 1,5-12 mM y el HC fue >32 mg/ml. Estas diferencias fueron estadísticamente significativas entre los 3 antimicrobianos utilizados (p<0,001).Conclusiones: El SC-II mostró actividad antimicrobiana a bajas concentraciones, superiores a CHX, pero menores a HC (que pudo ser afectado por el efecto tampón de la técnica de microdilución en caldo). Se sugiere seguir con los estudios de las propiedades del SC-II, como evaluación de biocompatibilidad y reacción con otros materiales para ser utilizados clínicamente en la terapia endodóntica.

Presence and molecular characterisation of Giardia and Cryptosporidium in alpacas (Vicugna pacos) from Peru.

The presence of Giardia and Cryptosporidium was investigated in 274 faecal samples of alpacas (Vicugna pacos) from 12 herds from Peru by immunofluorescence microscopy and PCR amplification and sequencing of fragments of the ssu-rRNA and ß-giardin genes from Giardia spp., as well as the ssu-rRNA gene from Cryptosporidium spp. A total of 137 samples (50.0%) were positive for Giardia spp., and 12 samples (4.4%) for Cryptosporidium spp. In ten samples (3.6%), co-infection by both pathogens was found. Herd prevalence was found to be 91.7% (11/12 herds) for Giardia and 58.3% (7/12 herds) for Cryptosporidium. Regarding the age of the animals, although Giardia was detected in animals as young as 1 week, the prevalence increased with age, reaching 80% by 8 weeks. Similarly, the highest percentage of Cryptosporidium detection (20%) was also found in the 8 week-old group. By PCR, 92 of the 274 analysed samples were positive for Giardia. Sequencing of the amplicons showed the existence of Giardia duodenalis assemblage A in 67 samples; G. duodenalis assemblage E in 24 samples; and inconsistent results between the two molecular markers used in a further sample. Cryptosporidium was only detected by PCR in 3 of the 274 samples; Cryptosporidium parvum was identified in two samples and Cryptosporidium ubiquitum in one sample. This study is the first performing molecular characterisation of both parasites in Peruvian alpacas, and the first report of C. ubiquitum in this host. The identification of G. duodenalis assemblage A, C. parvum and C. ubiquitum, suggests that zoonotic transmission of these enteropathogens between alpacas and humans is possible.

Multiscale molecular dynamics of protein aggregation.

The 60's gave birth to the practical implementation of classical mechanics to unravel the dynamics and energetics of biomolecules. In the 70's the use of generalized force fields and more advanced integrative solutions to the microscopic understanding of nature (like hybrid QM/MM) were introduced. During the 80's, algorithms to obtain free energy values were further developed and in the 90's practical integration schemes of molecular mechanics force fields with other levels of detail (QM on one extreme and advances in implicit solvation on the other) were implemented in widely spread software. In the first decade of the XXIst century a considerable effort has been put in two seemingly discordant models for the simulation of biomolecules. On the one hand, extraordinary advances in computing technologies (both in terms of processor power and of new efficient parallel and distributed computing schemas) have allowed researchers to deal with bigger systems and longer simulations, reaching molecular processes including millions of particles or lying in the microsecond scale. On the other hand, the realization that the relevant answers to many biomolecular problems are not homogeneously distributed through the molecular structure, something already envisioned by the QM/MM pioneers more than three decades ago, has led researchers to find smart ways of putting different emphases on different ranges of the spatial or system time scale. In this context, e.g., molecular aggregation represents a paradigm for multiscalability, as molecular recognition can be understood with simple (semi-)macroscopic terms when the two fragments are far apart, while the atomic interactions need to be considered in full detail upon close distances. In this manuscript the current status of the techniques that use multiple scale representations of biomolecules are reviewed, and the findings are synthesized in a modular schema that can be extensively used when studying aggregation processes. It is shown that a smart alternative to brute force and massive computation of uninteresting regions in the all atom potential energy surface is the consideration of a simplified reference potential, explored thoroughly in the relevant regions, combined with a free energy perturbation approach that transforms this simple representation to a full atom representation.

Estudio clínico, serológico y patológico de una cepa velogénica del virus de Newcastle en codornices (Coturnix coturnix japonica) / Clinical, srelogical and pathological study of a Newcastle velogenic virus strain in quails (Coturnix, coturnix japonica)

Se determinó la susceptibilidad, el efecto patológico, y la respuesta serológica inducida por una cepa velogénica viscerotrópica del virus de la enfermedad de Newcastle (vvENC). Para este fin, se criaron 40 codornices japónicas (Coturnix coturnix japonica) hembras, donde 20 se inocularon vía nasal y ocular con una cepa de vvENC y 20 se usaron como grupo control. Para el análisis histopatológico y recuperación viral se tomó muestras de tejidos e hisopados de cloaca de las aves inoculadas y del grupo control; y para detectar anticuerpos contra el virus de la ENC mediante la prueba de inhibición de la hemaglutinación (IH) se tomó muestras de sangre durante 5 semanas posteriores al desafío. En el 40% de las aves inoculadas se registraron signos severos, así como mortalidad del 20% de las aves inoculadas. Del mismo modo, se registraron lesiones macroscópicas y microscópicas en los animales inoculados. El grupo desafiado registró un incremento en los niveles de anticuerpos a partir de los 7 días postinoculación (PGT 6.1), alcanzando el mayor PGT a los 14 días post inoculación (PGT 29.9), mientras que en el grupo de aves control no se registró seroconversiones. La recuperación viral se logró a partir de los hisopados de cloaca de las aves afectadas por la enfermedad. El grupo control no registró signos de enfermedad ni cambios histopatológicos.

The objective of the study was to determine the susceptibility, pathological effect, and serological response induced by a velogenic viscerotropic strain of Newcastle disease virus (vvNDV). For this purpose, 40 female quails (Coturnix coturnix japonica) were raised. Twenty were nasal and ocular inoculated with a vvNDV strain and 20 remained as a control group. Tissue samples and cloacae swaps of all birds were collected for histopathology analysis and virus isolation. Blood samples were collected during 5 weeks after the viral challenge to detect antibodies against NC virus using the hemaglutination inhibition (HI) test. In 40% of the inoculated birds was observed severe clinical signs and 20% mortality. The inoculated group registered an increase in the antibody level after day 7 post inoculation (MGT 6.1), reaching the highest level at 14 days post inoculation (MGT 29.9), whereas the control group did not register seroconversions. The viral isolation was obtained from cloacae swabs of the affected animals. The control group did not show signs of illness or histopathologycal changes.

Contribución al estudio del parasitismo gastrointestinal en guanacos (Lama guanicoe cacsilensis) / Contribution to the study of gastrointestinal parasitism in the guanaco (Lama Guanicoe Cacsilensis)

El propósito del presente estudio fue identificar las especies de parásitos gastrointestinales que afectan al guanaco peruano y determinar los niveles de parasitismo de las poblaciones evaluadas. Se obtuvieron 132 muestras de heces frescas de guanacos silvestres pertenecientes a nueve poblaciones ubicadas en seis departamentos del Perú: Comunidad Campesina de Huallhua (Ayacucho), Reserva Nacional de Calipuy (La Libertad), Comunidad Campesina de Chavín (Ica), Reserva Nacional Salinas y Aguada Blanca y distritos de Machaguay y Yarabamba (Arequipa), distrito de Quilahuani y Comunidad Campesina de Vila Vilani (Tacna), y distrito de La Capilla (Moquegua). Las muestras fueron procesadas mediante técnicas coproparasitológicas de flotación, sedimentación, cultivo de larvas, Baerman y biometría de larvas y ooquistes. Se identificaron ocho especies de nematodos: Graphinema aucheniae, Bunostomun sp., Ostertagia sp., Trichuris sp, Cooperia sp., Nematodirus sp., Mazamastrongylus peruvianus y Trichostrongylus sp. y cuatro especies de Eimeria: E. lamae, E. alpacae, E. punoensis y E. macusaniensis. Todas las poblaciones se encontraban con al menos un guanaco parasitado, presentando en general cargas bajas y variando las frecuencias de parasitismo gastrointestinal de una población a otra, dependiendo del hábitat y de la proximidad a herbívoros domésticos.

The aim of this study was to identify the species of gastrointestinal parasites affecting the Peruvian guanaco and to determine the levels of parasitism in the populations under evaluation. For this purpose, 132 fresh faecal samples were collected from nine populations of wild guanacos located in six departments of Peru: Huallhua Community in Ayacucho; Calipuy National Reserve in La Libertad; Chavín community in Ica; Salinas and Aguada Blanca National Reserve, and Machaguay and Yarabamba districts in Arequipa, Quilahuani district and Vila Vilani community in Tacna, and La Capilla district in Moquegua. Samples were processed by the coproparasitological techniques of flotation, sedimentation, larvae culture, and Baerman, and biometry of larvae and oocysts. Eight species of nematodes were identified: Graphinema aucheniae, Bunostomun spp., Ostertagia spp., Trichuris spp., Cooperia spp., Nematodirus spp., Mazamastrongylus peruvianus and Trichostrongylus spp., and four Eimeria species: E. lamae, E. alpacae, E. punoensis and E. macusaniensis. All guanaco populations had at least one animal with parasites, showing low parasite burden in general, and with a variation in the frequency of gastrointestinal parasitism from one population to another, depending on the habitat and the proximity to other domestic herbivores.

Determinación del sexo por ADN en cinco especies de guacamayos / Sex determination by ADN in five macaw species

La determinación del sexo en aves de especies silvestres es de vital importancia para tomar medidas de conservación. En especies donde no existe dimorfismo sexual, es necesario contar con una prueba de sexaje alternativo a los métodos quirúrgicos. En este sentido, con la finalidad de estandarizar una prueba para la determinación del sexo en guacamayos, se desarrolló y evaluó una prueba molecular que consistió en el análisis de ADN mediante la Reacción en Cadena de la Polimerasa (PCR). Para ello, se procedió a la extracción de ADN a partir de muestras de sangre cinco especies de guacamayos (Ara ararauna, Ara chloroptera, Ara macao, Ara militaris y Propyrrhura couloni). A través de la técnica de PCR y el uso de primers específicos, se amplificó regiones conservadas (exones) y secuencias de regiones no conservadas (intrones) del gen chd (Chromodomain-helicase-DNA-binding protein), presentes en ambos cromosomas sexuales (Z y W), que permiten diferenciar hembras (chd-ZW) de machos (chd-ZZ). Para la amplificación, se optimizaron las condiciones y los ciclos de PCR. Se pudo detectar dos fragmentos entre 300-400 pares de bases en aves hembras y solamente uno en especies machos. Se observó 100 por ciento (31/31) de correspondencia entre el sexaje por métodos convencionales y por análisis de ADN. La prueba molecular fue posteriormente utilizada para sexar a 28 guacamayos de sexo desconocido, incluyendo 11 aves de la especie peruana Propyrrhura couloni.

Determination of sex in wild birds is crucial in developing conservation plans for threatened species. The absence of sexual dimorphism in birds makes impossible to determine sex based on physical characteristics and sexing has traditionally depended upon surgical methods which are highly stressful for the animals. The present study had the purpose of developing of a noninvasive DNA test for sex determination in macaws (guacamayos). Blood samples from five species (Ara ararauna, Ara chloroptera, Ara macao, Ara militaris y Propyrrhura couloni) were studied. DNA was extracted and specific primers were used to amplify both exons and introns of the chd (chromo-helicase-DNA-binding) gene located on the sex chromosomes of all birds. Females are identified by chd-ZW on the W chromosome and males by chd-ZZ on the Z chromosome. The amplification was carried out by the optimization of the conditions and the PCR cycles. Two fragments of 300-400 bp were detected in female birds and one in males. Sex determined by conventional methods in 31 birds agreed with DNA results. The molecular test was also used to sex 28 macaws of unknown sex, including 11 Propyrrhura couloni.

Evaluación de un método numérico de medición del diámetro de la fibra de alpaca / Evaluation of a numerical method for measuring the diameter of the alpaca fiber

El presente estudio evalúa el método de medición de diámetro de la fibra de alpaca mediante el procesamiento de imagen digital, Digital Image Fibre Diameter Analisis (DIFDA), desarrollado en el Centro Internacional de la Papa, y lo compara con los valores obtenidos mediante dos métodos de medición convencionales: Lanámetro (Microscopio de Proyección) y OFDA (Optical Fibre Diameter Analysis). El DIFDA requiere de una evaluación del proceso de imágenes digitales obtenidas mediante un escáner de transparencias y negativos de una muestra de fibra de alpaca preparada en porta slides. Este método presenta una opción de procesamiento de imagen digital completa o otra de secciones dentro de la imagen digital, donde los resultados se expresan en promedio, desviación estándar y coeficiente de variación. Un total de de 206 muestras de fibra de alpacas del fundo Pacomarca, Puno, fueron evaluadas. Los valores promedio de diámetro de las fibras fueron de 21.74 ± 3.03, 21.64 ± 3.58 y 21.74 ± 4.01 según los métodos DIFDA, Lanámetro y OFDA, respectivamente; sin haber diferencia significativa entre promedios. El coeficiente de correlación de Pearson entre DIFDA con el lánametro fue de 0.87 y con OFDA fue de 0.84. La medición del diámetro puede realizarse procesando la imagen digital completa, a partir de tres secciones de 1000 x 1000 píxeles, o desde cinco secciones de 500 x 500 píxeles. Se concluye que no existe diferencia significativa entre los resultados de DIFDA y de los métodos Lanámetro y OFDA, por lo que su uso puede ser de utilidad en programas de mejoramiento animal que requieren medición continua del diámetro de fibras.

This study presents the statistical evaluation of a digital image alpaca fibre diameter measurement method, Digital Image Fibre Diameter Analysis (DIFDA). This method developed in the International Potato Center (CIP) requires an evaluation of the fibre sample digital image process, prepared in slides covers and taked from a scanner of negatives and films. DIFDA presents two options for fibre diameter measurement process: Digital image complete and sections of the digital image. The results of the fibre sample are mean fibre diameter, standard deviation and coefficient of variability. The objective was to test and compare the mean fibre diameter values obtained by DIFDA with the values from two conventionals methods: Projection Microscope and OFDA (Optical Fibre Diameter Analysis). In adition, DIFDA’s process options were evaluated. Two hundred six alpaca fibre samples from Pacomarca Farm, Puno, were evaluated. The mean fibre diameter samples values, measured by commercials methods, Projection Microscope and OFDA, were, 21.64 ± 3.58 and 21.74 ± 4.0, respectively. The mean fibre diameter reported for DIFDA was 21.74 ± 3.03. No statistical differences between the methods were detected. The Pearson’s correlation coefficient for DIFDA and Projection Microscope and for DIFDA and OFDA was 0.87 and 0.84, respectively. The process of DIFDA could be done in the complete digital image option, or using three or more sections for the dimention of 1000 x 1000 pixels, or five or more sections for the dimention of 500 x 500 pixels. The conclutions were that the DIFDA’s results showed no statistical significative differences between neither the Projection Microscope’s nor the OFDA’s results. Consequently, this measurement method would be used satisfactorily in breeding programs that requires continuos fibre measurement.

Seroprevalencia de Toxoplasma gondii en alpacas de comunidades de la provincia de Canchis, Cusco / Seroprevalence of Toxoplasma gondii in alpacas communities Canchis province, Cusco

El objetivo del estudio fue cuantificar la seroprevalencia de Toxoplasma gondii en alpacas de comunidades alpaqueras de los distritos de Maranganí, Pitumarca, Checacupe y San Pablo, en la provincia de Canchis, Cusco. Se recolectaron 272 muestras de sangre en marzo del 2003 para la detección de anticuerpos contra T. gondii mediante la prueba de inmunofluorescencia indirecta (IFI). Se encontró una seroprevalencia moderada de 35.7 ± 5.7 por cento. No se encontró asociación entre las variables distrito, sexo, raza y la respuesta a la prueba de IFI. Sin embargo, se encontró una asociación significativa entre la edad y la respuesta a la prueba. La seroprevalencia del presente estudio concuerda con resultados obtenidos en camélidos sudamericanos en otras zonas del sur del Perú.

The objective of the present study was to determine the seroprevalence of Toxoplasma gondii in alpacas of communities from the districts of Maranganí, Pitumarca, Checacupe and San Pablo, located in the province of Canchis, department of Cusco. A total of 272 blood samples were collected in March 2003, for the detection of antibodies against T. gondii by the indirect immunofluorescence test (IFAT). The resulting seroprevalence was 35.7 ± 5.7 per cent, without significant differences due to district, sex, and breed; however, there was a significant association between age and IFAT value. The results of the present study agreed with other studies conducted in South American camelids in different localities in the south of Peru.