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PubMed2004

Comparison of PCR prescreening to two cultivation procedures with PCR confirmation for detection of Mycobacterium avium subsp. paratuberculosis in U.S. Department of Agriculture fecal check test samples.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent in Johne's disease in cattle and causes diarrhea, decreased milk production, emaciation, and frequently death. The ability to detect MAP rapidly and accurately is an integral part of herd management. However, detection of this bacterium is complicated due to its slow division time and its ability to enter dormancy. Culture methods are considered the "gold standard," but they have their limitations. Many enzyme-linked immunosorbent assay methods and conventional PCR methods have been used as diagnostic tools. The present study compares the results of a PCR prescreen to two culture methods of detection paired with confirmatory PCR to determine the most accurate, rapid, and sensitive method using U.S. Department of Agriculture (USDA) fecal check samples. This study involving two laboratories (Marshfield Clinic Laboratories, using solid culture medium [Herrold's egg yolk agar], and TREK Diagnostic Systems Research and Development, using liquid culture medium [ESP Culture System II]) showed that the PCR prescreening method used in this study lacked specificity and sensitivity as a stand-alone test in fecal samples. However, the combination of liquid enrichment culture using the ESP II system, and PCR confirmation with the hspX primer set, was not only 100% sensitive and specific but also correlated with viable MAP and USDA culture results.

Animals↗
PubMed1997

Prepatent periods of different Oesophagostomum spp. isolates in experimentally infected pigs.

To define prepatent periods of different Oesophagostomum spp. isolates we carried out two separate experiments, one using two monospecific laboratory isolates and another using laboratory isolates as well as isolates obtained from pig herds having different management systems and with different anthelmintic treatment histories. Pigs were inoculated with 1,000-2,000 infective larvae. Fecal samples were collected daily beginning on days 15 and 16 postinoculation (p.i.). Fecal cultures were set up at different times to yield larvae that could be identified by DNA analyses. All pigs started to excrete eggs on days 18-24 p.i. The mean prepatent period was 20.2 +/- 1.4 days, with no significant difference being observed between species and isolates. Prepatent periods of 17-19 days were found for the monospecific laboratory isolates of O. dentatum and O. quadrispinulatum. These findings conflict with parasitology textbooks; therefore, suggestions as to the possible reasons for the observed short prepatent periods are given.

Animals↗
PubMed2005

Herd-level factors associated with isolation of Salmonella in a multi-state study of conventional and organic dairy farms I. Salmonella shedding in cows.

The objective of this study was to evaluate associations between herd characteristics and the isolation of Salmonella from dairy cows in Minnesota, Wisconsin, Michigan, and New York. Study farms were 129 conventional and organic farms enrolled without regard to previous history of Salmonella infection. Herds were sampled at 2-month intervals over a 1-year period. This is the largest study to date on Salmonella shedding in dairy cows and the only study evaluating herd-level risk factors using longitudinal sampling to characterize Salmonella shedding on dairy farms. Salmonella was isolated in fecal samples from 1026 (4.9%) of 20,089 cows. Over the course of the study, 113 (87.6%) of 129 farms had at least one positive cow sample. Multi-variable logistic regression using the generalized estimating equations approach was used to test the association between herd-level risk factors and the dependent variable of within-herd prevalence by visit (number of Salmonella-positive cows/number of cows sampled) after adjustment for effects of herd size, season, state of origin, and the multiple sampling occasions per herd. Factors retained in the final model included lack of use of tiestall or stanchion facilities to house lactating cows (OR=1.9; 95% CI: 1.1-3.3), not storing all purchased concentrate or protein feeds in an enclosed building (OR=2.5; 95% CI: 1.3-4.9), not using monensin in weaned calf or bred heifer diets (OR=3.2; 95% CI: 2.0-5.4), access of lactating or dry cows to surface water (e.g., lake, pond, river, or stream) (OR=2.3; 95% CI: 1.3-3.9), disposal of manure in liquid form (slurry or irrigation, as opposed to disposal of manure by broadcast/solid spreader only) on owned or rented land (OR=1.8; 95% CI: 1.3-3.9), and cows eating or grazing of roughage from fields where manure was applied in solid or liquid form and not plowed under during the same growing season (OR=1.8; 95% CI: 1.0-3.0). A seasonal association was also present as cows were more likely to be Salmonella-positive in summer, spring, and fall compared to winter. Herd size was not associated with Salmonella shedding in the final multi-variable model. The herd-level risk factors identified in this study could potentially be implemented in Salmonella control programs on dairy farms.

Animal Feed↗
PubMed2004

Suppression of Salmonella enterica serovar Enteritidis excretion by intraocular vaccination with fimbriae proteins incorporated in liposomes.

Liposome-associated fimbriae antigens (SEF14 and SEF21) were prepared for intraocular immunization to seek protective efficacy for intestinal infection with Salmonella enterica serovar Enteritidis. Chickens were immunized intraocularly with the antigens at 8 and 10 weeks of age. Evidence of an IgA and IgG responses were found in the intestinal tract and in sera of these chickens. Antibody-secreting lymphocytes were detected in the Harderian gland of immunized chickens as determined by enzyme-linked immunospot assay. Two weeks after the booster immunization, the chickens were challenged orally with 1x10(7) live Salmonella Enteritidis, and fecal samples were examined for bacterial excretion from the intestinal tract. Significantly less fecal excretion of bacteria was observed in immunized chickens for 15 days after challenge. The numbers of bacteria in the intestinal contents (caecum and rectum) were also significantly lower in immunized chickens than in unimmunized controls. Detection of S. Enteritidis-specific DNA by the polymerase chain reaction was consistent with the bacterial observations. Intraocular immunization with liposome-associated SEF14 and SEF21 therefore elicits both systemic and mucosal antibody responses, so that bacterial colonization in the intestinal tract and excretion of S. Enteritidis in the feces are suppressed by immunization.

Animals↗
PubMed1999

[Usefulness of a gene amplification technique (LCx MTB) in the diagnosis of tuberculosis: preliminary results with different sputum samples].

OBJECTIVE: To evaluate the use in non-sputum samples of a commercial molecular amplification kit (LCx MTB, Abbott Diagnostica) (LCx) for the diagnosis of tuberculosis. MATERIAL AND METHOD: Ninety-nine non-sputum samples from the same number of patients (bronchoalveolar, pleural and ascitic fluid, fecal samples, blood cultures, biopsies from different sites, cerebrospinal fluid, urine and gastric juices) and 14 sputum samples (10 from patients clinically suspected of having tuberculosis and 4 from patients diagnosed of tuberculosis and undergoing appropriate treatment for at least one month). All samples were LCx processed according to the manufacturer's instructions. The reference diagnosis was obtained by the Löwestein-jensen method and when results were inconsistent, we took into account the degree of clinical suspicion, response to treatment and histology. RESULTS: Seven of the 99 samples were positive by the LCx technique, and 6 of the 7 were also LJ positive; 1 could not be evaluated because of culture contamination. One LJ positive culture was LCx negative. Only one sample was positive by Ziehl-Neelsen (ZN) staining. Ninety-two samples were LCx negative, with 91 showing no growth at all. Sensitivity was 86% and specificity 98%. Atypical mycobacteria were detected in 4 cases, all of which were LCx negative. CONCLUSIONS: Diagnosis of tuberculosis by applying the LCx system to various types of samples other than sputum is simple, rapid, sensitive and specific.

Humans↗
PubMed1997

Fecal hydrogen production and consumption measurements. Response to daily lactose ingestion by lactose maldigesters.

The alteration of hydrogen (H2) metabolism, which accounts for the large decrease in breath H2 excretion following prolonged ingestion of malabsorbed carbohydrate (lactulose, lactose in lactose maldigesters) was studied in six lactose-maldigesting adults. Metabolic inhibitors of the three main H2-consuming reactions (methanogenesis, sulfate reduction, and acetogenesis) were used to independently measure H2 production and consumption in fecal samples obtained after 10 days of either dextrose or lactose feeding. Absolute fecal H2 production (net of production minus consumption) after 3 hr of incubation with lactose was approximately threefold lower after lactose adaptation (242 +/- 54 microliters) compared to dextrose adaptation (680 +/- 79 microliters, P = 0.006). Fecal H2 consumption was not affected by either feeding period. We conclude that decreased absolute H2 production, rather than increased H2 consumption, is responsible for the decrease in breath H2 observed with lactose feeding.

Adult↗
PubMed1986

Using density rather than mass to express the concentration of gastrointestinal tract constituents.

This experiment had two objectives: to develop an analytical technique to measure the density of digesta and fecal samples; to determine whether this measurement is physiologically relevant. To address the latter question, rats were provided with one of five high-fiber diets or a fiber-free control diet, and density determinations were made on samples from the cecum, proximal and distal colon, and from a passed fecal pellet. The density of colonic contents varied with the fiber component of the diet. The fiber-free control diet produced the densest stool at all sites studied (P less than 0.01), whereas wheat bran produced the least dense stool at every site (P less than 0.01). There were also density differences among bowel positions within diet treatments: the stool from those animals consuming the fiber-free, guar and pectin diets became denser as it moved distally; wheat bran, on the other hand, produced the opposite effect--the more distal the stool traveled, the less dense it became. Since the density of colonic contents varied with respect to both diet and anatomical site, these data suggest that the traditional technique of expressing the concentration of a constituent of interest (e.g., bile acid, volatile fatty acid) per gram of digesta or fecal matter may not always be appropriate. This is particularly true if exposure of the intestinal mucosal cells to a substance in the digesta or fecal stream is of interest, since density, rather than mass, reflects the diluting potential of the diet.

Animals↗
PubMed2006

Fecal S100A12: a novel noninvasive marker in children with Crohn's disease.

BACKGROUND: The calcium-binding protein S100A12 is related to calprotectin, a protein shown to be a useful marker of gut inflammation. S100A12 levels are elevated in serum and mucosa of children with inflammatory bowel disease (IBD) and may be implicated in the pathogenesis of IBD. The aims of this study were to validate an immunoassay for the detection of fecal S100A12, to assess its value as a new noninvasive marker of gut inflammation, and to investigate S100A12 levels in feces of children with IBD at diagnosis and during treatment. MATERIALS AND METHODS: Feces were collected from children with active IBD at diagnosis and during treatment for IBD and from normal healthy control subjects. Fecal and serum levels of S100A12 were measured by immunoassay. RESULTS: A sensitivity of 96% and a specificity of 92% were observed when 10 mg/kg fecal S100A12 was used as a cutoff. S100A12 levels were evenly distributed throughout fecal samples and were stable for 7 days when stored at room temperature. Fecal S100A12 was elevated in children with IBD compared with healthy control subjects, with levels closely correlated to disease activity and other serum inflammatory markers, particularly lower gut involvement. Fecal S100A12 levels fell during therapy in children entering remission with normal C-reactive protein levels. CONCLUSIONS: Fecal S100A12 is a novel noninvasive marker that distinguishes children with active IBD from healthy control subjects with high sensitivity and specificity. Fecal S100A12 possesses characteristics that are desirable for a noninvasive disease marker and therefore is a suitable candidate marker for IBD. Further evaluation is required to examine this marker in additional contexts.

Adolescent↗
PubMed1988

Coccidian parasites (Apicomplexa: Eimeriidae) from insectivores. IV. Four new species in Talpa europaea from England.

Moles from England were examined for coccidian oocysts and all 64 Talpa europaea were infected; of 64 infected hosts, 56 (88%) had multiple infections representing two to six coccidian species when examined. Oocysts in 31 of the 64 samples remainedunsporulated. Three eimerians and one isosporan were studied from the 33 fecal samples that had sporulated oocysts and these are described as new species; Cyclospora talpae Pellérdy & Tanyi, 1968, and Isospora sofiae (Golemansky, 1978) Levine & Ivens, 1979, are redescribed; and Cyclospora sp., similar to C. talpae, is discussed. Sporulated oocysts of C. talpae are ellipsoidal, 14.3 X 9.6 (12-19 X 6-13) microns with sporocysts ovoid, 9.4 X 5.7 (6-13 X 4-8) microns; it was found in 21 of the 33 (63.6%) sporulated samples. Sporulated oocysts of Cyclospora sp. are subspheroidal to ellipsoidal, 12.5 X 8.9 (10-14 X 6-12) microns with sporocysts ovoid, 8.6 X 5.3 (6-10 X 4-6) microns; it was found in 21 of the 33 (63.6%) sporulated samples. Sporulated oocysts of Eimeria avonensis n. sp. are elongate-ellipsoidal, 15.0 X 9.6 (13-20 X 7-12) microns with sporocysts ovoid, 6.6 X 3.6 (5-9 X 3-7) microns; it was found in 15 of the 33 (45.5%) sporulated samples. Sporulated oocysts of Eimeria berea n. sp. are subspheroidal, 12.1 X 10.5 (10-15 X 8-14) microns with sporocysts ovoid, 6.3 X 3.9 (5-10 X 2-5) microns; it was found in 8 of the 33 (24.2%) sporulated samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Evaluation of a New Latex Agglutination Test for Fecal Lactoferrin in Travelers' Diarrhea.

The detection and diagnosis of travelers' diarrhea by fecal culture method, often insensitive, expensive, and time consuming, could well be replaced with the latex agglutination test to detect fecal lactoferrin. As an initial screening, this test could be useful to differentiate between those patients who are more likely to have an invasive enteropathogen and those patients requiring antidiarrheal chemotherapy only. Fecal samples from 92 patients with travelers' diarrhea were tested for occult blood, fecal leukocytes, and fecal lactoferrin at stool dilutions of 1:50 and 1:200. The results were compared with findings from fecal cultures for enteropathogens. Statistical analyses were performed measuring the performance of the latex agglutination test for fecal lactoferrin to calculate its sensitivity, specificity, and positive and negative predictive values. Invasive pathogens were identified in 36 (39%), and a noninvasive pathogen was found in 18, or 20%, of the cases. At the stool dilution of 1:50, fecal lactoferrin showed more sensitivity than did leukocytes or occult blood in detecting the presence of invasive enteropathogens and, with a negative predictive value of 94%, was superior in predicting their absence. At the 1:200 dilution, a lower sensitivity of 55%, but a higher specificity of 82%, compared to 55% with the 1:50 dilution, was demonstrated. The determination of fecal lactoferrin might prove to be more useful and less expensive and time consuming as an initial screening for patients presenting with travelers' diarrhea. Further evaluation and antibacterial treatment could, therefore, be reserved for those with a positive lactoferrin test.

Journal Article↗
PubMed2006

Effects of Lactococcus lactis on composition of intestinal microbiota: role of nisin.

This study examined the ability of (i) pure nisin, (ii) nisin-producing Lactococcus lactis strain CHCC5826, and (iii) the non-nisin-producing L. lactis strain CHCH2862 to affect the composition of the intestinal microbiota of human flora-associated rats. The presence of both the nisin-producing and the non-nisin-producing L. lactis strains significantly increased the number of Bifidobacterium cells in fecal samples during the first 8 days but decreased the number of enterococci/streptococci in duodenum, ileum, cecum, and colon samples as detected by selective cultivation. No significant changes in the rat fecal microbiota were observed after dosage with nisin. Pearson cluster analysis of denaturing gradient gel electrophoresis profiles of the 16S rRNA genes present in the fecal microbial population revealed that the microbiota of animals dosed with either of the two L. lactis strains were different from that of control animals dosed with saline. However, profiles of the microbiota from animals dosed with nisin did not differ from the controls. The concentrations of nisin estimated by competitive enzyme-linked immunosorbent assay (ELISA) were approximately 10-fold higher in the small intestine and 200-fold higher in feces than the corresponding concentrations estimated by a biological assay. This indicates that nisin was degraded or inactivated in the gastrointestinal tract, since fragments of this bacteriocin are detected by ELISA while an intact molecule is needed to retain biological activity.

Agar↗
PubMed1974

Isolation of Clostridium absonum and its cultural and biochemical properties.

A new procedure for isolation of Clostridium absonum was devised. Sixtyseven strains of C. absonum were isolated from 135 soil samples, but no strain of C. absonum could be found from human fecal samples. The lecithinase, hemolysin, and lethal toxin in the culture filtrates of this species exhibited low avidity for C. perfringens type A antitoxin. The three activities were inseparable by the present method of purification. A reinvestigation of biochemical properties revealed that incomplete suppression of lecithinase reaction by C. perfringens type A antitoxin and no fermentation of raffinose, melibiose, and starch are useful criteria to differentiate C. absonum from C. perfringens, and that positive, although weak, gelatin liquefaction and fermentation of trehalose are useful to differentiate it from C. paraperfringens.

Antitoxins↗
PubMed2002

Sensitivity of test strategies used in the Voluntary Johne's Disease Herd Status Program for detection of Mycobacterium paratuberculosis infection in dairy cattle herds.

OBJECTIVE: To evaluate sensitivities at the herd level of test strategies used in the Voluntary Johne's Disease Herd Status Program (VJDHSP) and alternative test strategies for detecting dairy cattle herds infected with Mycobacterium paratuberculosis. DESIGN: Nonrandom cross-sectional study. SAMPLE POPULATION: 64 dairy herds from Pennsylvania, Minnesota, Colorado, Ohio, and Wisconsin. Fifty-six herds had at least 1 cow shedding M. paratuberculosis in feces; the other 8 herds were free from paratuberculosis. PROCEDURE: For all adult cows in each herd, serum samples were tested for antibodies to M. paratuberculosis with an ELISA, and fecal samples were submitted for bacterial culture for M. paratuberculosis. Sensitivities at the herd level (probability of detecting infected herd) of various testing strategies were then evaluated. RESULTS: Sensitivity at the herd level of the testing strategy used in level 1 of the VJDHSP (use of the ELISA to test samples from 30 cows followed by confirmatory bacterial culture of feces from cows with positive ELISA result) ranged from 33 to 84% for infected herds, depending on percentage of cows in the herd with positive bacterial culture results. If follow-up bacterial culture was not used to confirm positive ELISA results, sensitivity ranged from 70 to 93%, but probability of identifying uninfected herds as infected was 89%. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the testing strategy used in the VJDHSP will fail to identify as infected most dairy herds with a low prevalence of paratuberculosis. A higher percentage of infected herds was detected if follow-up bacterial culture was not used, but this test strategy was associated with a high probability of misclassifying uninfected herds.

Animals↗
PubMed1975

Survival of Ancylostoma caninum on bare ground, pea gravel, and concrete.

Studies were done to determine the survival of infective Ancylostoma caninum 3rd-stage larvae on 3 ground covers commonly used in dog run construction: bare ground, pea gravel, and concrete. Changes in numbers of recovered larvae were compared to meterologic data and the most significant weather variables were determined. Larvae were recovered 1 to 7 days on bare ground. Larvae survived longer in the fecal mass (mean of 3 days) than on the bare ground (mean of 2 days). Rain was the most significant variable, in that it was positive in its effects (higher larval count) early in the experiment (causing fecal mass breakdown and release of larvae) and negative (lower larval count) later in the experiment (spreading larvae away from test site). Larvae were also recovered 1 to 7 days on pea gravel. They were recovered for a mean 2.6 days from the fecal sample, a mean of 1.5 days from the rocks directly below the fecal mass, and a mean of 1.3 days from the remaining rocks. Here also, rain was the most significant weather factor. It was negatively significant (lower larval count) for the fecal mass (spreading of the larvae) and positive for those in the pebbles (increasing the moisture in the pebbles). Survival time of larvae on concrete was shorter than that on the other 2 substrates: from 0 to 2 days. Larvae were recovered a mean of 1.3 days from the fecal mass and a mean of 0., days from the surrounding concrete. Rain was positively significant early in the experiments in that it released trapped larvae from the fecal mass. Sunlight consistently was negatively significant (lower larval count) due to its lethality to the unprotected larvae.

Ancylostoma↗
PubMedDate not supplied

[Emergent viruses: SARS-associate coronavirus and H5N1 influenza virus].

Two viral agents with RNA genome are responsible for emerging illnesses: influenza virus A/H5N1 and Severe Acute Respiratory Syndrome virus (SARS). For the diagnosis of SARS virus infection, an epidemiological investigation is necessary to know whether the patient has been exposed to a risk in a country where the SARS virus is circulating or whether the patient had worked in a laboratory handling SARS virus. The detection of SARS virus is possible in various clinical samples (including urine) by viral culture or RT-PCR. The handling of those samples and RNA extraction must be performed in a BSL3 laboratory. The SARS virus RT-PCR is poorly sensitive, therefore the test should be performed on samples collected consecutively for several days. In front of a suspicion of A/H5N1, similar procedures are recommended. An epidemiologic investigation is necessary to specify whether the patient stayed in a country where A/H5N1 virus was circulating. Clinical samples needed for a specific diagnosis are: nasopharyngeal, throat-swab or fecal samples, cerebrospinal fluid and blood. The presence of A/H5N1 virus is confirmed by viral isolation or RNA detection by RT-PCR. RNA extraction must be performed in a BSL3 laboratory. For diagnosis of A/H5N1 virus infection, RT-PCR test amplifies specifically a fragment of H5 gene (Hemagglutinin). In french laboratories of medical virology, procedures are ready to diagnose the first case of A/H5N1 virus infection and cases of reemerging SARS virus infection.

Communicable Diseases, Emerging↗
PubMed1974

Validation of a dual-isotope plasma ratio method for measurement of cholesterol absorption in rats.

Several methods for measuring cholesterol absorption in the rat have been compared. After administration of an oral dose of labeled cholesterol ((14)C or (3)H) and an intravenous dose of colloidal labeled cholesterol ((3)H or (14)C) the ratio of the two labels in plasma or whole blood 48 hr or more after dosing compared closely to the ratio of areas under the respective specific activity-time curves. The area ratio method is independent of a time lag between the appearance of oral and intravenous label in the bloodstream. Both measures of cholesterol absorption agree fairly well with a method based on measuring the unabsorbed dietary cholesterol in a pooled fecal sample. The plasma isotope ratio method gave more reproducible results than the fecal collection method when the measurement was repeated in the same animals 5 days after the first measurement. Cholesterol absorption was overestimated by the use of Tween 20-solubilized labeled cholesterol for the intravenous dose. The plasma disappearance curves of injected labeled colloidal cholesterol and cholesterol-labeled chylomicrons infused intravenously over a 3.5-hr period in the same animal coincided within experimental error from the first day until 75 days after injection. The plasma isotope ratio method for cholesterol absorption gave the same results in rats practicing coprophagy as in those in which this practice was prevented. The addition of sulfaguanidine to the diet lowered cholesterol absorption as measured by the plasma isotope ratio to the same degree as that measured by the fecal collection method.

Animals↗
PubMed1984

Prevalence of gastrointestinal parasitism in Oklahoma swine.

The prevalence of swine gastrointestinal parasites was determined from all listed large swine operations (28) and a geographically representative sample (70) of the population of small hog farm operations in Oklahoma. Fecal samples (n = 975) were collected from 98 farms. From the 98 farms, parasites were recovered in pig feces as follows: Ascaris, 53.0%, strongyles, 53.1%, Trichuris, 35.7%, spirurids, 6.1%, Strongyloides, 19.4%, coccidia, 57.1%, and Balantidium, 55.1%. A higher percentage (16.5%) of hogs maintained on cement floors were positive for Ascaris than were those on either dirt lots (11.9%) or slatted floors (9.9%), but pigs on dirt lots were more often positive with a higher percentage of coccidia (21.0%) than those on either cement or slatted floors (8.5% and 6.0%, respectively). Prevalence of Trichuris was essentially the same (6.8% to 11.3%) in hogs from all 3 management practices.

Animals↗
PubMed1978

Identification of the dietary lectin, wheat germ agglutinin, in human intestinal contents.

Plant lectins are known to have potent biological actions of normal and malignant cells. High concentrations of these lectins are present in many types of high residue diets. The specific binding of wheat germ agglutinin, a dietary plant lectin, to N-acetylglucosamine was used as the basis for purification of this lectin by biospecific chitin affinity chromatography. Subsequently, methods were developed for the extraction, purification, and identification of wheat germ agglutinin from fecal samples. Biologically intact wheat germ agglutinin was detected in ileostomy effluent and fecal collections from human subjects consuming a diet containing wheat germ. These studies demonstrate that wheat germ agglutinin can traverse the human small intestine intact. It is feasible that orally ingested wheat germ agglutinin and other plant lectins which interact with a wide variety of cell membranes may alter intestinal epithelial or bacterial cell function in the human bowel.

Feces↗