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PubMed2005

The Salmonella enterica serotype Typhimurium lpf, bcf, stb, stc, std, and sth fimbrial operons are required for intestinal persistence in mice.

Salmonella enterica serotype Typhimurium causes human infections that can frequently be traced back through the food chain to healthy livestock whose intestine is colonized by the pathogen. Little is known about the genes important for intestinal carriage of S. enterica serotype Typhimurium in vertebrate animals. Here we characterized the role of 10 fimbrial operons, agf, fim, lpf, pef, bcf, stb, stc, std, stf, and sth, using competitive infection experiments performed in genetically susceptible (BALB/c) and resistant (CBA) mice. Deletion of agfAB, fimAICDHF, lpfABCDE, pefABCDI, bcfABCDEFG, stbABCD, stcABCD, stdAB, stfACDEFG, or sthABCDE did not reduce the ability of S. enterica serotype Typhimurium to colonize the spleen and cecum of BALB/c mice 5 days after infection. Similarly, deletion of agfAB, fimAICDHF, pefABCDI, and stfACDEFG did not result in reduced recovery of S. enterica serotype Typhimurium from fecal samples collected from infected CBA mice over a 30-day time period. However, S. enterica serotype Typhimurium strains carrying deletions in lpfABCDE, bcfABCDEFG, stbABCD, stcABCD, stdAB, or sthABCDE were recovered at significantly reduced numbers from the feces of CBA mice. There was a good correlation (R(2) = 0.9626) between competitive indices in the cecum and fecal samples of CBA mice at 30 days after infection, suggesting that the recovery of S. enterica serotype Typhimurium from fecal samples closely reflected its ability to colonize the cecum. Collectively, these data show that six fimbrial operons (lpf, bcf, stb, stc, std, and sth) contribute to long-term intestinal carriage of S. enterica serotype Typhimurium in genetically resistant mice.

Animals↗
PubMed2004

Detection of sorbitol-negative and sorbitol-positive Shiga toxin-producing Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, and Salmonella spp. in dairy farm environmental samples.

Six visits were conducted to four dairy farms to collect swab, liquid, and solid dairy farm environmental samples (165 to 180/farm; 15 sample types). The objective of the study was to determine on-farm sources of Campylobacter jejuni, Salmonella spp., Listeria monocytogenes, and Shiga toxin-producing Escherichia coli (STEC), which might serve as reservoirs for transmission of pathogens. Samples were analyzed using mostly U.S. Food and Drug Administration's Bacteriological Analytical Manual protocols; however, Salmonella spp., L. monocytogenes and STEC were co-enriched in universal pre-enrichment broth. Campylobacter jejuni were enriched in Bolton broth containing Bolton broth supplement. Pathogens were isolated on agar media, typed biochemically, and confirmed using multiplex polymerase chain reaction protocols. Campylobacter jejuni, Salmonella spp., L. monocytogenes, Sorbitol-negative (SN)-STEC O157:H7, and sorbitol-positive (SP)-STEC, respectively, were isolated from 5.06%, 3.76%, 6.51%, 0.72%, and 17.3% of samples evaluated. Whereas other pathogens were isolated from all four farms, SN-STEC O157:H7 were isolated from only two farms. Diverse serotypes of SP-STEC including O157:H7, O26:H11, O111, and O103 were isolated. None of the five pathogen groups studied were isolated from bulk tank milk (BTM). Most pathogens (44.2%) were isolated directly from fecal samples. Bovine fecal samples, lagoon water, bedding, bird droppings, and rat intestinal contents constituted areas of major concern on dairy farms. Although in-line milk filters from two farms tested positive for Salmonella or L. monocytogenes, none of the pathogens were detected in the corresponding BTM samples. Good manure management practices, including control of feral animals, are critical in assuring dairy farm hygiene. Identification of on-farm pathogen reservoirs could aid with implementation of farm-specific pathogen reduction programs.

Animal Husbandry↗
PubMed2001

Comparison of cultivation and PCR-hybridization for detection of Salmonella in porcine fecal and water samples.

A total of 150 fecal and water samples from four swine farms were tested for the presence of Salmonella enterica using different enrichment techniques as follows: (i) 92 fecal samples from nursery and farrowing barns at three swine farms were preenriched overnight in tryptic soy broth (TSB) at 37 degrees C followed by overnight enrichment in Rappaport-Vassiliadis 10 broth (RV10) at 42 degrees C; (ii) 24 water samples from the third farm were preenriched overnight in 3MC broth at 37 degrees C followed by overnight enrichment in RV10 at 42 degrees C; and (iii) 34 fecal samples from a fourth farm, a finishing farm, were enriched overnight in RV10 at 42 degrees C with no additional enrichment. Following each of the enrichment techniques, samples were subcultured onto modified semisolid Rappaport-Vassiliadis (MSRV) agar prior to transfer to Hektoen Enteric agar plates for the recovery of viable Salmonella bacteria. Presumptive Salmonella isolates were biochemically and serologically confirmed. For the PCR detection of Salmonella, a 1-ml portion was removed from each sample after the first overnight enrichment and the DNA was extracted using a Sepharose CL-6B spin column. Amplicons (457 bp) derived from primers to the invA and invE genes were confirmed as Salmonella specific on ethidium bromide-stained agarose gels by Southern hybridization with a 20-mer oligonucleotide probe specific for the Salmonella invA gene. Neither the standard microbiological method nor the molecular method detected all of the 65 samples that tested positive by both methods or either method alone. Salmonella bacteria were detected by both cultivation and PCR-hybridization in 68% (17 of 25) of the positive samples that were preenriched in TSB, in 73% (11 of 15) of the positive samples preenriched in 3MC broth, and in 24% (6 of 25) of the positive samples enriched in RV10. Agreement between Salmonella detection using cultivation with preenrichment and detection by PCR was 76% using the kappa statistic. However, agreement between Salmonella detection using cultivation without preenrichment and detection by PCR was about 6%; the PCR assay detected 80% (20 of 25) of the 25 positive samples, while Salmonella bacteria were recovered from only 44% (11 of 25) by cultivation. Our results indicate that the PCR-hybridization approach is equivalent to or better than cultivation for detecting Salmonella in swine feces or water samples from swine farms when using the medium combinations evaluated in this study.

Animal Husbandry↗
PubMed2005

Diagnosis of enteroviral meningitis by use of polymerase chain reaction of cerebrospinal fluid, stool, and serum specimens.

BACKGROUND: Because enteroviruses can be detected in various clinical samples during enteroviral meningitis, we analyzed the combined diagnostic utility of polymerase chain reaction (PCR) of cerebrospinal fluid (CSF), feces, and serum for detection of enterovirus in specimens obtained from adults with aseptic meningitis or encephalitis. METHODS: PCR results were analyzed for 34 adults for whom enteroviral meningitis was diagnosed on the basis of virus isolation and antibody detection in our hospital during 1999-2003. PCR results were also analyzed for 77 adults with meningitis or encephalitis of another defined cause for whom this assay was used for diagnostic evaluation during that period. RESULTS: Twenty-six (76%) of 34 CSF samples and 24 (96%) of 25 fecal samples collected from patients with enteroviral meningitis had positive PCR results. The diagnostic yield of the test was lower for CSF specimens obtained >2 days after clinical onset, compared with CSF collected < or =2 days after onset. Instead, PCR of feces was highly useful also later, because 12 of the 13 fecal specimens obtained 5-16 days after clinical onset had positive test results. None of 75 CSF samples and 2 of 48 fecal samples obtained from patients with nonenteroviral infection had positive PCR results. All serum samples were PCR negative. CONCLUSIONS: PCR of fecal specimens obtained throughout the course of enteroviral meningitis had the highest clinical sensitivity for detecting enterovirus. It is recommended that, in addition to performance of CSF PCR, fecal samples collected from patients with suspected enteroviral meningitis should be tested by PCR, especially when the duration of symptoms is >2 days.

Adult↗
PubMed1996

Application of 16S rRNA gene PCR to study bowel flora of preterm infants with and without necrotizing enterocolitis.

The purpose of the present study was to determine the extent to which bacteria not detected by culture contribute to the microbial flora of the bowel of preterm infants with and without neonatal necrotizing enterocolitis (NEC). Fecal samples from 32 preterm infants in special care baby units including samples from 10 infants with NEC were examined by culture and PCR amplification of the 16S rRNA gene (rDNA). The 16S rDNA V3 region was amplified with eubacterial primers, and the amplification products derived from the fecal sample DNA were compared with the products from individual cultured isolates by PCR and denaturing gradient gel electrophoresis (PCR-DGGE), allowing the DNA from uncultured bacteria to be identified. For the 22 infants without NEC weekly samples were examined for a mean of 5.3 postnatal weeks. The total number of types detected by culture combined with PCR-DGGE was 10.1 per infant, of which PCR-DGGE contributed 10.4% of the types identified. Additional types detected by PCR-DGGE were found in 14 (63.6%) of the 22 infants. The majority of the sequences associated with uncultured bacteria showed > 90% 16S rDNA sequence identity with sequences from culturable human enteric flora, and all were found in single infants with the exception of sequences indistinguishable by DGGE from seven infants. These sequences showed > 90% sequence identity with the 16S rDNA of Streptococcus salivarius and may have been derived from upper gastrointestinal or respiratory tract flora. In the present study uncultured bacteria detected by PCR-DGGE were no more frequent in fecal samples from infants with NEC than in samples from infants without NEC, although these findings do not exclude the possibility of unrecognized bacteria associated with the mucosa of the small intestine of infants with NEC.

Bacteria↗
PubMed2003

Evaluation of a human group a rotavirus assay for on-site detection of bovine rotavirus.

Neonatal diarrhea induced by bovine group A rotavirus causes significant economic loss in the dairy and beef industry due to increased morbidity and mortality, treatment costs, and reduced growth rates. The objective of this study was to evaluate a human group A rotavirus assay (ImmunoCardSTAT Rotavirus [ICS-RV]) as an on-site diagnostic test for bovine rotavirus. When used with a collection of bovine diarrhea samples submitted to the Virology Section of the Diagnostic Center for Population and Animal Health at Michigan State University and compared to a bovine group A rotavirus-specific reverse transcription-PCR (RT-PCR), the ICS-RV assay had a sensitivity and specificity of 87.0 and 93.6%, respectively. A commercially available group A rotavirus enzyme-linked immunosorbent assay (ELISA) (Pathfinder; Sanofi Diagnostics, Redmond, Wash.), when used with the same fecal sample collection and compared to the same RT-PCR, had a sensitivity and specificity of 78.3 and 67.7%, respectively. Subsequently, the ICS-RV assay, RT-PCR, and a different commercially available group A rotavirus ELISA (Rotaclone; Meridian Diagnostics, Cincinnati, Ohio) were used to evaluate fecal samples collected from neonatal calves experimentally infected with bovine rotavirus. When diarrheic fecal samples that were positive for bovine rotavirus by RT-PCR were evaluated, the ICS-RV assay and the Rotaclone assay detected bovine rotavirus 85 and 95% of the time, respectively. Based on these studies, the ICS-RV assay appears to be an excellent test for detecting group A bovine rotaviruses. This assay may be useful as an on-site diagnostic test for veterinarians as an aid in the management of bovine neonatal diarrhea.

Animals↗
PubMed2005

Activation of proestrogens from hops (Humulus lupulus L.) by intestinal microbiota; conversion of isoxanthohumol into 8-prenylnaringenin.

Hop, an essential ingredient in most beers, contains a number of prenylflavonoids, among which 8-prenylnaringenin (8-PN) would be the most potent phytoestrogen currently known. Although a number of health effects are attributed to these compounds, only a few reports are available about the bioavailability of prenylflavonoids and the transformation potency of the intestinal microbial community. To test these transformations, four fecal samples were incubated with xanthohumol, isoxanthohumol (IX), and 8-PN. Upon incubation with IX, present in strong ales up to 4 mg/L, 36% was converted into 8-PN in one fecal sample and the estrogenic properties of the sample drastically increased. In an experiment with 12 fecal cultures, this conversion was observed in one-third of the samples, indicating the importance of interindividual variability in the intestinal microbial community. Eubacterium limosum was identified to be capable of this conversion (O-demethylation) of IX into 8-PN, and after strain selection, a conversion efficiency of 90% was achieved. Finally, strain supplementation to a nonconverting fecal sample led to rapid and high 8-PN production at only 1% (v/v) addition. Up to now, the concentration of 8-PN in beer was considered too low to affect human health. However, these results show that the activity of the intestinal microbial community could more than 10-fold increase the exposure concentration. Because prenylflavonoids are present in many beers with IX being the major constituent, the results raise the question whether moderate beer consumption might contribute to increased in vivo levels of 8-PN and even influence human health.

Adult↗
PubMed1992

Evaluation of the abilities of three diagnostic tests based on the polymerase chain reaction to detect Mycobacterium paratuberculosis in cattle: application in a control program.

Three assays for the specific detection of Mycobacterium paratuberculosis by dot spot hybridization of polymerase chain reaction products were applied to fecal samples of dairy cattle. The first two tests used polymerase chain reaction primers and a DNA probe derived from M. paratuberculosis-specific sequences of the 16S rRNA gene and insertion element IS900, respectively. These two tests were carried out on spiked fecal samples to determine the detection limits. The 16S rRNA test was able to detect 10(7) bacteria per g of feces, and the IS900 test detected 10(4) to 10(5) per g of feces. Next, we studied the usefulness of these tests in a control program for paratuberculosis. Therefore, the tests and a third, commercially available, test (IDEXX Corp.) were used twice with an interval of 3 months on fecal samples of 87 cows from two dairy herds with a history of Johne's disease. We compared the results of these tests with those of culturing. This showed that the tests are specific but that the sensitivity ranged from 3 to 23%. Further improvement of the sensitivity is needed before the tests can be used in a control program to eradicate Johne's disease.

Animals↗
PubMedDate not supplied

[Evaluation of a new medium using red-cell lysate for the isolation of Campylobacter jejuni and Campylobacter coli].

A semiprepared medium containing horse globular lysate was evaluated for the isolation of Campylobacter under two incubation atmospheres (5% O2 and candle jar). Studying 19 known strains of C. jejuni, 151 human and 230 animal fecal samples. They were seeded on conventional medium (MC) and on the new medium (MLG) and incubated at 42 degrees C for 24 and 48 h. Campylobacter strains grew better in 5% O2 atmosphere after 48 h of incubation. All the strains that grew or were isolated on MC, could also grow in MLG incubation in candle jar. Campylobacter was isolated from human fecal samples with the same frequency (5.3%) in MC and MLG, independently of the incubation atmosphere employed. These bacteria were isolated in 35.2% and 34.3% of the animal fecal samples seeded on Skirrow modified medium and MLG, respectively. The concordance proportion between both media was 99.13%.

Aerobiosis↗
PubMed2002

Efficacy of a combination febantel-praziquantel-pyrantel product, with or without vaccination with a commercial Giardia vaccine, for treatment of dogs with naturally occurring giardiasis.

OBJECTIVE: To determine efficacy of treatment with a combination febantel-praziquantel-pyrantel product, with or without vaccination with a commercial Giardia vaccine, in dogs with naturally occurring giardiasis. DESIGN: Prospective trial. ANIMALS: 16 Beagles naturally infected with Giardia duodenalis. PROCEDURES: During phase 1, 6 dogs were treated with the parasiticide for 3 days (4 were also vaccinated). Four weeks later, all 6 dogs were treated with the parasiticide again for 5 days and were bathed and moved to clean cages after the last treatment (phase 2). Nine dogs were treated with the parasiticide for 3 (n = 4) or 5 (5) days and bathed and moved to clean cages after the last treatment (phase 3). Fecal samples were collected twice weekly for 24 days after treatment and tested for cysts with a quantitative zinc sulfate flotation technique and for Giardia antigen with an immunoassay. RESULTS: Dogs in phase 1 were all shedding cysts again by day 24. In phase 2, only 1 dog shed cysts after treatment, and shedding was transient (day 17). In phase 3, neither cysts nor antigen was detected in fecal samples from 2 of 4 dogs treated for 3 days and 4 of 5 dogs treated for 5 days. In 18 of 57 (31.6%) fecal samples, cysts were seen, but results of the immunoassay were negative. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that when a combination febantel-praziquantel-pyrantel product is used to treat dogs with giardiasis, bathing and changing the environment after treatment may be more important in preventing recurrence than duration of treatment.

Animals↗
PubMed1997

Fecal bacterial enzyme activities in infants increase with age and adoption of adult-type diet.

BACKGROUND: The present study was conducted to determine the effects of age and diet (breast milk, milk formula, weaning diet) on the activities of some bacterial enzymes in fecal samples collected from 3- to 24-month-old infants. These bacterial enzymes may generate toxic, mutagenic, or carcinogenic metabolites from plant glycosides (beta-glucuronidase, beta-glucosidase) or from urea (urease). METHODS: The subjects were 68 healthy children ages 3 (n = 20), 6 (n = 20), 12 (n = 17), and 24 (n = 11) months. None of the children had been given any antibiotics during 4 weeks before collection of the fecal samples. Health status, bowel habits, and food consumption of the children were determined by interview of one of the parents using a structured questionnaire. A spot fecal sample from each child was collected. RESULTS: The activities of beta-glucuronidase and beta-glucosidase increased from 0.52 and 1.17 nmol/min x mg protein (at 3 months) to 1.41 and 2.38 nmol/min x mg protein (at 2 years), respectively. However, at 2 years of age, the children had not yet reached the levels reported in earlier studies on adult populations. Urease activity increased from 4.18 nmol/min x mg protein (at 3 months) to 19.0 nmol/min x mg protein (at 2 years), reaching by 12 months of age the level observed in adults. Breast feeding as such had no consistent effect on fecal bacterial enzyme activities. beta-Glucuronidase and beta-glucosidase were higher in those 6- and 12-month-old children who consumed more meat, fish, sausages, or eggs. CONCLUSIONS: It can be concluded that the fecal enzyme activities in the gastrointestinal tract increase and that their profile changes with age and adoption of adult-type diet. It remains open, however, whether these changes are caused by the normal physiological aging process or by stepwise introduction of new foods to the diet.

Adult↗
PubMed2002

Production and application of new monoclonal antibodies specific for a fecal Helicobacter pylori antigen.

The aim of the present study was to establish monoclonal antibodies that could be used to produce a diagnostic test composed of one kind of monoclonal antibody recognizing a fecal Helicobacter pylori antigen. The need to develop such a test arose from disadvantages of the diagnostic test that uses a polyclonal antibody or plural kinds of monoclonal antibodies, such as the lower specificity for H. pylori antigen and the difficulty of reproduction with consistent quality. Mice were immunized with sonicated cells of the coccoid form of H. pylori, and fecal samples from H. pylori-positive subjects were screened by a direct sandwich enzyme immunoassay (EIA) for antibody production from 32 hybridoma clones. The three stable clones produced antibodies (21G2, 41A5, and 82B9) that reacted with the same soluble antigen. Gel filtration chromatography showed that the molecular masses of the cellular antigen and the fecal antigen were the same, 260 kDa. The antigen was labile in response to sodium dodecyl sulfate and heat treatments. A single-step direct sandwich EIA using a single monoclonal antibody, 21G2, was developed. The EIA could detect the antigen in 41 H. pylori clinical isolates and in fecal samples from seven H. pylori-positive subjects. Several kinds of Helicobacter species (Helicobacter felis, Helicobacter hepaticus, Helicobacter mustelae, and Helicobacter cinaedi) except H. pylori, major bacteria in feces (Campylobacter jejuni, Bacteroides vulgatus, Bifidobacterium breve, Bifidobacterium infantis, and Escherichia coli), and fecal samples from six H. pylori-negative subjects showed negative results. These results indicate that the new monoclonal antibodies and the new specific EIA would be useful as a noninvasive method of diagnosis of H. pylori infection.

Animals↗
PubMed1981

In vitro transformation of chenodeoxycholic acid and ursodeoxycholic acid by human intestinal flora, with particular reference to the mutual conversion between the two bile acids.

Nine fecal samples from four healthy subjects were examined for their ability to transform chenodeoxy-cholic acid (CDCA) and ursodeoxycholic acid (UDCA) in in vitro anaerobic broth cultures. Seven samples converted CDCA and UDCA into each other (more than 50% of CDCA was converted into UDCA while 10% or less of UDCA was converted into CDCA), and produced 7-keto-lithocholic acid and lithocholic acid equally from both acids. No alteration of the 7 beta-hydroxy group of UDCA was demonstrated by two fecal samples that failed to perform mutual 7-epimerization, suggesting the conversion of UDCA into lithocholic acid via CDCA. The 3 alpha-hydroxy groups of these substrate and metabolite bile acids were invariably partially epimerized to 3 beta-hydroxy groups by all the fecal samples. Evidence is presented for the prevalence of these 7- and 3-epimerizing organisms among the human intestinal flora.

Adult↗
PubMed2003

Prevalence of endoparasitic infection in children and its relation with cholera prevention efforts in Mexico.

OBJECTIVE: To investigate whether increased knowledge and use of public health measures promoted for cholera prevention is reflected in lower prevalence of parasitic infection in households in a community in the state of Tamaulipas, Mexico, that is close to the border with the United States of America. METHODS: Between 1994 and 1997, fecal samples from 438 children were collected through convenience sampling and then examined for helminth eggs/larvae and protozoan cysts as biologic indicators of household compliance with recommended cholera prevention measures. The suggested measures were to wash hands before meals and after defecation, to drink purified water, to wash fruits and vegetables, and to eat well-cooked food. In addition, information on the knowledge of and the use of cholera preventive measures was collected by interviews with adult informants in 252 households (186 of those households also provided a fecal sample for analysis). RESULTS: Parasitic infections occurred in 131 of the 438 children (30%), who resided in 79 of the 186 households (42%) that provided fecal samples. Giardia lamblia accounted for 12.5% of all infections. Infections with Hymenolepis nana, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularis, Ancylostoma/Necator, Strongyloides stercoralis, Entamoeba coli, Entamoeba hartmanni, Entamoeba histolytica, Endolimax nana, and Iodamoeba bütschlii were also noted. Infected children were older and more often had an infected sibling. Households with three or more children were also more likely to have an infected child. The primary caregivers in the households where at least one child had a parasitic infection were distinguished by their inability to list at least three cholera prevention measures from memory. CONCLUSIONS: The 42% household prevalence of parasitic infection was relatively high and indicates that some residents of this community may not have fully embraced the public health education efforts promoted for prevention of cholera. The occurrence of nonpathogenic protozoan parasites such as Endolimax nana, Entamoeba coli, Entamoeba hartmanni, and I. bütschlii are important bioindicators for the persistence of unhygienic behaviors that increase the risk of cholera and other infectious diseases dependent on fecal-oral transmission. Information obtained by similar studies can be useful for monitoring compliance with community health and hygiene programs and may indicate the need to intensify educational efforts for the prevention of diarrhea associated with enteric pathogens that cannot be controlled by drugs alone.

Adolescent↗
PubMed1988

Excreted steroids in primate feces over the menstrual cycle and pregnancy.

Techniques were established for the extraction and measurement of 17 beta-estradiol (E2) and progestins (P4) from feces of Old World primates. Studies were conducted to show the sensitivity of these measures, means of preserving fecal samples in the field, effects of urinary contamination, and means to eliminate these effects. Our results show that excreted steroid measures can be used to distinguish between mid-follicular and luteal phases in the menstrual cycle, and to identify pregnancy by Day 20 of gestation; the steroid measures can also be used to identify ovulatory levels of E2 and to establish the length of the menstrual cycle. Urine was shown to contaminate the fecal sample and to confound the estimate of steroid levels in feces; prolonged storage (less than 6 h) was shown to change the steroid estimate. Both urinary contamination and storage-dependent changes were eliminated by the addition of ethanol to the sample. Preliminary results also suggest that effects of dietary fiber on steroid hormone levels are minimal when controlled quantitatively by adjusting for water content of the fecal sample. We conclude that these measurements of excreted steroids provide a valid, noninvasive measure of physiological state of the hypothalamic-pituitary-ovarian axis among free-ranging animals in the field.

Animals↗
PubMed1999

Investigations on airborne microorganisms in animal stables. 2. Report: further characterization of airborne Clostridium perfringens.

Toxovars of 97 airborne C. perfringens isolates and 10 C. perfringens isolates from fecal samples of a calf stable were determined by an EIA procedure. Most airborne and fecal isolates belonged to toxovar A (88.7% and 80.0% respectively). Eight point two% of airborne C. perfringens were identified as toxovar C and 3.1% as toxovar D. Toxovar B was not found in the airborne state. Twenty% of fecal C. perfringens belonged to toxovar D. Toxovar B and C was not isolated from fecal samples. In addition, all fecal and air-borne isolates of C. perfringens toxovar D strains were analyzed in SDS-PAGE for their polypeptide pattern. All isolates from both sources exhibited the same polypeptide pattern after electrophoretic analysis in SDS-PAGE. Both results, determination of toxovars as well as polypeptide pattern analysis in SDS-PAGE, suggest that a major source of airborne C. perfringens in animal stables is animal feces.

Air Microbiology↗
PubMed1993

Detection of microsporidian spores in clinical samples by indirect fluorescent-antibody assay using whole-cell antisera to Encephalitozoon cuniculi and Encephalitozoon hellem.

Three polyclonal mouse antisera, to Encephalitozoon cuniculi, Nosema algerae, and Nosema corneum, and two polyclonal rabbit antisera, to E. cuniculi and Encephalitozoon hellem, were used in an indirect fluorescent-antibody assay (IFA) with Enterocytozoon bieneusi, E. cuniculi, and Encephalitgozoon. hellem spores (spores of the last two were taken from culture). Enterocytozoon bieneusi cannot be cultured. By IFA, antisera to E. cuniculi and E. hellem reacted strongly and equally with each other's spores. The mouse antisera reacted strongly with the homologous species, but for these there was segmental and particulate or "dot" staining of heterologous microsporidian spores, indicating cross-reactions with more selected antigens. In fecal samples, cross-reactions with both mouse and rabbit antisera were sometimes seen with different yeast species, with species of streptococci, and species of gram-negative rods. There were no cross-reactions to staphylococci. Enterocytozoon bieneusi was easily identified in duodenal and colonic biopsies, duodenal and colonic fluids, and feces of symptomatic AIDS patients by IFA. In a study of 12 AIDS patients with diarrhea, the new IFA identified microsporidia in all of 11 fecal samples, three colon fluids, six duodenal fluids, and three duodenal biopsy touch preparations. Although the fecal sample of 1 of the 12 was negative, the patient's duodenal fluid contained microsporidian spores by IFA.

Animals↗
PubMed1996

Rapid identification of Salmonella serovars in feces by specific detection of virulence genes, invA and spvC, by an enrichment broth culture-multiplex PCR combination assay.

In order to make a rapid and definite diagnosis of Salmonella enteritis in children, an enrichment broth culture-multiplex PCR combination assay was devised to identify Salmonella serovars directly from fecal samples. Two pairs of oligonucleotide primers were prepared according to the sequences of the chromosomal invA and plasmid spvC genes. PCR with these two primers would produce either one amplicon (from the invA gene) or two amplicons (from the invA and spvC genes), depending on whether or not the Salmonella bacteria contained a virulence plasmid. The fecal sample was diluted 10- to 20-fold into gram-negative enrichment broth and incubated to eliminate inhibitory compounds and also to allow selective enrichment of the bacteria. One or two amplicons were obtained, the expected result if Salmonella bacteria were present. The detection limit of this PCR was about 200 bacteria per reaction mixture. The primers were specific, as no amplification products were obtained with 18 species and 22 isolates of non-Salmonella bacteria tested which could be present in the feces or cause contamination. In contrast, when 23 commonly seen Salmonella serovars (38 isolates) were tested, all were shown to carry the invA gene and seven concomitantly harbored the spvC gene of the virulence plasmid. This assay was applied to the diagnosis of Salmonella enteritis in 57 children who were suffering from mucoid and/or bloody diarrhea. Of the 57 children, 38 were PCR positive and 22 were culture positive. There were two culture-positive samples that were not detected by PCR. Thus, this PCR assay showed an efficiency of 95% (38 of 40), which is much higher than the 60% (24 of 40) by culture alone. Not only is this method more sensitive, rapid, and efficient but it will cause only an incremental increase in the cost of stool processing, since enrichment cultivation of fecal samples from diarrheal patients using gram-negative enrichment broth is a routine practice for identification in many diagnostic microbiology laboratories. This PCR method, therefore, has clinical application.

Bacterial Proteins↗