Analysis of Doberman Pinscher and Toy Poodle samples with targeted next-generation sequencing.

Next-generation sequencing (NGS) technologies have enabled the identification of many causal variants of genetic disorders, the development of parentage tests and the analysis of multiple traits in domestic animals. In this study, we evaluated the performance of a Canine Targeted Genotyping-by-Sequencing (GBS) custom panel (Thermo Fisher Scientific, Waltham, Ma, USA) in a cohort of 95 dog DNA samples, comprising 76 Doberman Pinschers and 19 Toy Poodles from Argentina. The used panel included 383 targets (228 parentage SNVs, 137 genetic disorder markers and 18 trait markers). While paternity analysis showed correct duo (97.4%; LOD > 2.98E+13) and trio (100%; LOD > 2.20E+15) parentage assignment, the panel resulted still insufficient for excluding close relatives in inbred populations. In this sense, close relatives were wrongly assigned as parents in 12.6% of duos and 0.3% of trios. We detected 17 polymorphic markers (genetic disorders, n = 4; hair type, n = 3; coat color, n = 10) and estimated their allele frequencies in the studied breeds. The accuracy of targeted GBS results were evaluated for three markers that were associated with Progressive rod-cone degeneration, von Willebrand disease type 1 and dilated cardiomyopathy by pyrosequencing and Sanger sequencing genotyping, showing 94-100% concordance among assays. The targeted GBS custom panel resulted cost-effective strategy to study the prevalence of genetic disorders and traits in a large number of samples and to analyze genetic interactions between previously reported variants. Once assays based on AgriSeq technology were standardized, their uses are a good strategy for large-scale routine genetic evaluation of animal populations.

Infiltración comisural dorsal en el tratamiento del pulgar y los dedos en resorte. Estudio de una cohorte prospectiva / Out-of-sheath corticosteroid injections through the dorsal webspace for trigger finger and trigger thumb. A prospective cohort study

Antecedentes y objetivo: Las infiltraciones de corticoides son efectivas en el tratamiento de los dedos en gatillo pero el dolor percibido por el paciente durante la inyección es un efecto acompañante siempre presente. El objetivo de este estudio fue evaluar la efectividad y el dolor percibido durante una infiltración corticoidea depositada fuera de la vaina tendinosa utilizando la técnica comisural dorsal. Material y método: Se incluyeron 126 pacientes consecutivos. Se realizó una infiltración esteroidea subcutánea (fuera de la vaina) a través de la comisura dorsal. En los casos en que los signos o síntomas persistieron, se ofreció una segunda infiltración. Se registró el dolor percibido durante la infiltración mediante la escala visual analógica, el cuestionario DASH antes del tratamiento y al final de seguimiento, la tasa de éxito y las complicaciones. Resultados: Fueron 86 mujeres y 40 hombres con una edad media de 61 años. La puntuación media del dolor durante la infiltración fue de 3,8. Doce pacientes se perdieron durante el seguimiento. El éxito global fue del 68% y el éxito tras una única inyección fue del 54%. El mejor resultado se obtuvo en el dedo anular. Los pacientes que no habían sido operados previamente del síndrome del túnel carpiano respondieron mejor. No se objetivaron complicaciones. Conclusiones: La infiltración esteroidea fuera de la vaina tendinosa y utilizando la técnica comisural dorsal es efectiva y segura en el tratamiento de los dedos en resorte. Parece ser menos dolorosa que los resultados publicados para la técnica palmar sobre la línea media, aunque esto debe evaluarse en un estudio diseñado para ello.(AU)

Background and objective: Steroid injections are effective in the treatment of trigger digits but the pain during the injection is an always-present accompanying effect. The aim of this study was to assess the effectiveness and perceived pain during an out-of-sheath corticosteroid injection through the dorsal webspace in the treatment of trigger digits. Material and method: A total of 126 consecutive patients were included. A subcutaneous (out-of-sheath) corticosteroid injection was performed through the dorsal webspace in all digits. In cases where signs or symptoms persisted, a second injection was offered. Visual analog scale for pain during the injection, DASH questionnaire, success rate and complications were collected. Results: There were 86 women and 40 men with a mean age of 61 years. The mean visual analog scale for pain during the injection was 3.8. Twelve patients were lost to follow-up. The overall success was 68% and success after a single injection was 54%. The best result was achieved on the ring finger. Patients who were not previously operated on carpal tunnel syndrome responded better. No complications were noted. Conclusions: The extra-sheath corticosteroid injection through the dorsal webspace is effective and safe. It seems to be less painful than the reported scores for the palmar midline technique although it should be assessed in a comparative study.(AU)

Out-of-sheath corticosteroid injections through the dorsal webspace for trigger finger and trigger thumb. A prospective cohort study / Infiltración comisural dorsal en el tratamiento del pulgar y los dedos en resorte. Estudio de una cohorte prospectiva

Background and objective: Steroid injections are effective in the treatment of trigger digits but the pain during the injection is an always-present accompanying effect. The aim of this study was to assess the effectiveness and perceived pain during an out-of-sheath corticosteroid injection through the dorsal webspace in the treatment of trigger digits. Material and method: A total of 126 consecutive patients were included. A subcutaneous (out-of-sheath) corticosteroid injection was performed through the dorsal webspace in all digits. In cases where signs or symptoms persisted, a second injection was offered. Visual analog scale for pain during the injection, DASH questionnaire, success rate and complications were collected. Results: There were 86 women and 40 men with a mean age of 61 years. The mean visual analog scale for pain during the injection was 3.8. Twelve patients were lost to follow-up. The overall success was 68% and success after a single injection was 54%. The best result was achieved on the ring finger. Patients who were not previously operated on carpal tunnel syndrome responded better. No complications were noted. Conclusions: The extra-sheath corticosteroid injection through the dorsal webspace is effective and safe. It seems to be less painful than the reported scores for the palmar midline technique although it should be assessed in a comparative study.(AU)

Antecedentes y objetivo: Las infiltraciones de corticoides son efectivas en el tratamiento de los dedos en gatillo pero el dolor percibido por el paciente durante la inyección es un efecto acompañante siempre presente. El objetivo de este estudio fue evaluar la efectividad y el dolor percibido durante una infiltración corticoidea depositada fuera de la vaina tendinosa utilizando la técnica comisural dorsal. Material y método: Se incluyeron 126 pacientes consecutivos. Se realizó una infiltración esteroidea subcutánea (fuera de la vaina) a través de la comisura dorsal. En los casos en que los signos o síntomas persistieron, se ofreció una segunda infiltración. Se registró el dolor percibido durante la infiltración mediante la escala visual analógica, el cuestionario DASH antes del tratamiento y al final de seguimiento, la tasa de éxito y las complicaciones. Resultados: Fueron 86 mujeres y 40 hombres con una edad media de 61 años. La puntuación media del dolor durante la infiltración fue de 3,8. Doce pacientes se perdieron durante el seguimiento. El éxito global fue del 68% y el éxito tras una única inyección fue del 54%. El mejor resultado se obtuvo en el dedo anular. Los pacientes que no habían sido operados previamente del síndrome del túnel carpiano respondieron mejor. No se objetivaron complicaciones. Conclusiones: La infiltración esteroidea fuera de la vaina tendinosa y utilizando la técnica comisural dorsal es efectiva y segura en el tratamiento de los dedos en resorte. Parece ser menos dolorosa que los resultados publicados para la técnica palmar sobre la línea media, aunque esto debe evaluarse en un estudio diseñado para ello.(AU)

Out-of-sheath corticosteroid injections through the dorsal webspace for trigger finger and trigger thumb. A prospective cohort study. / Infiltración comisural dorsal en el tratamiento del pulgar y los dedos en resorte. Estudio de una cohorte prospectiva.

BACKGROUND AND OBJECTIVE: Steroid injections are effective in the treatment of trigger digits but the pain during the injection is an always-present accompanying effect. The aim of this study was to assess the effectiveness and perceived pain during an out-of-sheath corticosteroid injection through the dorsal webspace in the treatment of trigger digits. MATERIAL AND METHOD: A total of 126 consecutive patients were included. A subcutaneous (out-of-sheath) corticosteroid injection was performed through the dorsal webspace in all digits. In cases where signs or symptoms persisted, a second injection was offered. Visual analog scale for pain during the injection, DASH questionnaire, success rate and complications were collected. RESULTS: There were 86 women and 40 men with a mean age of 61 years. The mean visual analog scale for pain during the injection was 3.8. Twelve patients were lost to follow-up. The overall success was 68% and success after a single injection was 54%. The best result was achieved on the ring finger. Patients who were not previously operated on carpal tunnel syndrome responded better. No complications were noted. CONCLUSIONS: The extra-sheath corticosteroid injection through the dorsal webspace is effective and safe. It seems to be less painful than the reported scores for the palmar midline technique although it should be assessed in a comparative study.

Efectos de la rehabilitación pulmonar sobre los síntomas asociadosa las exacerbaciones en pacientes / Effects of pulmonary rehabilitation on symptoms associated with exacerbations in patients with COPD

Las exacerbaciones de enfermedad pulmonar obstructiva crónica (EPOC) son responsables del deterioro físico de los pacientes, del ingreso y reingreso hospitalario, así como de su muerte. Esta revisión evidencia los beneficios de la rehabilitación pulmonar en la reducción de episodios de exacerbaciones en la EPOC. La metodología de búsqueda abarcó un total de 2.473 artículos, de los cuales fueron usados 14 para esta revisión según el cumplimiento de criterios establecidos. Los resultados obtenidos fueron significativos en las siguientes variables vinculadas directamente con la frecuencia de los episodios de exacerbación: disnea, saturación de oxígeno, volumen espiratorio forzado en el primer segundo, días de hospitalización, número de reingresos hospitalarios, capacidad de ejercicio, depresión y calidad de vida. La rehabilitación pulmonar mostró efectos positivos en las variables mencionadas, disminuyendo los episodios de exacerbaciones de la EPOC como las limitaciones en las actividades de los pacientes (AU)

Exacerbations of chronic obstructive pulmonary disease (COPD) are responsible for the physical deterioration of patients, hospital admission and readmission, as well as their death. This review demonstrates the benefits of pulmonary rehabilitation in reducing exacerbation episodes in COPD. A search was carried obtained a total of 2473 articles, of which 14 were used for this review according to the fulfillment of established criteria. The results obtained were significant in relation to the variables directly related to the frequency of exacerbation episodes: dyspnea, oxygen saturation, forced expiratory volume in the first second, days of hospitalization, number of hospital readmissions, exercise capacity, depression and quality of life. Pulmonary rehabilitation causes positive effects on the aforementioned variables, reducing episodes of COPD exacerbations as well as limitations in patients’ activities (AU)

External Marginal Gap Evaluation of Different Resin-filling Techniques for Class II Restorations-A Micro-CT and SEM Analysis.

CLINICAL RELEVANCE: Secondary caries are the main reason for the failure of restorations, class II being the most affected. Techniques that promote less gap percentage are important. Flowable bulk fill composites used at such locations have been shown to decrease gap formation while being a faster procedure than an incremental technique.

Dorsal web injection technique in the treatment of trigger finger and trigger thumb. Anatomical study. / Infiltración comisural dorsal en el tratamiento del pulgar y los dedos en resorte. Estudio anatómico.

BACKGROUND AND OBJECTIVE: Corticosteroid injection is an effective treatment for trigger digits but the pain during the injection is an ever-present side effect. Since the palmar skin has a high density of sensitive receptors, injecting through dorsal skin could be less painful. Our aim was to assess whether a dorsal technique through the dorsal web is safe for extra-sheath injection of trigger fingers and thumb. MATERIAL AND METHOD: This is an anatomical study in sixteen cadaveric hands. An injection through the dorsal web was performed on each digit. After careful resection of the palmar skin, the distance between the needle and the main anatomical structures was measured. The risk of major injury was considered high when the mean distance from the needle to the neurovascular bundle was below 1mm. RESULTS: The mean distance from the needle to the neurovascular bundle was 1.77mm. Two neurovascular injuries in 112 injections were observed, one nerve and one artery. The safest digit was the thumb while the most dangerous was the index finger. At the ring finger, the technique was safer when it was carried out from the dorso-radial. CONCLUSIONS: A subcutaneous injection near the flexor tendon sheath can be carried out through the dorsal web with a low, but present, risk of neurovascular injury. It could be useful for injection in the treatment of trigger finger and trigger thumb but it should be assessed in a clinical study.

Polledness in Argentinean Creole cattle, five centuries surviving.

Polledness has been shown to have autosomal Mendelian inheritance, with the polled locus being dominant to the horned locus. This trait was mapped to the BTA1 centromeric end in several breeds. One of the distinctive attributes of Creole cattle, such as the Argentinean Creole, is the presence of long, lyre-shaped horns. However, polled native animals were reported before the introduction of modern selected European breeds. Here, we studied the origin of the polled mutation, either independent or introgressed, in a Creole line from the Creole cattle founder group at the IIACS-INTA Leales Experimental Station (northwest Argentina). The study sample (65 animals: 26 horned and 39 polled) was genotyped using high-density SNP microarrays and three previously reported genetic markers (P202 ID , P80kb ID and PG ). A genome-wide association study, selection signatures, linkage disequilibrium analysis and copy number variations were used to detect the responsible region and the segregating haplotypes/alleles. The interval mapped in the Leales herd (1.23-2.13 Mb) overlapped with the region previously reported in several European cattle breeds, suggesting that the same locus could be segregating in this population. The previously reported variants PF and PG were not detected, thus dismissing the Holstein-Friesian and Nellore origins of the polled phenotype in this native breed. Conversely, the presence of the Celtic variant PC suggests an almost complete co-segregation. The cluster analysis rejected the hypothesis of recent introgression, which is compatible with the historical record of polled Creole cattle in northwest Argentina.

Experimental analysis of the minimally invasive plate osteosynthesis technique applied with non-locking screws and screw locking elements.

Minimally invasive plate osteosynthesis (MIPO) is an effective surgical technique in the repair of humeral and tibial shaft fractures. There is some controversy as to the minimum number of screws required to ensure correct stability to promote healing, especially when dealing with low quality bones. This work compared different systems assembled on synthetic models simulating a comminuted fracture. Group 1 comprised a locking compression plate with four non-locking screws placed at the holes furthest from the fracture. Group 2 differed from group 1 only in the additional use of two screw locking elements (SLE). Group 3 had four rather than two SLE and, finally, Group 4 used 4 locking screws. The compression and torsion tests with static and cyclic loads showed that, in MIPO, two locking screws or two non-locking screws with SLE could be used per segment without any significant loss in stiffness after 1000 cycles, with system stability guaranteed in both cases. However, lower strength and significant loss of stiffness were observed when non-locking screws were used alone.

Mechanical properties and corrosion behavior of Mg-HAP composites.

Mg and Mg-HAP composites containing 5, 10 and 15 wt% of hydroxyapatite have been produced following a powder metallurgy route that consists of mixing raw powders and consolidation by extrusion. The microstructure, texture, mechanical behavior and resistance to corrosion under a PBS solution have been studied. Addition of HAP increases the microhardness of the composites, however the yield strength under compression slightly decreases. Texture analyses reveal a fiber texture for pure Mg that is weakened increasing the HAP fraction. This texture promotes twinning and softening of Mg and Mg-5HAP during the initial deformation stages. Mg-10HAP and Mg-15HAP present a strain-hardening dependence showing no softening. The volume fraction of HAP particles weakens the texture and favors the activation of secondary slip systems. Corrosion experiments in PBS solution have shown that Mg-5HAP exhibits the best resistance to corrosion. Texture and porosity appear to be the main material features controlling the corrosion rates of Mg-HAP composites under the present conditions.

Suitability of DCPs with screw locking elements to allow sufficient interfragmentary motion to promote secondary bone healing of osteoporotic fractures.

This paper analyses the suitability of a system comprising a Dynamic Compression Plate (DCP) and Screw Locking Elements (SLEs) to allow sufficient interfragmentary motion to promote secondary bone healing in osteoporotic fractures. Four fixation systems were mounted on bone-simulating reinforced epoxy bars filled with solid rigid polyurethane foam. Group 1, used for comparison purposes, represents a system comprised of a Locking Compression Plate (LCP) and eight locking screws. Groups 2 and 3 represent a system comprised of a DCP plate with eight cortical screws and two SLEs placed on the screws furthest from (group 2) and nearest to (group 3) the fracture. Group 4 represents the system comprised of a DCP plate with SLEs placed on all eight cortical screws. Cyclic compression tests of up to 10,000 load cycles were performed in order to determine the parameters of interest, namely the stiffnesses and the interfragmentary motion of the various configurations under consideration. Tukey's multiple comparison test was used to analyse the existence or otherwise of significant differences between the means of the groups. At 10,000 cycles, interfragmentary motion at the far cortex for group 2 was 0.60±0.04 mm and for group 3 0.59±0.03 mm (there being no significant differences: p=0.995). The mean interfragmentary motion at the far cortex of the LCP construct was 70% less than that of the two groups with 2SLEs (there being significant differences: p=1.1×10(-8)). In the case of group 4 this figure was 45% less than in groups 2 and 3 (there being significant differences: p=5.6×10(-6)). At 10,000 cycles, interfragmentary motion at the near cortex for group 2 was 0.24±0.06 mm and for group 3 0.24±0.03 mm (there being no significant differences: p=1.000). The mean interfragmentary motion at the near cortex of the LCP construct was 70.8% less than that of the two groups with 2SLEs (there being significant differences: p=0.011). In the case of group 4 this figure was 66.7% less than in groups 2 and 3 (there being significant differences: p=0.016). The mean stiffness at 10,000 cycles was 960±110 N mm(-1) for group 2 and 969±53 N mm(-1) for group 3 (there being no significant differences: p=1.000). For group 1 (the LCP construct) the mean stiffness at 10,000 cycles was 3144±446 N mm(-1), 3.25 times higher than that of groups 2 and 3 (there being significant differences: p=0.00002), and 1.6 times higher than that of the DCP+8SLEs construct (1944±408 N mm(-1), there being significant differences: p=0.007). It is concluded that using the DCP+2SLEs construct sufficient interfragmentary motion is ensured to promote secondary bone healing. However, if too many SLEs are used the result may be, as with the LCP, an excessively rigid system for callus formation.

Screw locking elements: a means to modify the flexibility of osteoporotic fracture fixation with DCPs without compromising system strength or stability.

This paper analyses whether it is possible to use dynamic compression plates (DCPs) and screw locking elements (SLEs) to vary the flexibility of osteoporotic fracture fixation without compromising the strength and stability of the construct. Compression, torsion and four-point bending static strength tests were conducted. Cyclic load tests of up to 10,000 load cycles were also carried out to determine stiffness performance. Four fixation systems were mounted onto polyurethane bone models. Group 1 consists of the DCP and six cortical screws. Group 2, idem, but with the addition of two SLEs. Group 3, idem, but with the addition of six SLEs. Group 4 used the locking compression plate (LCP) and locking screws. The results indicated no significant difference (p>0.05) in the strength of groups 2-4. It was also observed that the torsional stiffness of group 3 (0.30 Nm/°) was higher than that of group 2 (0.23 Nm/°) and similar to that of group 4 (0.28 Nm/°). Compression stiffness of group 4 (124 N/mm) was higher than that of group 2 (102 N/mm), but lower than that of group 3 (150 N/mm). No notable differences were observed for structural bending stiffness. It is concluded that by using the DCP with SLEs it is possible to modify the stiffness of the fixation construct for the repair of osteoporotic fractures and, in this way, facilitate the conditions suitable on secondary bone healing.

Chronic kidney disease after orthotopic liver transplantation in recipients receiving tacrolimus.

INTRODUCTION: Chronic kidney disease (CKD) is common in liver transplant recipients receiving calcineurin inhibitors. METHOD AND POPULATION: The goals of this case-control study were to identify risk factors associated with CKD and its effect on mortality in 294 liver transplant recipients receiving calcineurin inhibition with tacrolimus. RESULTS: Hepatitis C virus (HCV) was the most common indication (42%) for transplantation. CKD 4 and 5 (estimated glomerular filtration rate (eGFR) of <=29 ml/min/1.73 m2) developed in 10.8% of recipients during a mean follow-up of 52 months. The incidence density of CKD was 2.56 per 100 patient-years. End-stage renal disease developed in 2.7%. By univariate analysis, CKD patients were older (mean±sd, 57±10 vs. 51±11, p<0.05) with hypertension (56 vs. 32%, p<0.05), had lower preoperative hematocrit (31±6 vs. 34±5, p<0.05), alanine aminotransferase (median (95% confidence limit) 46 (34­80) vs. 68 (56­77), <0.05) and eGFR (56±28 vs. 91±35 ml/ min/1.73 m2, p<0.05), had higher preoperative prothrombin time (16.1 (14.6­17.2) vs. 14.8 (14.5­15.1) seconds, p<0.05), and required more perioperative renal replacement therapy (RRT) (41% vs. 6.5%, p<0.05) compared to controls. Perioperative need for RRT (hazard ratio (95% CI) 2.72 (1.05­7.03)) and lower preoperative eGFR: 60­89 (4.08 (1.23­13.5)), 30­59 (4.26 (1.18­15.36)), and<=29 (5.91 ((1.28­27.19)) vs. eGFR>=90 ml/min/1.73 m2 were independently associated with development of CKD adjusting for important covariates. The development of CKD (2.36 (1.22­4.59)) was independently associated with late mortality with an attributable risk of 12.8%. CONCLUSION: Data demonstrate that CKD is an important clinical event associated with increased risk for death after primary liver transplantation.

Biomechanical evaluation of a new system to improve screw fixation in osteoporotic bones.

In this paper an experimental analysis is undertaken of the affect a new screw-to-bone fixation system has on the stiffness of fixation systems of osteoporotic fractures based on osteosynthesis plates. The proposed system, which we have named the screw locking element (SLE), is made with elements manufactured from a biocompatible polymer material known as polyetheretherketon (PEEK) which act like a lock nut, holding the end of the threaded screw shank after this has passed through both bone corticals. Seventy-two osteoporotic synbone simulated fracture models were instrumented with one of four constructs: locking compression plate with 6 locking screws (LCP), dynamic compression plate with 6 cortical screws (DCP), DCP with 2 SLEs or DCP with 6 SLEs (DCP+6SLEs). Each group of 18 simulated fracture models were further split into 3 subgroups of 6. One subgroup was tested under cyclic cantilever bending, another under cyclic compression and the third under cyclic torsion. Loss of stiffness was determined in each test every 1,000 load cycles, between 0 and 30,000 cycles. Regardless of the load type, it was seen that the DCP system had the highest stiffness loss percentages of all the tested systems. The inclusion of SLEs significantly decreased the stiffness loss of the DCP system. Unlike the cyclic compression loads, where the LCP performed slightly better, on terminating the cantilever bending and torsion load cycles no statistically significant difference was noted (Tukey test, p>0.05) between the percentage stiffness loss of the DCP+6SLEs system and the LCP system. It is concluded that the proposed SLEs enable DCPs to lower the high failure rate that these exhibit in osteoporotic fracture repairs, at significantly lower costs than those resulting from the use of LCPs.

A new device for the fixation of anterior cruciate ligament tendon grafts. Design and experimental study.

In this paper the design and experimental analysis is presented of a new fixation device of anterior cruciate ligament (ACL) grafts of the knee. This device is inserted into the bone tunnel, after the graft, in the same way as an interference screw. However, the fixation device described in this paper has been designed in such a way that, after the insertion of a threaded element in its interior, some of its components expand in a radial direction, pressing against the walls of the bone tunnel and thereby increasing the fixation of the graft. This expansion device can be used in both the femur and the tibia. The device proposed in this paper was compared with an interference screw for load failure and fixation stiffness in experiments performed using porcine bones. The failure load was significantly higher in the new expansion device group (633+/-202 N) than in the interference screw group (471+/-179 N). The stiffness obtained when the new device was used (59+/-20 N/mm) was also significantly higher than that obtained using the interference screw (37+/-19 N/mm) (t-test, P<0.05). According to these results, this new device could be considered a good alternative to improve fixation of anterior cruciate ligament grafts.

Quantification of Cryptosporidium parvum in anaerobic digesters treating manure by (reverse-transcription) quantitative real-time PCR, infectivity and excystation tests.

The survival of Cryptosporidium parvum oocysts in anaerobic digesters treating manure was investigated for mesophilic, thermophilic, and a combined treatment (mesophilic-thermophilic-mesophilic) under different retention times of oocysts in the reactors. C. parvum DNA was extracted with an optimised protocol, and its amount determined by quantitative real-time PCR (qPCR). Results indicated noteworthy differences in DNA content after the different treatments. DNA was not degraded during the process. However, excystation and infectivity tests showed a reduction of viable oocyst numbers of > or = 2 and > or = 5 log units after the thermophilic treatment in two different experiments. Thus qPCR-targeting DNA can overestimate the number of oocysts that survive and remain viable after anaerobic digestion. However, targeting DNA is suitable to indicate the presence or absence of oocysts. Reverse transcription qPCR (RT-qPCR) targeting C. parvum hsp70 mRNA successfully indicated the presence of viable fraction of oocysts.

Mesophilic-thermophilic-mesophilic anaerobic digestion of liquid dairy cattle manure.

The potential of a mesophilic-thermophilic-mesophilic anaerobic digestion system was investigated with respect to improvement of both digestion and sanitation efficiencies during treatment of liquid cattle manure. The pilot plant produced a high methane yield from liquid dairy cattle manure of 0.24 m3 (kg VSfed)(-1) Considering the low system loading rate of 1.4-1.5 kg VS (m3 d)(-1), digestion efficiency compared to conventional processes did not appear improved. The minimum guaranteed retention time in the tubular thermophilic reactor was increased compared to a continuously stirred tank reactor. Levels of intestinal enterococci in raw liquid manure as determined with cultivation methods were reduced by 2.5 -3 log units to a level of around 10(2) cfu/mL. This sanitizing effect was achieved both during mesophilic-thermophilic-mesophilic and thermophilic-mesophilic treatment, provided the thermophilic digester was operated at 53-55 degrees C. A change in feeding interval from 1 h to 4 h did not significantly alter methane yield and sanitation efficiency. It was proposed that a two-stage, thermophilic-mesophilic anaerobic digestion system would be able to achieve the same sanitizing effect and equal or better digestion efficiency at lower costs.

Hygienization by anaerobic digestion: comparison between evaluation by cultivation and quantitative real-time PCR.

In order to assess hygienization by anaerobic digestion, a comparison between evaluation by cultivation and quantitative real-time PCR (qPCR) including optimized DNA extraction and quantification was carried out for samples from a full-scale fermenter cascade (F1, mesophilic; F2, thermophilic; F3, mesophilic). The system was highly effective in inactivating (pathogenic) viable microorganisms, except for spore-formers. Conventionally performed cultivation underestimated viable organisms particularly in F2 and F3 by a factor of at least 10 as shown by data from extended incubation times, probably due to the rise of sublethally injured (active but not cultivable) cells. Incubation should hence be extended adequately in incubation-based hygiene monitoring of stressed samples, in order to minimize contamination risks. Although results from qPCR and cultivation agreed for the equilibrated compartments, considerably higher qPCR values were obtained for the fermenters. The difference probably corresponded to DNA copies from decayed cells that had not yet been degraded by the residual microbial activity. An extrapolation from qPCR determination to the quantity of viable organisms is hence not justified for samples that had been exposed to lethal stress.

Evaluating real-time PCR for the quantification of distinct pathogens and indicator organisms in environmental samples.

We evaluated quantitative real-time PCR (qPCR) and RTqPCR (for RNA species) for their ability to quantify microorganisms and viruses in problematic environmental samples such as cattle manure, digester material, wastewater and soil. Important developments included a standard spiking approach which compensated for methodological bias and allowed sample-to-sample comparison and reliable quantification. Programme CeTe was developed to calculate endogenous concentrations of target organisms (nucleic acid copies) for each sample separately from the generated standard curves. The approach also permitted assessment of the detection limit of the complete method, including extraction. It varied from sample to sample, due to different extraction efficiencies and variable co-extraction of PCR inhibitors. False negative results were thereby avoided. By using this approach we were able to optimise a DNA extraction protocol from the different tested sample types. Protocols for the extraction of RNA species from environmental samples were also optimised. DNA was (almost) not degraded after lethal shock (autoclaving) in the sterile environment. In contrast, the parallel selective cultivation and qPCR results for various microbial parameters from an anaerobic digester chain suggested that DNA from decaying organisms was readily recycled in metabolically active environments. It may, therefore, be used to determine viable organisms in samples exhibiting substantial metabolic turnover. It is proposed that our standard spiking approach, including data evaluation by the program CeTe, should be considered in future standardisation and norms for the quantification of nucleic acid containing organisms in environmental and product samples.