Bayesian estimation of diagnostic accuracy of fecal culture and PCR-based tests for the detection of Salmonella enterica in California cull dairy cattle.

Epidemiological studies of low prevalence disease problems are often hindered by the high cost of diagnostic testing. The objective of this study was to evaluate PCR screening of both individual and pooled fecal samples from culled dairy cows for the invA gene of Salmonella followed by culture to determine if the sensitivity and specificity were comparable to the results from traditional culture methods applied to individual samples. Cows from six different dairies were sampled in all four seasons. A total of 240 individual cow fecal samples, 24 fecal pools and 24 pools of 24-hour tetrathionate enrichment broth were tested. Diagnostic sensitivity of PCR screening followed by culture of PCR positive or indeterminate samples (i.e PCR-CUL method) was lower than that of culture (CUL) when applied to individual fecal samples (94.8%, 99.5%), however the specificity was comparable (99.6% and 97.7% respectively). For pools of five fecal samples and pools of five, 24 h tetrathionate broth samples, the specificity of both tests were comparable (∼98%); however, their sensitivity was only comparable in pooled fecal samples (∼93%) but greater for culture compared to PCR-CUL in pooled broth samples (∼99% versus ∼93%). Compared to culture results from testing of individual fecal samples, testing pooled fecal samples by culture had a relative sensitivity of 74% and relative specificity of 96%, testing pooled fecal samples by PCR-CUL resulted in relative sensitivity of 90% and relative specificity of 96%. Testing of pooled 24-hour enrichment broth by PCR-CUL increased the relative sensitivity and specificity to 100%. PCR testing followed by culture of positive or indeterminate samples is a time saving alternative to traditional methods. In addition, pooling of samples may be a useful method for decreasing cost if study aims can accommodate a moderate loss of relative sensitivity

Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota.

Studies involving gut microbiome analysis play an increasing role in the evaluation of health and disease in humans and animals alike. Fecal sampling methods for DNA preservation in laboratory, clinical, and field settings can greatly influence inferences of microbial composition and diversity, but are often inconsistent and under-investigated between studies. Many laboratories have utilized either temperature control or preservation buffers for optimization of DNA preservation, but few studies have evaluated the effects of combining both methods to preserve fecal microbiota. To determine the optimal method for fecal DNA preservation, we collected fecal samples from one canine donor and stored aliquots in RNAlater, 70% ethanol, 50:50 glycerol:PBS, or without buffer at 25 °C, 4 °C, and -80 °C. Fecal DNA was extracted, quantified, and 16S rRNA gene analysis performed on Days 0, 7, 14, and 56 to evaluate changes in DNA concentration, purity, and bacterial diversity and composition over time. We detected overall effects on bacterial community of storage buffer (F-value = 6.87, DF = 3, P < 0.001), storage temperature (F-value=1.77, DF = 3, P = 0.037), and duration of sample storage (F-value = 3.68, DF = 3, P < 0.001). Changes in bacterial composition were observed in samples stored in -80 °C without buffer, a commonly used method for fecal DNA storage, suggesting that simply freezing samples may be suboptimal for bacterial analysis. Fecal preservation with 70% ethanol and RNAlater closely resembled that of fresh samples, though RNAlater yielded significantly lower DNA concentrations (DF = 8.57, P < 0.001). Although bacterial composition varied with temperature and buffer storage, 70% ethanol was the best method for preserving bacterial DNA in canine feces, yielding the highest DNA concentration and minimal changes in bacterial diversity and composition. The differences observed between samples highlight the need to consider optimized post-collection methods in microbiome research

Measurement of Fecal Testosterone Metabolites in Mice: Replacement of Invasive Techniques

Testosterone is the main reproductive hormone in male vertebrates and conventional methods to measure testosterone rely on invasive blood sampling procedures. Here, we aimed to establish a non-invasive alternative by assessing testosterone metabolites (TMs) in fecal and urinary samples in mice. We performed a radiometabolism study to determine the effects of daytime and sex on the metabolism and excretion pattern of radiolabeled TMs. We performed physiological and biological validations of the applied EIA to measure TMs and assessed diurnal fluctuations in TM excretions in male and female mice and across strains. We found that males excreted significantly more radiolabeled TMs via the feces (59%) compared to females (49.5%). TM excretion patterns differed significantly between urinary and fecal samples and were affected by the daytime of ³H-testosterone injection. Overall, TM excretion occurred faster in urinary than fecal samples. Peak excretion of fecal TMs occurred after 8 h when animals received the 3H-testosterone in the morning, or after 4 h when they received the 3H-testosterone injection in the evening. Daytime had no effect on the formed TMs; however, males and females formed different types of TMs. As expected, males showed higher fecal TM levels than females. Males also showed diurnal fluctuations in their TM levels but we found no differences in the TM levels of C57BL/6J and B6D2F1 hybrid males. Finally, we successfully validated our applied EIA (measuring 17β-hydroxyandrostane) by showing that hCG (human chorionic gonadotropin) administration increased TM levels, whereas castration reduced them. In conclusion, our EIA proved suitable for measuring fecal TMs in mice. Our non-invasive method to assess fecal TMs can be widely used in various research disciplines like animal behavior, reproduction, animal welfare, ecology, conservation, and biomedicine

RNA Viral Community in Human Feces: Prevalence of Plant Pathogenic Viruses

The human gut is known to be a reservoir of a wide variety of microbes, including viruses. Many RNA viruses are known to be associated with gastroenteritis; however, the enteric RNA viral community present in healthy humans has not been described. Here, we present a comparative metagenomic analysis of the RNA viruses found in three fecal samples from two healthy human individuals. For this study, uncultured viruses were concentrated by tangential flow filtration, and viral RNA was extracted and cloned into shotgun viral cDNA libraries for sequencing analysis. The vast majority of the 36,769 viral sequences obtained were similar to plant pathogenic RNA viruses. The most abundant fecal virus in this study was pepper mild mottle virus (PMMV), which was found in high concentrations—up to 10(9) virions per gram of dry weight fecal matter. PMMV was also detected in 12 (66.7%) of 18 fecal samples collected from healthy individuals on two continents, indicating that this plant virus is prevalent in the human population. A number of pepper-based foods tested positive for PMMV, suggesting dietary origins for this virus. Intriguingly, the fecal PMMV was infectious to host plants, suggesting that humans might act as a vehicle for the dissemination of certain plant viruses

Environmental sampling to assess the bioburden of Mycobacterium avium subspecies paratuberculosis in drylot pens on California dairies.

Mycobacterium avium subspecies paratuberculosis (MAP) is a bacterium that can cause substantial economic losses in infected dairy herds due to reduced milk production and increased cow-replacement costs. In order to control MAP in dairies with drylot pens, a standardized environmental sampling protocol to quantify MAP in fecal slurry was developed based on an existing protocol for freestall pens. Specifically, following a 24 h hold of the flush, a grab sample of approximately 10 ml of fecal slurry was collected every 1 m along the flush lane of the drylot pens, avoiding individual cow fecal pats. To determine the reliability and repatability of the new environmental sampling protocol for estimation of MAP bioburden at the pen level, two collectors simultaneously collected fecal slurry samples every day for 3 days from six drylot cow pens on two Central California dairies. During the study period no cow movement between pens was allowed with the exception of sick cows. The study herds had MAP seroprevalence of 5.8% and 3.2%, respectively, based on whole pen serum ELISA results. Variance components models for quantitative real-time PCR (qPCR) results showed samples collected from different pens on different dairies accounted for greater variablitiy in MAP concentration (65%), while samples collected by different collectors had the least variability (0.1%). In contrast, variability in MAP concentration in environmental samples collected on different days had 25% variability. The intraclass correlation coefficient showed high reliability (93%) of environmental sampling simultaneously by different collectors. In contrast, the reliability of environmental sampling at different days was 65%, which was similar to the reliability for sampling by different collectors on different days. Investigators can expect high reliability when employing the new environmental sampling protocol along with qPCR testing of environmental samples from drylot pens

Association of Chorioamnionitis with Aberrant Neonatal Gut Colonization and Adverse Clinical Outcomes.

ObjectiveChorioamnionitis (inflammation of the placenta and fetal membranes) and abnormal gastrointestinal colonization have been associated with an increased risk of sepsis and death in preterm infants, but whether chorioamnionitis causes abnormal pioneering gastrointestinal colonization in infants is not known. We determined the relationship between chorioamnionitis, altered infant fecal microbiome indicating abnormal gastrointestinal colonization, and adverse outcomes.Study designPreterm infants ≤ 28 weeks at birth were enrolled from 3 level III NICUs in Cincinnati, Ohio and Birmingham, Alabama. Sequencing for 16S microbial gene was performed on stool samples in the first 3 weeks of life. Chorioamnionitis was diagnosed by placental histology. Late onset sepsis and death outcomes were analyzed in relation to fecal microbiota and chorioamnionitis with or without funisitis (inflammation of the umbilical cord).ResultsOf the 106 enrolled infants, 48 infants had no chorioamnionitis, 32 infants had chorioamnionitis but no funisitis (AC), and 26 infants had chorioamnionitis with funisitis (ACF). The fecal samples from ACF infants collected by day of life 7 had higher relative abundance of family Mycoplasmataceae (phylum Tenericutes), genus Prevotella (phylum Bacteroidetes) and genus Sneathia (phylum Fusobacteria). Further, AC and ACF infants had higher incidence of late-onset sepsis/death as a combined outcome. Presence of specific clades in fecal samples, specifically, order Fusobacteria, genus Sneathia or family Mycoplasmataceae, were significantly associated with higher risk of sepsis or death.ConclusionThe results support the hypothesis that specific alterations in the pioneering infant gastrointestinal microbiota induced by chorioamnionitis predispose to neonatal sepsis or death

Evaluation of Ankom F58 Filter Bags Compared to Dacron Bags and Beakers for Analysis of Acid Detergent Fiber

Feed and fecal samples were analyzed to compare three methods of determining acid detergent fiber. Each sample was weighed into both Dacron and Ankom F58 fiber bags and then analyzed using an Ankom fiber analyzer. Results were then compared to the Van Soest beaker method. Ankom F58 bags helped reduce washout of small particles associated with Dacron bags, but fecal samples needed to be incubated in detergent for an extended amount of time to isolate acid detergent fiber material. Utilizing a technique that produces correct acid detergent fiber values is important for producers because these values are used as a proxy for calculating total digestible nutrients of feedstuffs