Use of polimerase chain reaction for sexing south american camelids

Se reporta el desarrollo y optimizaciones de técnicas moleculares (PCR simple, múltiple y semi-anidada) para determinar el sexo de camélidos sudamericanos (CSA) amplificando la secuencia del gen Zinc Finger Protein (ZF). La técnica utilizó ADN obtenido de 28 muestras de sangre de alpacas, llamas y vicuñas, 20 muestras de heces de vicuñas y guanacos conservadas en etanol al 96%, y 22 embriones de alpaca colectados entre 72 y 96 horas postmonta y preservados en etanol. Las muestras de ADN de sangre y heces fueron extraídas usando kits comerciales, y las de embriones aplicando tres métodos (ebullición, proteinasa K y fenol-cloroformo). Una vez optimizada la PCR simple para la detección de los genes ZFY y ZFX, se implementó la PCR múltiple para ADN de sangre y heces y la PCR semi-anidada para ADN de embriones. La técnica de PCR múltiple determinó el sexo correctamente en el 100% de las muestras de ADN sanguíneo, en el 87.5% de muestras de ADN de heces colectadas en 2008 y en el 50% de las muestras de ADN de heces colectadas en 2004 y preservadas durante cuatro años antes del análisis. La prueba de PCR semi-anidada, sin embargo, no pudo ser optimizada.The objective of this study was to develop a PCR technique to determine the sex of South American camelids (CSA) using Zinc Finger Protein (ZF) sequences from blood and fecal samples, as well as cells from alpaca embryos. A total of 28 alpaca, llama and vicuña blood samples, 20 vicuña and guanaco fecal samples, and 22 alpaca embryos collected between 72 and 96 hours postcopula were used. The fecal and embryo samples were preserved in 96% and 70% ethanol respectively. DNA was extracted from blood and feces using commercial kits. Three methods (boiling, proteinase K and phenol-cloroform) were used to extract DNA from alpaca embryos. Two PCR techniques were developed to analyze DNA: multiplex (for fecal and blood sample DNA) and heminested PCR (for embryo cell DNA). The multiplex PCR accurately determined the sex in 100% of the DNA samples extracted from blood, in 87.5% of the samples extracted from fresh feces and in 50% of the 4-year old fecal samples. The heminested PCR, however, could not be optimized

Field test to evaluate colostrum quality in alpaca

Las concentraciones de inmunoglobulinas (Igs) calostrales en la mayoría de especies productivas determinan los niveles de Igs en sus crías, y las fallas en la transferencia pasiva ocasionan susceptibilidades a infecciones en el recién nacido. El presente estudio evaluó dos pruebas de campo (grado de viscosidad visual y uso de refractómetro) para determinar la calidad del calostro de la alpaca en 77 muestras. Asimismo, se determinó la concentración de Igs mediante una prueba de inmunodifusión radial en 26 muestras de calostro y en 77 muestras de suero sanguíneo de crías obtenidas entre las 36 a 48 horas del nacimiento. Las muestras de calostro se analizaron visualmente para determinar grados de viscosidad (1 a 5), y con el refractómetro de azúcar Brix para determinar sólidos totales. El 60% de las muestras calostrales presentó grados de 2-5 de viscosidad y lecturas promedio de 37.3% por el refractómetro de Brix, encontrándose una correlación altamente significativa entre viscosidad y lecturas por el refractómetro Brix (p<0.0001; r2 = 0.69). El IgG sérico en crías fue de 2679 ± 603 mg/dL y de IgG calostral fue de 28 337 ± 5593 mg/dL, encontrándose un solo animal con fallas de transferencia pasiva (valores séricos: 750 mg/dL). Las concentraciones de IgG calostrales tienen una correlación significativa con las lecturas del refractómetro (p<0.0007), no así con las concentraciones de IgG séricas de las crías (p=0.15). Similarmente, la correlación entre las lecturas de refractometría Brix y los valores séricos de IgG de crías fue baja (p=0.338). La alta correlación entre la escala del % Brix y las IgG calostrales muestran que el refractómetro de azúcar mide correctamente los niveles de IgG, por lo que sería un buen indicador de la calidad del calostro en condiciones de campo. Sin embargo, la baja correlación entre la escala de Brix y las IgG séricas de crías limita el valor de la utilización del refractómetro para predecir el estatus de transferencia pasiva en los neonatos.In the majority of livestock species, the concentration of maternal immunoglobulin (Ig) in colostrum determines the Ig level in their offspring, and the failure of passive transfer of maternal immunoglobulins results in their susceptibility to disease. This study evaluates two methods (visual assessment and Brix refratometry) for determining alpaca colostrum quality in the field. Evaluations of 26 fresh colostrum samples from 77 alpaca mothers were compared with the results obtained by radial immunodiffusion assay for 77 blood serum samples from 36-48 hours old offspring, and 26 colostrum samples. The colostrum was collected from the dams immediately postpartum and pre-suckling and blood was taken from the crias by jugular venipuncture. Grades of calostral viscosity were assessed visually, with 60% at 2-5, and total calostral solids measured by Brix sugar refractometry averaged 37.3%. The radial immunodiffusion test yielded newborn IgG serum levels of 2679 ± 603.4 mg/dL and colostral IgG levels of 28337± 5593 mg/dL, and only one animal registered failure of immune transfer with serum levels of 750 mg/dL. Results obtained by visual assessment of the colostrum coincide with those obtained by refractometry (p=0.0007), but differ from the serum IgG concentrations in the newborn animals (p=0.15), as do the Brix refractometry readings (p=0.338). Due to the lack of low IgG levels in the colostrum samples, it was impossible to determine if a relationship exists between observed viscosity and colostral IgG concentration. Nonetheless, the positive correlation (p<0.001) between viscosity and Brix refractometry readings point to the need for further research

CARACTERIZACIÓN FENOTÍPICA Y ANÁLISIS DE ADN MITOCONDRIAL DE LLAMAS DE MARCAPOMACOCHA, PERÚ.

The k’ara llama population of Marcapomacocha district, Yauli province, department of Junín, Peru is known for the preponderance of ancestral coloration in the herds. In order to document the apparently unique characteristics of these animals, phenotypic and DNA analyses were carried out on 50 llamas (5 male and 45 female aged 1 to >4 years) from the region. The coat coloration pattern of the animals was uniform, with tones varying from yellow brown to dark red brown, similar to that of the ancestral Peruvian guanaco (Lama guanicoe cacsilensis), while the chest, abdomen and inner surface of the legs are white with a grey-black head and white outlining of the lips, eyes and ears. Biometric analysis of 30 adults (4 years of age and greater) yielded the following results: withers height 123.2 ± 12.2 cm; rump height 119.5 ± 8.5 cm; chest width 36.5 ± 2.7 cm; chest girth 136.4 ± 5.5 cm; ear length 19.6 ± 2.7, upper and lower neck circumferences 42.8 ± 2.7 and 63.9 ± 4.7 cm respectively. Body length averaged 118.5 ± 5.3 cm and body weight was 152.5 ± 12.3 kg. Based on a survey of the literature, the Marcapomacocha llamas are taller, longer and heavier than k´ara llamas from other regions of Peru. Analysis of a diagnostic segment of the cytochrome b gene revealed that all 50 llamas had the ancestral guanaco haplotype, possibly indicating that no hybridization with alpacas has occurred.Las llamas k’ara de Marcapomacocha, provincia de Yauli, departamento de Junín, Perú, se distinguen por presentar un alto porcentaje de animales con coloración ancestral con una semblanza muy semejante al guanaco peruano, Lama guanicoe cacsilensis. Con el objetivo de documentar estas características, aparentemente únicas de esta población, se describen medidas biométricas y análisis del ADN mitocondrial en 50 llamas (5 machos y 45 hembras de uno a más de cuatro años de edad). El patrón de coloración de las llamas muestra tonalidades desde marrón amarillento hasta rojizo oscuro, con el pecho, abdomen y la parte interna de las piernas de color casi blanco y la cabeza gris a negra con blanco alrededor de los labios, ojos y bordes de las orejas. El análisis biométrico de los 30 animales mayores a 4 años fue: altura a la cruz 123.2 ± 12.2 cm; altura a la grupa 119.5 ± 8.5 cm, ancho de pecho 36.5 ± 2.7 cm, perímetro torácico 136.4 ± 5.5 cm, largo de orejas 19.6 ± 2.7, perímetro de cuello al nivel superior 42.8 ± 2.7 cm y al nivel inferior 63.9 ± 4.7 cm, longitud corporal 118.5 ± 5.3 cm y peso promedio de 152. 5 ± 12.3 kg. Al comparar estos datos con los existentes en la literatura, se constata que las llamas de Marcapomacocha son más altas, más largas y más pesadas que las llamas k´ara de otras regiones del Perú. El análisis de un segmento diagnóstico del gen de citocromo b (ADN mitocondrial) reveló que las 50 llamas tenían el haplotipo ancestral guanaco, indicando reducida posibilidad de hibridización con la alpaca

Dyspnea Review for the Palliative Care Professional: Treatment Goals and Therapeutic Options

Although dyspnea is frequently encountered in the palliative care setting, its optimal management remains uncertain. Clinical approaches begin with accurate assessment, as delineated in part one of this two-part series. Comprehensive dyspnea assessment, which encompasses the physical, emotional, social, and spiritual aspects of this complex symptom, guide the clinician in choosing therapeutic approaches herein presented as part two. Global management of dyspnea is appropriate both as complementary to disease-targeted treatments that target the underlying etiology, and as the sole focus when the symptom has become intractable, disease is maximally treated, and goals of care shift to comfort and quality of life. In this setting, current evidence supports the use of oral or parenteral opioids as the mainstay of dyspnea management, and of inhaled furosemide and anxiolytics as adjuncts. Nonpharmacologic interventions such as acupuncture and pulmonary rehabilitation have potential effectiveness, although further research is needed, and use of a simple fan warrants consideration given its potential benefit and minimal burden and cost

Dyspnea Review for the Palliative Care Professional: Assessment, Burdens, and Etiologies

Dyspnea is a common symptom experienced by many patients with chronic, life-threatening, and/or life-limiting illnesses. Although it can be defined and measured in several ways, dyspnea is best described directly by patients through regular assessment, as its burdens exert a strong influence on the patient's experience throughout the trajectory of serious illness. Its significance is amplified due to its impact on family and caregivers

Microbiological, pathological and microelement analyses in vicuñas affected with "dandruff"

Se describen 75 estudios histopatológicos en biopsias de piel (33 afectados y 42 no afectados con “caspa”), 85 análisis microbiológicos en raspados de piel (44 afectados y 41 no afectados) y 70 determinaciones séricas de zinc, selenio, cobre y molibdeno (41 afectados y 29 no afectados) de tres poblaciones de vicuñas silvestres capturadas en “Chakus” en el 2009 en las comunidades campesinas de Huaytará, Ayaví, Santa Rosa de Tambo y en una población captiva multicomunal, en Huancavelica. Los animales afectados no tenían alteraciones clínicas, pero los vellones a la postesquila presentaron escamas blanquecinas dispersas o acumuladas y fuertemente adheridas, usualmente, al dorso lateral y algunas veces por todo el vellón. Todas las muestras de piel, con mayor severidad en las afectadas, mostraron moderada hiperqueratosis ortoqueratótica laminar asociada con dermatosis inespecífica, moderada-severa dilatación de folículos pilosos y moderada-severa atrofia de vaina interna de la raíz folicular pero con ausencia de agentes patógenos e inflamación. El 63.3% (28/44) de raspados de pieles afectadas y el 41.5% (17/ 41) de las no afectadas contenían especies saprofíticas de Ulocladium spp., Penicillum spp., Hialofomicetos, Geotrichum candidum y Aspergilus flavus. Los niveles sanguíneos, en las 70 muestras (afectados y no afectados) presentaron 10 veces la concentración esperada para selenio, principalmente en vicuñas captivas en el área multicomunal (afectados 3.23 ± 1.31 μg/mL y no afectados 3.56 ± 2.27 μg/mL), posiblemente debido al sobrepastoreo de los pastizales con presencia de especies seleníferas de Astragalus spp. (“garbanzo” o “garbancillo”). Todos los animales mostraron deficiencia de cobre y los animales afectados de Huaytará y todos los de Santa Rosa de Tambo presentaron deficiencia de zinc.In recent years important economic losses have resulted from what is described as “dandruff” in vicuña fiber. With the goal of analyzing the possible cause/s was conducted an histopathological analysis of 75 skin biopsies (33 affected/42 unaffected), microbiological analysis of 85 skin/fiber scrapings (44 affected/41 unaffected), and microelement analysis (zinc, selenium, copper, molybdenum) of 70 serum samples (41 affected/ 29 unaffected), collected from three wild populations in the communities of Huaytará, Ayaví and Santa Rosa de Tambo, Huancavelica, Peru, as well as from the captive herd held jointly by these communities. The affected vicuñas were clinically normal and the presence of “dandruff” was generally detected after shearing. In these fleeces, white scales scattered or accumulated and firmly adhered to the fibers were found, especially on the flanks and backs of the animals, but also widely dispersed throughout the fleece. Histopathological analysis of the skin biopsies revealed that both affected and unaffected animals had moderate to severe dermatosis (hyperkeratosis – orthokeratosis), with moderate to severe atrophy of the inner root sheath of the follicle, but without evidence of inflammation. Microbiological analysis determined the presence of fungus species in 63.3% (28/44) of the affected and 41.5% (17/41) of unaffected animals, including Ulocladium spp., Penicillum spp., Hialofomicetos, Geotrichum candidum and Aspergilus flavus. Microelement analysis revealed 10 fold selenium concentration as compared to normal values, especially in the captive population (affected: 3.23 ± 1.31 μg/ mL; unaffected: 3.56 ± 2.27 μg/mL) possibly due to overgrazing of pastures with presence of Astragalus spp., a common seleniferous plant in the region. All animals showed cooper deficiency. Also, all animals from Santa Rosa de Tambo and affected animals from Huaytará were zinc deficient

CONTRIBUTION TO THE STUDY OF GASTROINTESTINAL PARASITISM IN THE GUANACO (LAMA GUANICOE CACSILENSIS)

El propósito del presente estudio fue identificar las especies de parásitosgastrointestinales que afectan al guanaco peruano y determinar los niveles de parasitismode las poblaciones evaluadas. Se obtuvieron 132 muestras de heces frescas deguanacos silvestres pertenecientes a nueve poblaciones ubicadas en seis departamentosdel Perú: Comunidad Campesina de Huallhua (Ayacucho), Reserva Nacional de Calipuy(La Libertad), Comunidad Campesina de Chavín (Ica), Reserva Nacional Salinas y AguadaBlanca y distritos de Machaguay y Yarabamba (Arequipa), distrito de Quilahuani yComunidad Campesina de Vila Vilani (Tacna), y distrito de La Capilla (Moquegua). Lasmuestras fueron procesadas mediante técnicas coproparasitológicas de flotación, sedimentación,cultivo de larvas, Baerman y biometría de larvas y ooquistes. Se identificaronocho especies de nematodos: Graphinema aucheniae, Bunostomun sp., Ostertagia sp.,Trichuris sp, Cooperia sp., Nematodirus sp., Mazamastrongylus peruvianus yTrichostrongylus sp. y cuatro especies de Eimeria: E. lamae, E. alpacae, E. punoensis yE. macusaniensis. Todas las poblaciones se encontraban con al menos un guanacoparasitado, presentando en general cargas bajas y variando las frecuencias de parasitismogastrointestinal de una población a otra, dependiendo del hábitat y de la proximidada 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

DNA extraction methods from vicuña fecal samples (Vicugna vicugna mensalis)

Actualmente es posible determinar el estatus de especies silvestres mediante el análisis de ADN extraído de muestras no invasivas como las heces. Aunque dichos análisis suelen ser dificultosos debido principalmente a la composición y conservación de las heces, se ha demostrado en muchas especies que con el desarrollo de métodos adecuados de extracción es posible maximizar la fiabilidad y eficacia de este procedimiento. Por tanto, el objetivo del presente estudio fue optimizar un método de extracción de ADN de heces de vicuña Vicugna vicugna mensalis, especie clasificada como casi amenazada en el Perú, con la finalidad de obtener ADN de suficiente calidad para análisis moleculares. Se recolectaron 51 muestras de heces durante los meses de agosto y septiembre de 2008 en dos comunidades vicuñeras ubicadas a más de 4600 msnm en los departamentos de Junín y Moquegua. Luego de observar la defecación, las muestras fueron recolectadas en forma inmediata, conservadas en etanol y transportadas al laboratorio para la extracción del ADN. El kit comercial QIAamp® DNA Stool Mini Kit (QIAGEN) fue modificado para incluir un vigoroso y extenso proceso de agitación y los resultados fueron comparados con dos protocolos comúnmente utilizados: fenol/cloroformo/alcohol isoamílico y el mismo kit QIAGEN. La cantidad del ADN extraído fue observado en electroforesis horizontal en geles de agarosa, y la calidad evaluada mediante la amplificación y visualización de los tamaños esperados de los microsatélites YWLL46 y LCA19. Aunque se logró extraer ADN con los tres métodos, solamente el método modificado permitió obtener ADN de suficiente calidad para ser utilizado en pruebas moleculares.At present, the use of DNA extracted from non-invasive samples, especially feces, is common practice in the discipline of conservation genetics. Even though DNA analysis is often difficult mainly due to the composition and preservation of feces has been shown in many species with the development of suitable extraction methods is possible to maximize the reliability and effectiveness of such procedures. The objective of this study has been optimization of a protocol for the extraction of DNA from vicuña feces (Vicugna vicugna mensalis), species classified as Near Threatened in Peru, in order to obtain DNA of sufficient quality for molecular analysis. A total of 51 samples were taken from vicuña dung piles in two communities located 4600 meters above sea level in the dry Puna ecosystems of the regions of Junin and Moquegua between August and September 2008. After observing defecation, samples were collected immediately, preserved in ethanol and transported to the laboratory for DNA extraction. Modification of the protocol of the QIAamp ® DNA Stool Mini Kit (QIAGEN) to include an extended period of vigorous shaking was found to producer higher quality DNA than was obtained using both the kit protocol and PCI (Phenol/chloroform/isoamyl alcohol). The amount of extracted DNA was visualized using horizontal agarose gel electrophoresis, and the quality was tested analyzing microsatellite markers LCA 19 and YWLL 46 on polyacrylamide gels. Although DNA was obtained by all three methods, only the modified protocol produced DNA of sufficient quality for use in molecular analyses

Visualising harms in publications of randomised controlled trials: consensus and recommendations

OBJECTIVE: To improve communication of harm in publications of randomised controlled trials via the development of recommendations for visually presenting harm outcomes. DESIGN: Consensus study. SETTING: 15 clinical trials units registered with the UK Clinical Research Collaboration, an academic population health department, Roche Products, and The BMJ. PARTICIPANTS: Experts in clinical trials: 20 academic statisticians, one industry statistician, one academic health economist, one data graphics designer, and two clinicians. MAIN OUTCOME: measures A methodological review of statistical methods identified visualisations along with those recommended by consensus group members. Consensus on visual recommendations was achieved (at least 60% of the available votes) over a series of three meetings with participants. The participants reviewed and critically appraised candidate visualisations against an agreed framework and voted on whether to endorse each visualisation. Scores marginally below this threshold (50-60%) were revisited for further discussions and votes retaken until consensus was reached. RESULTS: 28 visualisations were considered, of which 10 are recommended for researchers to consider in publications of main research findings. The choice of visualisations to present will depend on outcome type (eg, binary, count, time-to-event, or continuous), and the scenario (eg, summarising multiple emerging events or one event of interest). A decision tree is presented to assist trialists in deciding which visualisations to use. Examples are provided of each endorsed visualisation, along with an example interpretation, potential limitations, and signposting to code for implementation across a range of standard statistical software. Clinician feedback was incorporated into the explanatory information provided in the recommendations to aid understanding and interpretation. CONCLUSIONS: Visualisations provide a powerful tool to communicate harms in clinical trials, offering an alternative perspective to the traditional frequency tables. Increasing the use of visualisations for harm outcomes in clinical trial manuscripts and reports will provide clearer presentation of information and enable more informative interpretations. The limitations of each visualisation are discussed and examples of where their use would be inappropriate are given. Although the decision tree aids the choice of visualisation, the statistician and clinical trial team must ultimately decide the most appropriate visualisations for their data and objectives. Trialists should continue to examine crude numbers alongside visualisations to fully understand harm profiles