Molecular phylogeny of the pinworms of mice, rats and rabbits, and its use to develop molecular beacon assays for the detection of pinworms in mice

Though pinworm infestation has been prevalent since the early years of laboratory animal medicine, the genomes of these parasites have not yet been sequenced. The authors used high-fidelity polymerase chain reaction to amplify a large portion of the ribosomal gene complex of four pinworm species commonly found in lab rodents and rabbits (Aspiculuris tetraptera, Passalurus ambiguus, Syphacia muris and Syphacia obvelata). They determined DNA sequences for these complexes and carried out phylogenetic analysis. Using this information, the authors developed real-time molecular beacon assays for pinworm detection, comparing the new diagnostic approach with traditional methods such as perianal tape testing, fecal flotation and direct examination of intestinal content.     

Sensitivity of double centrifugation sugar fecal flotation for detecting intestinal helminths in coyotes (Canis latrans)

Fecal analysis is commonly used to estimate prevalence and intensity of intestinal helminths in wild carnivores, but few studies have assessed the reliability of fecal flotation compared to analysis of intestinal tracts. We investigated sensitivity of the double centrifugation sugar fecal flotation and kappa agreement between fecal flotation and postmortem examination of intestines for helminths of coyotes (Canis latrans). We analyzed 57 coyote carcasses that were collected between October 2010 and March 2011 in the metropolitan area of Calgary and Edmonton, Alberta, Canada. Before analyses, intestines and feces were frozen at -80 C for 72 hr to inactivate Echinococcus eggs, protecting operators from potential exposure. Five species of helminths were found by postmortem examination, including Toxascaris leonina, Uncinaria stenocephala, Ancylostoma caninum, Taenia sp., and Echinococcus multilocularis. Sensitivity of fecal flotation was high (0.84) for detection of T. leonina but low for Taenia sp. (0.27), E. multilocularis (0.46), and U. stenocephala (0.00). Good kappa agreement between techniques was observed only for T. leonina (0.64), for which we detected also a significant correlation between adult female parasite intensity and fecal egg counts (R(s)=0.53, P=0.01). Differences in sensitivity may be related to parasite characteristics that affect recovery of eggs on flotation. Fecal parasitologic analyses are highly applicable to study the disease ecology of urban carnivores, and they often provide important information on environmental contamination and potential of zoonotic risks. However, fecal-based parasitologic surveys should first assess the sensitivity of the techniques to understand their biases and limitations.     

Rapid seven-hour fecal coliform test.

A rapid 7-h fecal coliform (FC) test for the detection of FC in water has been developed. This membrane filter test utilizes a lightly buffered lactose-based medium (m-7-h FC medium) combined with a sensitive pH indicator system. FC colonies appeared yellow against a light purple background after incubation at 41.5 degrees C for 7 to 7.25 h. Comparison of FC test results showed that the mean verified FC count ratio (7-h FC count/24-h FC count) for surface water samples was 1.08. The mean FC count ratio (7-h FC count/24-h FC count) for unchlorinater wastewater ranged from 1.95 to 5.05. Verification of yellow FC colonies from m-7-h FC medium averaged 97%. Data from field tests on Lake Michigan bathing beach water samples showed that unverified 7-h FC counts averaged 96% of the 24-h FC counts. The 7-h FC test was found to be suitable for the examination of surface waters and unchlorinated sewage and could serve as an emergency test for detection of sewage or fecal contamination of potable water.     

Rapid seven-hour fecal coliform test.

A rapid 7-h fecal coliform (FC) test for the detection of FC in water has been developed. This membrane filter test utilizes a lightly buffered lactose-based medium (m-7-h FC medium) combined with a sensitive pH indicator system. FC colonies appeared yellow against a light purple background after incubation at 41.5 degrees C for 7 to 7.25 h. Comparison of FC test results showed that the mean verified FC count ratio (7-h FC count/24-h FC count) for surface water samples was 1.08. The mean FC count ratio (7-h FC count/24-h FC count) for unchlorinater wastewater ranged from 1.95 to 5.05. Verification of yellow FC colonies from m-7-h FC medium averaged 97%. Data from field tests on Lake Michigan bathing beach water samples showed that unverified 7-h FC counts averaged 96% of the 24-h FC counts. The 7-h FC test was found to be suitable for the examination of surface waters and unchlorinated sewage and could serve as an emergency test for detection of sewage or fecal contamination of potable water.     

Improved Membrane Filter Method for Fecal Coliform Analysis

A two-layer agar method has been developed which consistently yields higher recovery of fecal coliforms on membrane filters when compared to the existing membrane fecal coliform procedure. This method has been evaluated by three laboratories using samples of raw and chlorinated waste water, and reservoir, river, and marine waters. Verification of 1,013 fecal coliform colonies isolated from 61 water samples averaged 92% on this proposed procedure. Comparison with the Standard Methods membrane fecal coliform procedure revealed the two-layer agar method had an overall increased sensitivity to fecal coliform detection in these waters. It is therefore proposed that this procedure be evaluated as an alternative to the Standard Methods fecal coliform membrane Filter test in the examination of chlorinated secondary effluents, marine waters, and any natural waters that may contain pollutants with heavy metal ions.     

Serological method of detecting the antigens of the causative agent of Sonne dysentery in lysates of the fecal inoculates from patients]

Serological method of detection of Sh. sonnei antigens in the lysates of the patients, fecal cultures is suggested and approved. In the majority of cases of the results of bacteriological and serological methods of study of the feces coincided. Data confirming the specificity of the antibody neutralization test (ANT) in Sonne dysentery are presented. In connection with detection of the screening action of the Vi-antigen of typhoid bacilli there were elaborated additional methods for verifying the specificity of the ANT results. It is recommended to keep agar plates after selection of suspicious colonies during the bacteriological test; the lysate of the microbial crop should be additionally subjected to the ANT, this considerably increasing the percentage of laboratory confirmations of dysentery caused by Sh. sonnei.     

First evidence of enterobiasis in ancient Egypt

The oldest and most common parasite for which we have direct evidence, in the New World, is Enterobius vernicularis. Numerous archaeological sites, especially in the arid American southwest, have yielded fecal samples positive for pinworm ova, some of these dating back 10,000 yr. Reports of pinworm from the Old World are scarce. This article reports the first evidence of pinworm infection from Roman-occupied (30 BC-AD 395) Egypt.     

Primary fecal culture used as template for PCR detection of diarrheagenic E. coli virulence factors

The PCR technique applied to primary fecal cultures (PFC-PCR) was compared to the usual method employing isolated colonies (IC-PCR) in order to assess its sensitivity in the detection of virulence markers of diarrheagenic Escherichia coli in fecal samples obtained from children with diarrhea. Among the 149 samples analysed, PFC-PCR detected 81(54.4%) samples presenting one or two virulence markers, while IC-PCR detected only 59 (39.6%) positive samples. The markers detected in order of frequency were: pAA, LT-I, eaeA, ST-I, daaE, and ipaH. The PFC-PCR method of detection of diarrheagenic E. coli virulence markers proved to be reliable and more sensitive (p<0.05) than the usual method employing isolated colonies. It has also the advantage of being faster and less expensive than the detection methods in current use.     

A case of human cryptosporidiosis in Czechoslovakia

A case of human cryptosporidiosis, the first one reported in Czechoslovakia, is described. The disease was diagnosed by the presence of Cryptosporidium oocysts in the feces. The methods used independently to identify oocysts were the fecal flotation technique employing a saturated solution of sucrose and the microscopic examination of stained fecal smears. The patient was a 4-year-old boy with watery diarrhea of 5 days' duration who was kept on a diet and treated with a suspension of Endiaron N Spofa. Excretion positivity for Cryptosporidium oocysts in the feces was detectable at 3 and 5 days after appearance of first clinical manifestations. Bacteriological examination was repeatedly negative. This finding leaves little doubt as to existence of human cryptosporidiosis in Czechoslovakia, but what remains obscure is its overall contribution to the etiology of diarrheal diseases encountered in the population.     

Efficacy of an enzyme-linked immunosorbent assay as a diagnostic tool for schistosomiasis mansoni in individuals with low worm burden

IgM-ELISA is an immunoenzymatic method useful for detection of IgM antibodies against a fraction of Schistosoma mansoni adult worm antigen (AWA) that is soluble in trichloroacetic acid (AWA-TCA). This method was applied to three groups of individuals with different clinical and epidemiological characteristics, and the results compared with those obtained by other diagnostic methods: immunofluorescence test for detection of IgM antibodies (IgM-IFT) or IgG antibodies (IgG-IFT), ELISA for detection of IgG antibodies (IgG-ELISA), and two parasitological methods, Kato-Katz and miracidium hatching. The IgM-ELISA presented a sensitivity of 98%, when the parasitologic fecal examination was defined as reference diagnostic method, and a specificity of 98 and 97.3%, respectively for the group of clinically healthy individuals and other helminth carriers. A comparative analysis between the results of IgM-ELISA and those obtained by other serologic tests showed a good degree of agreement, with Kappa indices ranging from 0.95 to 0.98. The diagnostic efficacy of 97.8%, as determined with schistosomiasis patients with low parasitic burden, suggests the excellent performance of the IgM-ELISA and its usefulness for the diagnosis of schistosomiasis when applied in low endemic areas.     

Evaluation of Pfizer selective enterococcus and KF media for recovery of fecal streptococci from water by membrane filtration.

Pfizer selective enterococcus (PSE) and KF agars were compared for their recovery of fecal streptococci from sewage effluent on membrane filters. The results showed that PSE agar is highly selective for the enterococci. The tan color resulting from esculin hydrolysis, which was not always visible on the surfaces of the colonies, is not considered a necessary differential characteristic on PSE agar since more than 90% of all colonies recovered on membrane filters were confirmed as fecal streptococci and 86% were confirmed as enterococci. The detection of esculin hydrolysis on membrane filters was not improved by using the new Millipore type HC filter. KF agar recovered significantly greater numbers of organisms but was not as selective, with 83% of the typical colonies being confirmed as fecal streptococci and 54% as enterococci. An attempt to improve the selectivity of KF agar while retaining its inclusiveness by incubation at 45 C was not successful.     

Variation in Eimeria oocyst count and species composition in weanling beef heifers

Rectal fecal samples were collected daily on 10 consecutive days in November 2004 from 11 weaned beef heifers to assess daily variation in fecal oocyst count and species composition. Subsequent samples were collected from the same animals on 15 April 2005 and 9 June 2005. Oocyst numbers were determined by the modified McMaster's test, and species were identified by examination of oocysts recovered with the Wisconsin sugar flotation technique. Soil samples were collected from the heifer pasture on 8 June 2005, and oocysts were quantified and identified to species. Mean fecal oocyst counts varied little at all sampling dates ranging from 134-377 oocysts/g. Ten Eimeria spp. were identified in fecal samples collected in November and April and 11 in June. Eimeria bovis was the most common species identified at all samplings. Mean species composition showed little variation during the 10-day sampling period in November, remained similar in April, and varied slightly in June. Twelve Eimeria spp. were identified in soil samples in proportions similar to those seen in fecal samples. The results indicate that clinically normal weanling beef heifers are likely to be infected with a diverse, but relatively stable, community of Eimeria spp.     

Quantitative and qualitative comparison of density-based purification methods for detection of Cryptosporidium oocysts in turbid environmental matrices

Purification methods for Cryptosporidium oocysts are usually selected on the basis of recovery yield, but the amount of particulate debris in environmental matrices could limit efficiency of oocyst detection by microscopic examination or PCR detection. Previous studies have shown that the standard immunomagnetic separation (IMS) procedure would not be the most suitable method for oocyst purification from turbid matrices. We compared the capacity of Percoll-sucrose flotation and six other density-based purification methods to achieve selective separation of Cryptosporidium oocysts from particulate debris. Rate of oocyst recovery and particulate loading in the purified suspensions were chosen as comparison criteria for the different purification methods. In most earlier studies, the chemical treatments employed to obtain a purified oocyst suspension modify the surface properties of oocysts in spiked samples. Assuming this produces unrealistic conditions affecting the evaluation of purification methods, we performed the present study with native oocysts. Flotation and gradient procedures were tested with and without formaldehyde ethyl acetate (FEA) separation. FEA separation was found to be unsuitable. Filtration and Percoll gradient did not allow selective oocyst separation from debris. Among the purification methods suitable for routine microscopic examination, Percoll-sucrose flotation provided the best recovery rates. For automated enumeration systems or PCR detection, potassium bromide and especially Nycodenz gradients appeared to be the most suitable purification methods. Potassium bromide and Nycodenz gradients provided the best balance between oocyst recovery and particulate load.     

Pixel by pixel: real-time observation and quantification of passive flotation speeds of three common equine endoparasite egg types

The efficacy of anthelmintic treatments against populations of endoparasites infecting livestock throughout the world is decreasing. To mitigate this, the use of fecal egg counts is recommended to determine both the necessity, and to ensure the appropriate choice, of anthelmintic treatment. Traditionally, and in order to facilitate easier identification and/or enumeration, samples are analysed after separating eggs from other fecal particulates by exposing them to a solution with a density higher than that of the eggs, but lower than the remaining fecal contents. While many parasite egg flotation protocols exist, little is known about the characteristics of these eggs with respect to their movement through a flotation solution. In this study, we have demonstrated a novel method for the observation and quantification of microscopic (65–100 µm) objects as they experience unassisted flotation. This also represents, to our knowledge for the first time, that the flotation of parasite eggs has been observed and their movement characteristics quantified as they float through solution. Particle tracking and video analysis software were utilised to automatically detect and track the movement of individual eggs as they floated. Three 30 s videos and one 2 min video of each egg type were analysed. If the first 30 s of video were discounted, the differences in mean flotation speed among all videos was statistically significant between egg types (P = 0.0004). Strongyle type eggs (n = 201) moved the fastest with a mean 51.08 µm/s (95% confidence interval: 47.54–54.62). This was followed by Parascaris spp. (n = 131) and Anoplocephala perfoliata eggs (n = 322), with mean speeds of 44.43 µm/s (95% confidence interval: 39.47–49.4) and 31.11 µm/s (95% confidence interval: 29.6–32.61), respectively. This method for evaluating the mean speed of passive flotation may represent a first step towards further optimizing fecal egg flotation and be of interest to parasitologists and veterinary practitioners.     

四川绵阳地区野生猕猴肠道寄生虫感染的调查研究

对四川绵阳地区野生猕猴抽样100只,分别采用饱和食盐水漂浮法和沉淀集印法检查粪便中寄生虫卵,监测肠道寄生虫的感染率和感染强度。结果显示,感染当地野生猕猴的肠道寄生虫主要有8种,总感染率为78．0％,分别为：吸虫50．0％,毛尾线虫48．0％,蛲虫41．0％,钩虫32．0％,蛔虫26．0％,泡状带绦虫16．0％,球虫4．0％,后圆线虫4．0％,感染强度为毛尾线虫“＋＋＋＋”,蛲虫“＋＋＋＋”,吸虫“＋＋＋”,钩虫“＋＋＋”,蛔虫“＋＋”,泡状带绦虫“＋”,球虫“＋”,后圆线虫“＋”。表明该地区野生猕猴寄生虫感染较普遍,感染强度较大。     

粪便DNA突变检测在快速筛选大肠癌中的应用

大肠癌是发病率和死亡率都很高的常见疾病, 对高危人群进行大规模的普查筛选, 可以降低大肠癌的发生率和死亡率。粪便DNA突变检测是新近发展的可用作大肠癌普查的技术, 与常规结肠镜检测和大便隐血实验相比, 具有特异性高、灵敏度高和易于被患者接受等优点。文章阐述了粪便DNA突变检测的相关基因、肿瘤特异性DNA提取方法和检测方法等, 并对其在快速筛选大肠癌中的应用进行了展望。大肠癌是发病率和死亡率都很高的常见疾病, 对高危人群进行大规模的普查筛选, 可以降低大肠癌的发生率和死亡率。粪便DNA突变检测是新近发展的可用作大肠癌普查的技术, 与常规结肠镜检测和大便隐血实验相比, 具有特异性高、灵敏度高和易于被患者接受等优点。文章阐述了粪便DNA突变检测的相关基因、肿瘤特异性DNA提取方法和检测方法等, 并对其在快速筛选大肠癌中的应用进行了展望。     

Sensitivity of Direct Plating for Detection of High Levels of <Emphasis Type="Italic">E. coli</Emphasis> O157:H7 in Bovine Fecal Samples

To assess the sensitivity of direct plating of bovine fecal samples for detection of Escherichia coli O157:H7, calves (n = 28) were orally inoculated with 10⁹ colony-forming units (cfu) per calf of a mixture of three strains of nalidixic acid-resistant E. coli O157:H7, and fecal samples were collected for analysis. One-gram samples from inoculated calves were mixed with 9 mL of Gram-negative broth with vancomycin, cefixime, and cefsoludin. From this suspension, serial dilutions were made (10⁻¹ to 10⁻⁴) and spread plated in triplicate on Sorbitol MacConkey agar with nalidixic acid for enumeration of E. coli O157:H7 in fecal samples. Direct plating samples were streaked for isolation on Sorbitol MacConkey agar with cefixime, and tellurite (SMACct). After incubation overnight at 37°C, morphologically typical colonies from direct streak plates were plated onto blood agar and incubated overnight at 37°C; then an indole test was performed on each colony. Indole-positive colonies were confirmed by O157 agglutination and were then plated on SMAC agar with 20 μg/mL nalidixic acid (SMACnal) to confirm nalidixic acid resistance. Overall sensitivity of detection was 32.5% (110/338 samples). Sensitivity to detect fecal samples shedding at above 5 × 10⁴ cfu/g was 83% (71/86 samples). Based on these data, direct plating of fecal samples might be an effective way to identify cattle that are likely to be shedding E. coli O157 at high levels.     

Transmission Patterns of Pinworms in Two Sympatric Congeneric Primate Species

Understanding pathogen transmission is essential to addressing the dynamics of infectious diseases in animal populations. Directly transmitted parasites spread in host populations via 1) contact with infected individuals and 2) contact with contaminated substrates. Although studies exist that support social or ranging effects on transmission, it is less clear how these factors interact. We test the hypothesis that a combination of social, ranging, diet, and intrinsic factors account for Trypanoxyuris minutus (pinworm) infections in sympatric howler species Alouatta palliata and A. pigra. We collected 211 howler fecal samples from 34 adults living in four groups, two of each species, in Tabasco (Mexico), and calculated pinworm prevalence and eggs per gram of feces (EPG). We followed each group for 80 h to determine ranging, diet, frequency of contact, and conspecific proximity. Prevalence of Trypanoxyuris minutus was high, with 82% of all individuals infected. Logistic modeling indicated that pinworm prevalence was positively associated with proximity and the proportion of group members contacted by focal individuals. Although EPG results should be interpreted cautiously owing to variable egg excretion, this index was also positively associated with proximity and the proportion of group members that were contacted, as well as with dietary diversity and use of non-tree foods. Neither intrinsic factors such as species and sex, nor group and population level variables, such as group and home range size, home range overlap, and intensity of range use, were significant predictors of pinworm infection. We conclude that both sociality and feeding behavior are key factors in infection dynamics of Trypanoxyuris minutus in sympatric Alouatta palliata and A. pigra, confirming that contact with infected conspecifics and contaminated substrates are important mechanisms for directly transmitted parasites.     

Identification of strains isolated as total and fecal coliforms and comparison of both groups as indicators of fecal pollution in tropical climates

This study was undertaken to better characterize the groups of total coliforms (TC) and fecal coliforms (FC) and to evaluate both groups as indicators of fecal contamination of drinking well water in a tropical climate (The Ivory Coast, West Africa). Isolated colonies obtained as TC or FC on membrane filters were identified using the API-20E system. From the well water samples, 58 golden-green colonies with a metallic sheen isolated on Endo medium (TC) were identified as Escherichia coli (55%), Enterobacter (26%), Klebsiella (14%), Proteus (3%), and Citrobacter (2%). Among 132 colonies isolated on Endo medium as non-TC (not showing the characteristic golden metallic sheen), 10% were identified as E. coli. The 196 blue colonies isolated on M-FC medium at 44.5 degrees C (FC) were identified as E. coli (66%), Klebsiella (12%), Enterobacter (10%), Citrobacter (5%), Salmonella (3%), Serratia (3%), Proteus (2%), and Yersinia (0.5%). Among 24 nonblue colonies on M-FC medium, none were identified as E. coli. Of the colonies isolated from human feces, E. coli represents 92% of the TC and 89% of the FC. Although these results are limited, they tend to confirm the greater specificity of the fecal coliform technique over that of total coliform for the detection of fecal contamination of untreated well water. From the results presented here and the observations of other workers, it is suggested that the use of FC instead of TC should be considered as the method of choice for determining drinking water pollution of untreated groundwater supplies.     

Modification of M-FC medium by eliminating rosolic acid.

Eliminating rosolic acid from M-FC medium improves the MFC procedure by allowing higher fecal coliform colony recoveries with greater ease in counting. Samples of unchlorinated and chlorinated domestic sewage, creek, lake, and river water were analyzed for fecal coliforms by standard procedures. Results of 200 comparisons of fecal coliform counts on M-FC medium without and with rosolic acid showed that higher counts were obtained 71% of the time when rosolic acid was excluded without an overgrowth of background colonies. Results from analyzing chlorinated sewage showed that eliminating rosolic acid improved the recovery of fecal coliform bacteria by 49%. A total of 1,675 blue colonies and 766 nonblue colonies were verified. Of the 1,675 blue colonies, 1,566 were confirmed as fecal coliform bacteria, for a verification of 93.5%. The percent verification of nonblue colonies as noncoliform bacteria was 84.2% (644/766).