Coprological diagnosis: what's new?

Analysis of faecal samples for the presence of parasite eggs, larvae, cysts and oocysts is the most widely used diagnostic procedure both in veterinary and human parasitology. After its fundation by C. J. Davaine in 1857, several copromicroscopic techniques have been developed. The McMaster technique developed and improved at the McMaster laboratory of the University of Sidney (whose name derives from one of the great benefactors in veterinary research in Australia, the McMaster family) is the most universally used technique for estimating the number of parasite elements in faeces. In the literature, however, many variations of the McMaster technique are to be found, and there is a clear need for the standardization of this technique. Recently, we have conducted a study in order to evaluate the influence of flotation solution, sample dilution, and the choice of McMaster slide area (volume) on the reliability of the McMaster technique in estimating the egg counts of gastrointestinal strongyles and Dicrocoelium dendriticum in a composite sample of faeces from naturally infected sheep. The study used 14 flotation solutions (having specific gravities between 1.200 and 1.450), 6 sample dilutions (1:10, 1:15, 1:20, 1:30, 1:40 and 1:50), and 4 McMaster slide areas (volumes) (McM 0.15 ml, McM 0.3 ml, McM 0.5 ml and McM 1.0 ml). The type of flotation solution used significantly influenced the EPG in the GI strongyle and in the D. dendriticum egg counts. All the sucrose based solutions at s.g. between 1.200 and 1.350 floated more GI strongyle eggs than the others. With respect to D. dendriticum, only six solutions were capable of floating eggs and the potassium jodomercurate solution (s.g.1.440) floated more eggs than the others. The reliability of the McMaster technique regarding sample dilution was high for both GI strongyle and D. dendriticum EPG at 1:10 and 1:15, and then progressively decreased with increasing dilution. The reliability of the McMaster technique regarding the choice of the McMaster slide area (volume) was high for both GI strongyle and D. dendriticum EPG at the McMaster slide area (volume) of 1.0 ml, i.e., the total area of the McMaster slide. The EPG counts resulting from choosing any of the other three McMaster slide areas (volumes), i.e. McM 0.15 ml, McM 0.3 ml, or McM 0.5 ml, produced unreliable over-estimates. In order to improve the sensitivity of copromicroscopic diagnosis, at our laboratory a novel technique (flotation-translation) was established based upon a new apparatus, the FLOTAC. This technique allows, after the centrifugation, the real count of the parasite elements in 1 gram of feaces.     

Ancylostoma caninum: calibration and comparison of diagnostic accuracy of flotation in tube, McMaster and FLOTAC in faecal samples of dogs

We performed a calibration of flotation in tube, McMaster and FLOTAC to determine the optimal flotation solution (FS) and the influence of faecal preservation for the diagnosis of Ancylostoma caninum in dogs, and compared the accuracy of the three copromicroscopic techniques. Among nine different FS, sodium chloride and sodium nitrate performed best for detection and quantification of A. caninum eggs. Faecal samples, either fresh or preserved in formalin 5%, resulted in higher A. caninum egg counts, compared to frozen samples or preserved in formalin 10% or sodium acetate–acetic acid–formalin. FLOTAC consistently resulted in higher A. caninum eggs per gram of faeces (EPG) and lower coefficient of variation (CV) than McMaster and flotation in tube. The best results in terms of mean faecal egg counts (highest value, i.e. 117.0 EPG) and CV (lowest value, i.e. 4.8%) were obtained with FLOTAC using sodium chloride and faecal samples preserved in formalin 5%. Our findings suggest that the FLOTAC technique should be considered for the diagnosis of A. caninum in dogs.     

A simple, robust and semi-automated parasite egg isolation protocol

Large-scale parasite quantification is required for improving our understanding of the epidemiology and genetics of host-parasite interactions. We describe a protocol that uses a low-density salt solution for flotation and centrifugation of nematode eggs. Subsequently, sucrose flotation and precipitation are used to obtain clear egg preparations. Most traditional quantification protocols such as the McMaster technique are unsuited for the standardized processing of large numbers of samples and the analysis of large amounts of feces per sample. Consequently, they are suited only for small-scale surveys. Our protocol, which can be used to analyze up to 6 g of feces, results in clear egg preparations that are concentrated in wells of a microtiter plate and that are suited for digital recording and automated counting. Starting from a fecal suspension in the first flotation solution to a digital recording requires approximately 40 min per 24 samples.     

FLOTAC, a novel apparatus for a multivalent faecal egg count technique

Faecal egg count (FEC) techniques are widely used for parasitological diagnosis in humans and animals. They are also used to measure the prevalence and intensity of infections for epidemiological surveys, to quantify the efficacy of chemotherapies, and to detect anthelmintic resistance. They assess the number of parasitic elements (eggs, larvae, oocysts) present in the faecal samples, expressed per gram of faeces, and are based on the microscopic examination of an aliquot of faecal suspension from a known volume of a faecal sample. In the present paper a novel multivalent FEC technique, the FLOTAC technique, is described. It is based on flotation in centrifuge and translation of the apical portion of the floating suspension, and gives eggs/larvae/oocyst counts in quantities of faeces up to 1 gram. A novel apparatus, the FLOTAC, has been developed and patented in order to carry out this technique. It is a cylindrical-shaped device, made of unbreakable plastic, and holds two sample flotation chambers, which are 5 ml each for a total volume of 10 ml, with two ruled grids (18 x 18 mm). Each grid contains 12 equidistant ruled lines that are transparent, and hence permit the counting of parasitic elements under them. The FLOTAC apparatus is also very useful in order to recover parasitic elements after flotation.     

Annual follow-up of the gastrointestinal parasitosis of white-tailed deer Odocoileus virginianus (Artiodactyla: Cervidae) in captivity in Yucatán, México]

Gastrointestinal parasites, and egg and oocyst output in the faeces of captive white-tailed deer (Odocoileus virginianus yucatanensis) were recorded in Yucatan, Mexico. Feces were obtained from from January through December 1995 (ten samples every two weeks per place). Samples were processed by flotation and the McMaster techniques. Faecal cultures for L3 larvae were made by the Corticelli-Lai technique. Oocysts in faeces were cultured in 2% potassium dicromate. Seven genera were determined (Haemonchus spp., Cooperia spp, Isospora spp., Eimeria spp., Trichuris spp., Strongyloides spp. and Moniezia spp.) which represent five orders. The most frequent genera were Haemonchus, Isospora and Eimeria. The genus Isospora is reported for the first time in deer of this region, although it was not possible to explain the source of this parasite. The frequency and level of faecal egg and oocyst outputs were variable during the year and increased during the rainy season. There was a positive correlation between relative humidity, environmental temperature and rainfall with the coccidia and strongylida orders. In the central zone of Yucatan the meteorological conditions during the rainy season are favourable for the development of gastrointestinal parasitism which enable an increased risk of infection for deer.     

Annual follow-up of the gastrointestinal parasitosis of the tepezcuintle,Agouti paca (Rodentia: Agoutidae) in captivity in the Mexican tropic

Previous reports showed that the tepezcuintle (Agouti paca) is commonly infested by gastrointestinal parasites (GIP), mainly Eucoccidiida and helminths. However, there is no available information on the frequency of those parasites and their faecal egg excretions at different moments during the year. These information would provide a valuable baseline for the establishment of control strategies against GIP in tepezcuintles under captivity. The objectives of the present study were to determine the prevalence of GIP orders and genera that infest tepezcuintles under captivity and, to describe the dynamics of faecal egg and oocyst excretion in a year. Ten tepezcuintles were sampled (faeces) twice every month for twelve months. The faecal samples were processed by the flotation and McMaster techniques. Two orders of parasites were determined: Strongylida and Eucoccidiida. Two genera of nematodes were also determined: Strongyloides and Trichuris. The prevalence of Strongylida eggs, Eucoccidiida oocysts and Trichuris sp. eggs reached 10-20% of animals in certain months. The most important genus was Strongyloides, found in 60 to 100% of the animals year round. The average excretion of eggs in the group was 45 to 372 eggs per gram. Tepezcuintles kept under captivity in Yucatan are parasited with Strongyloides sp throughout the year, but only occasionally had oocysts of Eucoccidiida and eggs of Strongylida and Trichuris sp.     

Prevalence of Libyostrongylus douglassii in commercially reared ostriches in the highveld region of Zimbabwe

A total of 435 freshly dropped faecal samples were collected from 11 randomly selected ostrich farms during September and November 2002 to determine the prevalence of Libyostrongylus douglassii (ostrich wireworm) in the highveld region of Zimbabwe. Samples, which consisted of 339 samples from breeder birds and 96 samples from pre-slaughter grower birds were screened for nematode eggs using the modified McMaster technique before being individually cultured in an incubator at 28 degrees C. Cultures were examined for the presence of L. douglassii third stage larvae (L3). Using faecal egg counts, eight of 11 farms (72.7%) were positive for L. douglassii in breeders but no eggs were detected in the growers. The faecal culture method detected wireworm larvae in the breeding stock of all farms that were surveyed (100%) and five of the eight farms (62.5%) which had grower birds. Libyostrongylus douglassii was detected in all farms (100%) based on the faecal culture method. Libyostrongylus douglassii was detected for the first time in 7 of 11 farms (64%) surveyed. Data from questionnaires designed to assess farm management practices showed that four out of seven (57.1%) of the ostrich producers were unaware of the importance of wireworms in ostriches. The farms did not have a regular deworming programme for their birds and no faecal samples were sent routinely to the veterinary laboratory for screening of wireworms. Wireworm infections were not taken into consideration by farmers during buying and selling of birds.     

Evaluating faecal egg count reduction using a specifically designed package “eggCounts” in R and a user friendly web interface

The seemingly straightforward task of analysing faecal egg counts resulting from laboratory procedures such as the McMaster technique has, in reality, a number of complexities. These include Poisson errors in the counting technique which result from eggs being randomly distributed in well mixed faecal samples. In addition, counts between animals in a single experimental or observational group are nearly always over-dispersed. We describe the R package “eggCounts” that we have developed that incorporates both sampling error and over-dispersion between animals to calculate the true egg counts in samples of faeces, the probability distribution of the true counts and summary statistics such as the 95% uncertainty intervals. Based on a hierarchical Bayesian framework, the software will also rigorously estimate the percentage reduction of faecal egg counts and the 95% uncertainty intervals of data generated by a faecal egg count reduction test. We have also developed a user friendly web interface that can be used by those with limited knowledge of the R statistical computing environment. We illustrate the package with three simulated data sets of faecal egg count reduction experiments.     

Evaluation of a double centrifugation technique for the detection of Anoplocephala eggs in horse faeces

Faecal samples of 250 horses from farms with a known history of tapeworm infection were examined comparatively for cestode eggs using a double centrifugation/combined sedimentation-floatation technique. From each faecal sample, three 5?g and three 15?g subsamples were processed, each using either saturated NaCl solution, specific gravity (sp. g.) 1.2 [NaCl]; concentrated sugar solution, sp. g. 1.26 [sugar]; or concentrated ZnSO4 solution, sp. g. 1.3 [ZnSO4] for floatation. In total, faeces from 187 horses (?=?74.8%) tested 'positive' for Anoplocephala eggs. Percentages of samples testing 'positive' for Anoplocephala ova were: 57.2% for 5?g faeces/NaCl, 66% for 15?g faeces/NaCl, 66% for 5?g faeces/sugar, 72.8% for 15?g faeces/sugar, 55.6% for 5?g faeces/ZnSO4, and 61.2% for 15?g faeces/ZnSO4, respectively. Processing of 15?g faecal samples resulted in a significant (P?相似文献   

Field Validity and Feasibility of Four Techniques for the Detection of Trichuris in Simians: A Model for Monitoring Drug Efficacy in Public Health?

Background

Soil-transmitted helminths, such as Trichuris trichiura, are of major concern in public health. Current efforts to control these helminth infections involve periodic mass treatment in endemic areas. Since these large-scale interventions are likely to intensify, monitoring the drug efficacy will become indispensible. However, studies comparing detection techniques based on sensitivity, fecal egg counts (FEC), feasibility for mass diagnosis and drug efficacy estimates are scarce.

Methodology/Principal Findings

In the present study, the ether-based concentration, the Parasep Solvent Free (SF), the McMaster and the FLOTAC techniques were compared based on both validity and feasibility for the detection of Trichuris eggs in 100 fecal samples of nonhuman primates. In addition, the drug efficacy estimates of quantitative techniques was examined using a statistical simulation. Trichuris eggs were found in 47% of the samples. FLOTAC was the most sensitive technique (100%), followed by the Parasep SF (83.0% [95% confidence interval (CI): 82.4–83.6%]) and the ether-based concentration technique (76.6% [95% CI: 75.8–77.3%]). McMaster was the least sensitive (61.7% [95% CI: 60.7–62.6%]) and failed to detect low FEC. The quantitative comparison revealed a positive correlation between the four techniques (Rs = 0.85–0.93; p<0.0001). However, the ether-based concentration technique and the Parasep SF detected significantly fewer eggs than both the McMaster and the FLOTAC (p<0.0083). Overall, the McMaster was the most feasible technique (3.9 min/sample for preparing, reading and cleaning of the apparatus), followed by the ether-based concentration technique (7.7 min/sample) and the FLOTAC (9.8 min/sample). Parasep SF was the least feasible (17.7 min/sample). The simulation revealed that the sensitivity is less important for monitoring drug efficacy and that both FLOTAC and McMaster were reliable estimators.

Conclusions/Significance

The results of this study demonstrated that McMaster is a promising technique when making use of FEC to monitor drug efficacy in Trichuris.     

A low‐cost,automated parasite diagnostic system via a portable,robotic microscope and deep learning

Manual hand counting of parasites in fecal samples requires costly components and substantial expertise, limiting its use in resource‐constrained settings and encouraging overuse of prophylactic medication. To address this issue, a cost‐effective, automated parasite diagnostic system that does not require special sample preparation or a trained user was developed. It is composed of an inexpensive (~US$350), portable, robotic microscope that can scan over the size of an entire McMaster chamber (100 mm²) and capture high‐resolution (~1 μm lateral resolution) bright field images without need for user intervention. Fecal samples prepared using the McMaster flotation method were imaged, with the imaging region comprising the entire McMaster chamber. These images are then automatically segmented and analyzed using a trained convolution neural network (CNN) to robustly separate eggs from background debris. Simple postprocessing of the CNN output yields both egg species and egg counts. The system was validated by comparing accuracy with hand‐counts by a trained operator, with excellent performance. As a further demonstration of utility, the system was used to conveniently quantify drug response over time in a single animal, showing residual disease due to Anthelmintic resistance after 2 weeks.     

Methods in coccidiosis research: separation of oocysts from faeces.

Factors which may be important in the large-scale extraction of coccidial oocysts from faeces ha.ve been investigated with Eimeria tenella. Age of bird, inoculum, feeding status at the time of inoculation, period of collection, feeding status during collection, collection medium, homogenization and sieving, flotation, washing, sporulation and further purification have all been considered. The aim has been to establish a method to produce the maximum number of oocysts of a required degree of purity and viability, with the expenditure of the minimum amount of physical effort, time, animals and chemicals. In our method, groups of chickens 3-4 weeks of age are inoculated with 5000 oocysts of E. tenella and food is supplied ad lib. Over the period 5-8 days after inoculation, faeces are collected in trays containing 2% (w/v) potassium dichromate solution, while food intake is restricted. The faecal material is homogenized, passed once through 40 and 100 mesh sieves, centrifuged and the oocysts recovered from the sediment by 3 flotations in saturated salt solution. Following washing, oocysts are sporulated by forced aeration at 30 degrees C and may be further purified by hypochlorite treatment, or passage in 5% Tween 80 solution through a glass bead column followed by sucrose density gradient centrifugation. Routine passages along these lines over a 5 year period have given a recovery of 46% of the oocysts excreted by over 7000 birds.     

A comparison of sampling methods for bibionid larvae (Diptera: Bibionidae) in grassland

Three extraction methods were compared for the recovery of bibionid larvae from grassland soil samples. Only wet-sieving followed by flotation in a saturated salt solution yielded bibionids. No larvae were recovered either with modified Tullgren funnels or by slow immersion of soil cores into a saturated salt solution. The efficiency with which larval bibionid populations can be estimated is poor. Generally, smaller cores yielded more larvae per volume of soil. Most bibionids were found in the top 4 cm of soil. It is concluded that 10 cm diameter and 6–8 cm deep soil cores are an acceptable compromise between efficiency and sampling effort but sample size will largely be determined by the resources available for processing of samples.     

First report of Eimeria myoxi in the garden dormouse (Eliomys quercinus Linnaeus, 1766) from Doñana Natural Area (Andalusia,SW Spain)

This work reports for the first time the presence and molecular characterization of Eimeria myoxi in the garden dormouse (Eliomys quercinus) from the Doñana Natural Area (Andalusia, SW Spain). Fresh faecal samples were collected from a total of 28 garden dormice, which were caught following current guidelines for the ethical use of animals in research, and processing by a standard flotation technique with saturated saline solution. Then, wet drops were examined microscopically, and the number of oocysts was semi-quantified. Eimeria oocysts were observed in 16 of the 28 (57.1%) faecal samples, showing most of them a very low number of oocysts (≤1 oocyst per microscopic field × 400). The unsporulated oocysts visualized in 16 faecal samples were subspherical and of length 19.2 ± 1.2 μm and width 17.4 ± 1.1 μm, being morphologically compatible with E. myoxi. This finding was supported by molecular analysis of the small subunit ribosomal RNA (SSU-rRNA) gene, identifying the same species in 22 of the 28 (78.6%) dormice, including 15 samples in which oocyst size was compatible with E. myoxi. Moreover, the subsequent analyses of the apicoplast open reading frame 470 (ORF470) and the mitochondrial cytochrome c oxidase subunit I (COI) genes confirmed the molecular identification of the isolates as E. myoxi. The phylogeny analyses were consistent with previous phylogenetic studies and support the existence of three lineages of rodent-infecting Eimeria species.     

Flotation as a bioseparation process for fungi removal

Summary Collectorless flotation has been investigated at a laboratory scale, as a separation technique applied effectively to biomass fine particles (dead free) of Penicillium and Rhizopus; these were previously used for cadmium (a priority toxic pollutant) biosorption from dilute aqueous solutions. The basic parameters of dispersea-air flotation were studied, i.e. solution pH, flotation time, ethanol addition (used as a convenient frother), Cd and biomass concentration.     

A short-run new analytical ultracentrifugal micromethod for determining low-density lipoprotein sub-fractions using Schlieren refractometry

We have developed a new analytical ultracentrifugal micromethod for the determination of serum low-density lipoprotein (LDL) subclasses directly from ultracentrifugal Schlieren scans. We have used special software for the analysis of this type of single-spin density-gradient ultracentrifugation. The flotation of LDL patterns was obtained by underlayering a physiological salt solution with serum or isolated lipoprotein fractions raised to a density of 1.3 g/mL in the spinning ultracentrifugation capillary band-forming cell. The repeated analysis of Schlieren curves of the same sample from 10 to 100 microL in the 60-100 min full-speed interval time resulted in quite reproducible results. We obtained quantitative results by measuring the Schlieren areas between the sample curves and the reference baseline curve by using computerised numerical and graphic techniques. The decomposition of the integrated curve was carried out using a nonlinear regression program followed by deconvolution algorithm analysis in order to determine the parameters of the composing Gaussian subclasses. The LDL particle concentrations were calculated from the area under the integral of the Gaussian curve using a calibration data constant. The flotation range of the LDL Schlieren curves in the cell was identified with serum from which LDL had been removed by means of precipitation reagents and with centrifugation of isolated LDL aliquots. With this technique, we measured the concentration of LDL and analysed its polydispersity without the need for preceding sequential isolation of the LDL. On the basis of the Schlieren curves, the LDL samples were either physically paucidisperse, having a symmetrical peak within a narrow density range, or were polydisperse, showing an asymmetrical pattern distributed over a broader density region. The described method proved to be useful for a clear and immediate visual presentation of the concentration values of the LDL and for the identification of the heterogeneity of LDL variants without the need for the preparative isolation of that density class.     

盐爪爪根部微生物分布特征及盐浓度对碳源代谢分析的影响

【背景】耐盐植物盐爪爪广泛分布于盐碱荒漠区域,其根际存在明显的盐离子富集,形成"盐岛效应",研究此过程中微生物如何演替和适应,对揭示盐爪爪根际微生物的分布及其耐盐特性、"盐岛"的形成具有重要科学意义。【目的】揭示盐爪爪根际、根区及其周围环境土壤中微生物分布特征,确定不同盐浓度处理对根际微生物Biolog Eco微平板碳源代谢多样性的影响。【方法】采用Biolog Eco微平板技术,对采自新疆和硕地区的盐爪爪根际、根区和环境土壤微生物碳源利用情况进行分析,并采用不同浓度NaCl溶液对盐爪爪根际土壤样品进行稀释加样,评估盐浓度对根际微生物活性及多样性分析的影响。【结果】样品微生物代谢活性总体呈现根际根区环境的趋势;随着与根部距离的增加,样品中利用氨基酸、胺类碳源微生物所占比例明显降低,而利用吐温和肝糖等多聚化合物类碳源微生物所占比例明显增加;多样性分析表明,随着与根部距离的增加,Shannon指数(H)随之降低且存在明显差异。不同浓度NaCl溶液对采用BiologEco微平板技术分析盐爪爪根际微生物的代谢活性和代谢多样性有着明显的影响,其中采用5%NaCl溶液作为稀释加样液的根际微生物活性最高。【结论】盐爪爪根部微生物组成和分布有着明显的演变规律,且采用Biolog Eco微平板技术分析微生物代谢多样性时需进行培养条件的优化。     

Polyethyleneimine-induced flocculation and flotation of cyanobacterium Anabaena flos-aquae for gas vesicle production

Gas vesicles are hollow, proteinaceous structures found in some strains of cyanobacteria. They have been used to increase the oxygen supply and improve the cultivation of shear-sensitive mammalian cells. However, the production and, especially, collection of cells and gas vesicles were laborious and ineffective. In this study we examined the use of the cationic polymer, polyethyleneimine (PEI), for improving the cell harvesting by flocculation and flotation. PEI was examined to determine the appropriate molecular weight, pH range, and dosage. The dose of 20–30 mg/l of PEI with molecular weight of 25,000 in the pH range of 6.0–8.5 was found to provide effective and efficient cell flocculation and flotation. As the PEI dose increased, the rate of flotation increased but the clearance (collection) efficacy declined slightly. The culture samples used in this study were taken from light-limiting continuous culture systems at different dilution rates (0.05–0.24 h⁻¹). Without PEI addition, the cells at dilution rates lower than 0.1 h did not float while those at higher dilution rates would float slowly. With PEI addition, the flocculated cells at the dilution rate of 0.05 h⁻¹ sank and those of higher dilution rates would float and the flotation rate increased with increasing specific growth rate. Nonetheless, PEI flocculation and flotation (or sedimentation) could be used to harvest cells at a wide range of growth states.     

Taenia solium taeniasis/cysticercosis in the Menoua division (West Cameroon)

The present study was carried out between August 1999 and April 2000 with the objective of determining the prevalence of Taenia solium taeniasis in two village communities of Bafou and Bamendou in the Menoua division (West Cameroon). Four (0.13%) out of 3,109 faecal samples were positive for Taenia spp. eggs using the flotation technique. Three of the four worms expelled were T. solium whereas the other one was T. saginata. Two cases of cysticercosis were diagnosed in one of the families with a T. solium carrier. Furthermore, coprological and serological investigations for T. solium taeniasis and cysticercosis were carried out among butchers and/or tongue inspectors (n = 137) of the city of Dschang. The results were compared with those of a control group (n = 198). Taenia spp. eggs were not detected by microscopic examination. The prevalence of cysticercosis in the two groups was relatively similar (3.6 and 4.5% respectively).     

Fecundity and egg output by Toxocara canis in the red fox, Vulpes vulpes

The role of the red fox Vulpes vulpes in the dissemination of eggs of Toxocara canis into the environment is considered with reference to female worm fecundity and egg output in the faeces of infected foxes collected from four localities in southern England. A significant positive correlation was found between female worm size and the number of eggs in the uterus but there was no significant relationship between T. canis worm numbers and egg output in fox faeces. Reliable estimates of worm burdens in foxes could not, therefore, be determined from faecal egg counts alone. The highest mean egg output of 2145.0 epg recorded from adult foxes indicated that fox cubs are not necessarily the main sources of environmental contamination with T. canis eggs. Saturated magnesium sulphate was found to be a more effective flotation solution than zinc sulphate and sodium chloride for recovering eggs from fox faecal samples.