Comunicación corta. Infección por Sarcocystis: una causa importante de decomiso de canales en ovino adulto en España

The frequency, distribution and impact of small ruminant Sarcocystis infection in the European Union is largely unknown; this study reports the prevalence of macroscopic Sarcocystis sp. cysts and associated carcass condemnation, in 6065 adult, cull, small ruminants from 145 farms in Spain. Macrocysts were detected in 12% of sheep from 60% flocks, and in none of the 345 goats examined. Most affected sheep had cysts in more than one body part and as a result, 79% of sheep carcasses with cysts were totally condemned. Consequently, it is estimated that Sarcocystis spp. infection could be costing the Spanish sheep industry € 20 million yr-1. Three types of cysts were identified according to size, shape and location: narrow, filament-shaped measuring 2-10 × ≤1 mm, present striated muscles only, and two wider types measuring 2-20 × 2-6 mm, including oval-shaped oesophageal cysts and more elongated cysts in striated muscles. Narrow and wide macrocysts were found in the same sheep and are compatible with Sarcocystis gigantea and Sarcocystis medusiformis, respectively, as described in New Zealand in the 1970s. However, cyst size and morphology varies with age and location. Moreover, S. medusiformis has not been reported in Europe and species-specific diagnosis is necessary to ascertain the ethiology of macrocysts in this study. Apenas existe información sobre la frecuencia, distribución e impacto actual de la sarcocistiosis en la Unión Europea; este trabajo describe la prevalencia de quistes macroscópicos de Sarcocystis spp. y el porcentaje de decomisos asociados a los quistes, en 6.065 pequeños rumiantes adultos de desvieje, de 145 granjas de España. Se observaron quistes en 12% de ovejas de 60% de rebaños, y en ninguna de las 345 cabras examinadas. La mayoría de las ovejas afectadas presentó quistes en varias zonas corporales decomisándose el 79% de las canales afectadas. Según esto, las pérdidas por la infección por Sarcocystis spp. para la industria ovina española se estimaron en 20 millones de euros año–1. Se observaron tres tipos de quistes según el tamaño, forma y localización: estrechos, filiformes, de 2-10 × ≤ 1 mm, en músculo estriado solamente y dos tipos de quistes anchos, de 2-20 × 2-6 mm, ovales en esófago y los mas elongados en musculatura estriada, que podrían corresponderse con las especies Sarcocystis gigantea y Sarcocystis medusiformis, descritas en Nueva Zelanda en la década de 1970. Sin embargo, sería necesario un diagnóstico específico para confirmar la etiología de los quistes de este estudio, ya que el tamaño y morfología de éstos varía según la edad y la localización, y S. medusiformis no está descrita en Europa

Prevalencia de Leishmaniosis en perros abandonados del ámbito periurbano de Murcia: evaluación de diferentes técnicas de diagnóstico

This study investigates the prevalence and validity of diagnostic techniques of Canine Leishmaniosis (CanL), caused by the protozoan Leishmania infantum, in 43 abandoned dogs of periurban areas of Murcia Region. This infection, transmitted by phlebotomine sandflies, is often asymptomatic in dogs, even without antibodies production against the parasite. Dogs were examined to detect compatible symptoms. Blood and lymphoid tissue samples were taken to analyze parasite’s presence by optical microscopy (OM) and in vitro culture, as well as DNA detection by Polymerase Chain Reaction (PCR) technique, and anti-Leishmania antibodies detection in serum with Enzyme-Linked ImmunoSorbent Assay (ELISA) technique. The percentage of symptomatic dogs was 16%, and positive results at the different diagnostic techniques were 37% at OM, 64% at PCR, 25% at culture and 40% at ELISA. The percentage of positive-PCR samples is similar to that described in asymptomatic dogs in endemic areas whereas OM and ELISA results are substantially higher than other studies in endemic areas, including a previous study in Murcia. The reasons of these results may be explained by the presence of symptomatic dogs in this study and the long time dedicated to OM impronts of up to three different tissues per animal, which raised diagnostic sensitivity. These results confirm the high prevalence of CanL in periurban areas of Murcia Region and the good sensitivity of optical microscopy in CanL diagnosis in the hands of an experienced observer.El presente trabajo investiga la prevalencia y la validez de técnicas de diagnóstico de la Leishmaniosis canina causada por el protozoo Leishmania infantum, en 43 perros abandonados del ámbito periurbano de la Región de Murcia. Esta infección, transmitida por artrópodos flebotominos, cursa en muchos animales de forma asintomática, en los que predomina una respuesta inmunológica celular sin producción de anticuerpos frente al parásito. Los perros se examinaron para detectar síntomas compatibles con la enfermedad, y se tomaron muestras de sangre y tejido linfoide (bazo, ganglio linfático y médula ósea) para analizar la presencia del parásito por microscopía óptica y cultivo in vitro, así como la detección de ADN parasitario mediante la reacción en cadena de la polimerasa (PCR) y de anticuerpos anti-Leishmania en suero mediante la técnica “Enzyme-Linked ImmunoSorbent Assay” (ELISA). El porcentaje de perros con síntomas fue del 16%, y el de resultados positivos a las distintas técnicas diagnósticas fue 37% en la microscopía óptica, 64% a PCR, 25% al cultivo y 40% al ELISA. El porcentaje de muestras positivas a la PCR es similar al descrito en perros asintomáticos de otras zonas endémicas, mientras que el de muestras positivas por microscopía óptica y ELISA fueron superiores al de otras zonas endémicas, incluido un estudio anterior en Murcia. Esto podría deberse a la presencia en este estudio de perros con síntomas y al elevado tiempo dedicado a la observación microscópica de preparaciones de hasta tres tejidos por animal, lo que aumentaría la sensibilidad del diagnóstico. Los resultados de este trabajo corroboran la elevada prevalencia de la Leishmaniosis en el ámbito periurbano de la Región de Murcia y la utilidad de la microscopía en el diagnóstico de la infección como una técnica que, si bien requiere experiencia del observador, es sencilla y económica

Application of virtual microscopy for the teaching of parasitic diseases in the veterinary degree

La creación de materiales digitales con muestras escaneadas y el uso de la plataforma de tele-enseñanza en la asignatura de Enfermedades parasitarias ha servido para dinamizar la docencia de esta asignatura tradicionalmente difícil por su requerimiento de habilidades memorísticas y analíticas, dotándola de un carácter colaborativo y más cercano a la realidad clínica, y ha permitido incorporar otras herramientas de evaluación, como la evaluación por pares, donde el alumnado aprende corrigiendo a sus compañeros. además, su uso ha resultado atractivo al alumnado, muy acostumbrado a manejar contenidos digitales en la web, y ha tenido un efecto positivo para la superación de la asignaturaThe creation of digital materials with scanned samples and the use of the tele-teaching platform in the subject of parasitic diseases has served to boost the teaching of this traditionally difficult subject due to its requirement of memory and analytical skills, giving it a collaborative character and closer to the clinical reality, and has allowed incorporating other assessment tools, such as peer evaluation, where students learn by correcting their peers. In addition, its use has been attractive to students, very used to handling digital content on the web, and has had a positive effect for the passing of the subjec

Estudio cropológico de parasitosis en gatos del área periurbana de la ciudad de Murcia y sus implicaciones zoonósicas

A coprological study was carried out in 61 cats from Murcia city´s periurban areas in southeast Spain, to estimate the prevalence and abundance of parasite species. Most of them were stray cats captured in the street by the local authority. Cats signalment was recorded and they were clinically examined to assess their health status and clinical signs. Faecal samples were collected directly from the rectum. They were first examined for macroscopic parasitic structures such as adult nematodes and cestode proglottids. Faeces were then processed by Bailinger´s technique and sedimentation and quantitative and qualitative flotation using Sheather and zinc sulphate solutions were employed to detect microscopic parasitic structures. Overall prevalence (95% confidence interval) of infected cats considering macroscopic and microscopic analysis was 59% (47-71%). However, prevalence was 34% (23-46%) for the intestinal nematode Toxocara cati, 20% (10-30%) for the lungworm Aelurostrongylus abstrusus, 15% (6-24%) for intestinal nematode Ancylostomatidae, 13% (5-22%) for the cestode Taenia spp., 8% (1-15%) for the cestode Dipylidium caninum´s eggs, 18% (8-28%) for D. caninum´s proglottids, 8% (1-15%) for protozoan Isospora rivolta and 2% (0-5%) for the intestinal nematode Trichuris spp. The prevalence of T. cati was higher than other parasites except A. abstrusus and Ancylostomatidae, and the prevalence of Trichuris spp. was lower compared to other parasites including Taenia spp., D. caninum and I. rivolta (p<0.05). Moreover, prevalence and parasite abundance were not significantly associated to clinical signs or other variables except that prevalence of Taenia spp. infection was greater in pregnant queens compared to other cats (p<0.05). The study shows a high prevalence of parasitosis in cats in the periurban area of Murcia and urges for improving their control. An unexpected finding was the absence in faeces of species with high zoonotic potential including protozoa such as Giardia duodenalis, Cryptosporidium parvum y Toxoplasma gondii and this could be related to the limited sensitivity of classical coprological techniques employed and also to the fact that these parasites have a limited period of patency.Se realizó un estudio coprológico para estimar la prevalencia y abundancia de formas parasitarias en heces de 61 gatos de la zona periurbana de Murcia (España), mayoritariamente callejeros. Tras la exploración clínica de los animales, se recogieron muestras de heces del recto que se examinaron macroscópicamente para detectar proglótidos de cestodos y nematodos adultos. Seguidamente, se analizaron con la técnica de Bailinger, examinándose a continuación la muestra mediante tres métodos: estudio del sedimento, examen del mismo con una solución de Sheather (d=1,27) y con una solución de sulfato de zinc (d = 1,) para la detección microscópica de parásitos, de forma cualitativa y cuantitativa (con recuento en cámara de McMaster). La prevalencia (IC95%) de gatos parasitados fue 59% (47-71%) y varió según la especie parasitaria, siendo 18% (8-28%) de proglótidos de Dipylidium caninum, 34% (23-46%) de huevos de Toxocara cati, 20% (10-30%) de larvas de Aelurostrongylus abstrusus, 15% (6-24%) de huevos de Ancylostomatidae, 13% (5-22%) de huevos de Taenia spp., 8% (1-15%) de huevos de Dipylidium caninum y ooquistes de Isospora rivolta y 2% (0-5%) de huevos de Trichuris spp.. La prevalencia de T. cati fue superior a la de otros parásitos excepto A. abstrusus y Ancylostomatidae, y la prevalencia de Trichuris spp. fue inferior a la de estos dos últimos y de Taenia spp. (p<0.05). Los recuentos de parásitos fueron bajos por lo general, siendo los más elevados los de T. cati (6500 huevos/g de heces) seguidos de Ancylostomatidae (2450 h/g) e I. rivolta (1400 ooquistes/g). No se observaron diferencias significativas en la prevalencia y en la abundancia de parásitos entre las técnicas microscópicas. Tampoco se asoció la parasitosis a variables demográficas ni a la presencia de signos clínicos, excepto que la prevalencia de Taenia spp. fue mayor en hembras gestantes que en otros gatos (p>0.05). El estudio demuestra una elevada prevalencia de parasitos y justifica la necesidad de mejorar su control en la población estudiada. Destaca la ausencia en las heces de los protozoos zoonósicos Giardia duodenalis, Cryptosporidium parvum y Toxoplasma gondii que podría estar relacionado con la sensibilidad limitada de las técnicas coprológicas clásicas empleadas y también al hecho de que estos parásitos tienen un periodo de patencia limitado

A trichostatin A expression signature identified by TempO-Seq targeted whole transcriptome profiling

<div><p>The use of gene expression signatures to classify compounds, identify efficacy or toxicity, and differentiate close analogs relies on the sensitivity of the method to identify modulated genes. We used a novel ligation-based targeted whole transcriptome expression profiling assay, TempO-Seq®, to determine whether previously unreported compound-responsive genes could be identified and incorporated into a broad but specific compound signature. TempO-Seq exhibits 99.6% specificity, single cell sensitivity, and excellent correlation with fold differences measured by RNA-Seq (R² = 0.9) for 20,629 targets. Unlike many expression assays, TempO-Seq does not require RNA purification, cDNA synthesis, or capture of targeted RNA, and lacks a 3′ end bias. To investigate the sensitivity of the TempO-Seq assay to identify significantly modulated compound-responsive genes, we derived whole transcriptome profiles from MCF-7 cells treated with the histone deacetylase inhibitor Trichostatin A (TSA) and identified more than 9,000 differentially expressed genes. The TSA profile for MCF-7 cells overlapped those for HL-60 and PC-3 cells in the Connectivity Map (cMAP) database, suggesting a common TSA-specific expression profile independent of baseline gene expression. A 43-gene cell-independent TSA signature was extracted from cMAP and confirmed in TempO-Seq MCF-7 data. Additional genes that were not previously reported to be TSA responsive in the cMAP database were also identified. TSA treatment of 5 cell types revealed 1,136 differentially expressed genes in common, including 785 genes not previously reported to be TSA responsive. We conclude that TSA induces a specific expression signature that is consistent across widely different cell types, that this signature contains genes not previously associated with TSA responses, and that TempO-Seq provides the sensitive differential expression detection needed to define such compound-specific, cell-independent, changes in expression.</p></div

TempO-Seq assay sensitivity.

<p>(A) URR RNA was diluted in 10-fold steps with input of 100 ng down to 0.1 pg total RNA, plus no-input, in triplicate. Error bars indicate 1 standard deviation. Each color indicates a different gene, selected across the dynamic range. (B) MDA MB 231 cell lysates were diluted in 10-fold steps, for a range of 4,000 down to 0.004 cells in the assay. Genes were selected as for (A). (C) Mix 2 of the synthetic reference ERCC ExFold RNA Mixtures was diluted in 10-fold steps from 1x10^-3 down to 1x10^-6 of the supplied stock in URR as carrier, then assayed using a detector oligo pool specific for the ERCC RNAs. Average reads per sample ranged from 3.6K for the 1x10^-6 dilution to 340K for the 1x10^-3 dilution. Results from the 1 x 10⁻⁵ dilution are shown. (D) MDA MB 231 cells were diluted in 10-fold increments into a constant background of MCF7 cells (blue bars), or MCF-7 cells were diluted into a constant background of MDA MB 231 cells (green bars), then lysed and assayed for cell-specific transcripts. Of the 13 and 14 genes monitored, respectively, the fraction that were significantly above background is shown for each cell dilution. Read depth ranged from 3.6M/sample for 100%, 299K for 0.1%, and down to 64K for 0.00001% for both titrations.</p

Context independence.

<p>(A) The overlap between two DO pools is illustrated. Red indicates the DOs only in the 2,363-plex pool, blue indicates the DOs only in the 2,941-plex pool, and green indicates the 1,396 DOs that are in present in both pools. (B) Read counts for the 1,396 transcripts targeted in both pools are compared (R² = 0.98). Read depth for the 2,363-plex pool was 6.3M/sample, and for the 2,941-plex pool was 4.7M/sample.</p

Assay repeatability, background and differential expression.

<p>Reference RNAs (100 ng each) were run in the Whole Transcriptome TempO-Seq assay in triplicates of Universal Reference RNA (A), Reference Brain (B), or no RNA input (C). Data were normalized by scaling to the average of the replicates by sample type. Correlations (R²) between technical replicates of RNA were 0.97 (URR) and 0.98 (Brain). No-input sample total reads were 0.07% of the reads for Brain and 0.10% of the reads for URR. (D) Differential expression between URR and Brain is shown.</p

RT-PCR confirmation of selected TSA-responsive genes.

<p>For each gene, the average number of cycles needed to achieve 20% of the maximum yield at 45 cycles is listed. Control housekeeping genes are shown above and proposed TSA-responsive genes are shown below. Green highlighting indicates concordance in the direction of the TSA effect. Tan highlighting indicates a limitation impacting sensitivity (high cycle number, small fold difference, low expression, difference in cycle number less than 1 standard deviation of the controls). Yellow highlighting indicates marginal sensitivity (high cycle number). Blue highlighting indicates relative confidence in results to be compared.</p

TempO-Seq biochemical scheme.

<p>RNAs are targeted by annealing to DOs that contain target-specific sequences (green) as well as primer landing sites (red and yellow) that are shared across all DOs. Excess oligos are removed by a 3′ exonuclease, then the hybridized oligos are ligated and amplified using primers that contain sample tag (index) sequences (orange and purple bars), and adaptors required for sequencing. The amplified assay products are pooled for a library, purified/concentrated and sequenced.</p