LIQUID-CHROMATOGRAPHY THERMOSPRAY MASS-SPECTROMETRIC ASSAY FOR TRENBOLONE IN BOVINE BILE AND FECES

A liquid chromatography-thermospray mass spectrometric assay was developed and validated to confirm the presence of illegal residues of the synthetic androgenic growth promoter, trenbolone acetate, in cattle. The assay was specific for 17alpha-trenbolone, the major bovine metabolite of trenbolone acetate. Methods were developed for the determination of 17alpha-trenbolone in both bile and faeces, the most appropriate matrices for the control of trenbolone acetate abuse. The clean-up.procedure developed relied on enzymatic hydrolysis, followed by sequential liquid-liquid and liquid-solid extraction. The extracts were then subjected to immunoaffinity chromatography. 17alpha-Trenbolone was detected by selected ion monitoring at m/z 271 using positive ion thermospray ionisation. The limit of detection was approximately 0.5 ng/g in faeces and 0.5 ng/ml in bile.

Risk of disease from wildlife reservoirs: Badgers, cattle, and bovine tuberculosis

Livestock face complex foraging options associated with optimizing nutrient intake while being able to avoid areas posing risk of parasites or disease. Areas of tall nutrient-rich swards around fecal deposits may be attractive for grazing, but might incur fitness costs from parasites. We use the example of dairy cattle and the risks of tuberculosis transmission posed to them by pastures contaminated with badger excreta to examine this trade-off. A risk may be posed either by aerosolized inhalation through investigation or by ingestion via grazing contaminated swards. We quantified the levels of investigation and grazing of 150 dairy cows at badger latrines (accumulations of feces and urine) and crossing points (urination-only sites). Grazing behavior was compared between strip-grazed and rotation-grazed fields. Strip grazing had fields subdivided for grazing periods of

Arsenic rich iron plaque on macrophyte roots--an ecotoxicological risk?

Arsenic is known to accumulate with iron plaque on macrophyte roots. Three to four years after the Aznalcóllar mine spill (Spain), residual arsenic contamination left in seasonal wetland habitats has been identified in this form by scanning electron microscopy. Total digestion has determined arsenic concentrations in thoroughly washed 'root+plaque' material in excess of 1000 mg kg(-1), and further analysis using X-ray absorption spectroscopy suggests arsenic exists as both arsenate and arsenite. Certain herbivorous species feed on rhizomes and bulbs of macrophytes in a wide range of global environments, and the ecotoxicological impact of consuming arsenic rich iron plaque associated with such food items remains to be quantified. Here, greylag geese which feed on Scirpus maritimus rhizome and bulb material in areas affected by the Aznalcóllar spill are shown to have elevated levels of arsenic in their feces, which may originate from arsenic rich iron plaque.

Altered porphyrin excretion and histopathology of greylag geese (Anser anser) exposed to soil contaminated with lead and arsenic in the Guadalquivir Marshes, southwestern Spain

Greylag geese (Anser anser) in the Guadalquivir Marshes (southwestern Spain) can be exposed to sources of inorganic pollution such as heavy metals and arsenic from mining activities or Pb shot used for hunting. We have sampled 270 fecal excreta in different areas of the marshes in 2001 to 2002 to evaluate the exposure to Pb, Zn, Cu, Mn, and As and to determine its relationship with soil ingestion and with the excretion of porphyrins and biliverdin as biomarkers. These effects and the histopathology of liver, kidney, and pancreas were also studied in 50 geese shot in 2002 to 2004. None of the geese had ingested Pb shot in the gizzard. This contrasts with earlier samplings before the ban of Pb shot for waterfowl hunting in 2001 and the removal of Pb shot in points of the Doñana National Park (Spain) in 1999 to 2000. The highest exposure through direct soil ingestion to Pb and other studied elements was observed in samples from Entremuros, the area of the Doñana Natural Park affected by the Aznalcóllar mine spill in 1998. Birds from Entremuros also more frequently showed mononuclear infiltrates in liver and kidney than birds from the unaffected areas, although other more specific lesions of Pb or Zn poisoning were not observed. The excretion of coproporphyrins, especially of the isomer I, was positively related to the fecal As concentration, and the ratio of coproporphyrin III/I was positively related to fecal Pb concentration. Biliary protoporphyrin IX concentration was also slightly related to hepatic Pb concentration. This study reflects biological effects on terrestrial animals by the mining pollution in Doñana that can be monitored with the simple noninvasive sampling of feces.

Isolation and detection of Campylobacter jejuni from chicken fecal samples by immunomagnetic separation–PCR

Campylobacter jejuni (C. jejuni) is one of the leading causes of bacterial food-borne disease worldwide. The presence of Campylobacter in chicken feces poses a high risk for contamination of chicken meat and for Campylobacter infections in human. Detection of this bacterium in chicken fecal specimens before slaughter is therefore vital to prevent disease transmission. By combining two techniques – immunomagnetic separation (IMS) and polymerase chain reaction (PCR), this study developed a reliable and specific method for rapid detection of C. jejuni in chicken fecal samples. The specificity of the assay was assured by two selection steps: 1) Dynabeads®M-270 Amine microbeads (2.8 µm in diameter) coated with C. jejuni monoclonal antibodies were used as the primary selection to isolate bacteria from fecal samples. 2) A PCR assay amplifying the Hippuricase gene was performed as the specific selection to accurately confirm the presence of C. jejuni. Without pre-enrichment, this method was able to detect approximately 10 CFU of C. jejuni in 1 µl of spiked feces within 3 h.

Effect of isomalt consumption on faecal microflora and colonic metabolism in healthy volunteers

Due to its low digestibility in the small intestine, a major fraction of the polyol isomalt reaches the colon. However, little is known about effects on the intestinal microflora. During two 4-week periods in a double-blind, placebo-controlled, cross-over design, nineteen healthy volunteers consumed a controlled basal diet enriched with either 30 g isomalt or 30 g sucrose daily. Stools were collected at the end of each test phase and various microbiological and luminal markers were analysed. Fermentation characteristics of isomalt were also investigated in vitro. Microbiological analyses of faecal samples indicated a shift of the gut flora towards an increase of bifidobacteria following consumption of the isomalt diet compared with the sucrose diet (P

Seroepidemiology of norovirus-associated travelers' diarrhea

Background: Noroviruses (NoVs) are the most common cause of epidemic gastroenteritis, responsible for at least 50% of all gastroenteritis outbreaks worldwide and were recently identified as a leading cause of travelers' diarrhea (TD) in US and European travelers to Mexico, Guatemala, and India.

Methods: Serum and diarrheic stool samples were collected from 75 US student travelers to Cuernavaca, Mexico, who developed TD. NoV RNA was detected in acute diarrheic stool samples using reverse transcription-polymerase chain reaction (RT-PCR). Serology assays were performed using GI.1 Norwalk virus (NV) and GII.4 Houston virus (HOV) virus-like particles (VLPs) to measure serum levels of immunoglobulin A (IgA) and IgG by dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA); serum IgM was measured by capture enzyme-linked immunosorbent assay (ELISA), and the 50% antibody-blocking titer (BT50 ) was determined by a carbohydrate-blocking assay.

Results: NoV infection was identified in 12 (16%; 9 GI-NoV and 3 GII-NoV) of 75 travelers by either RT-PCR or fourfold or more rise in antibody titer. Significantly more individuals had detectable preexisting IgA antibodies against HOV (62/75, 83%) than against NV (49/75, 65%) (p = 0.025) VLPs. A significant difference was observed between NV- and HOV-specific preexisting IgA antibody levels (p = 0.0037), IgG (p = 0.003), and BT50 (p = <0.0001). None of the NoV-infected TD travelers had BT50  > 200, a level that has been described previously as a possible correlate of protection.

Conclusion: We found that GI-NoVs are commonly associated with TD cases identified in US adults traveling to Mexico, and seroprevalence rates and geometric mean antibody levels to a GI-NoV were lower than to a GII-NoV strain.

Effects of orally administered Bdellovibrio bacteriovorus on the well-being and Salmonella colonization of young chicks

Bdellovibrio bacteriovorus is a bacterium which preys upon and kills Gram-negative bacteria, including the zoonotic pathogens Escherichia coli and Salmonella. Bdellovibrio has potential as a biocontrol agent, but no reports of it being tested in living animals have been published, and no data on whether Bdellovibrio might spread between animals are available. In this study, we tried to fill this knowledge gap, using B. bacteriovorus HD100 doses in poultry with a normal gut microbiota or predosed with a colonizing Salmonella strain. In both cases, Bdellovibrio was dosed orally along with antacids. After dosing non-Salmonella-infected birds with Bdellovibrio, we measured the health and well-being of the birds and any changes in their gut pathology and culturable microbiota, finding that although a Bdellovibrio dose at 2 days of age altered the overall diversity of the natural gut microbiota in 28-day-old birds, there were no adverse effects on their growth and well-being. Drinking water and fecal matter from the pens in which the birds were housed as groups showed no contamination by Bdellovibrio after dosing. Predatory Bdellovibrio orally administered to birds that had been predosed with a gut-colonizing Salmonella enterica serovar Enteritidis phage type 4 strain (an important zoonotic pathogen) significantly reduced Salmonella numbers in bird gut cecal contents and reduced abnormal cecal morphology, indicating reduced cecal inflammation, compared to the ceca of the untreated controls or a nonpredatory ΔpilA strain, suggesting that these effects were due to predatory action. This work is a first step to applying Bdellovibrio therapeutically for other animal, and possibly human, infections.

High prevalence of Schistosoma japonicum and Fasciola gigantica in bovines from Northern Samar, the Philippines

The cause of zoonotic schistosomiasis in the Philippines is Schistosoma japonicum, which infects up to 46 mammalian hosts, including humans and bovines. In China, water buffaloes have been identified as major reservoir hosts for schistosomiasis japonica, contributing up to 75% of human transmission. In the Philippines, water buffaloes (carabao; Bubalus bubalis carabanesis) have, historically, been considered unimportant reservoirs. We therefore revisited the possible role of bovines in schistosome transmission in the Philippines, using the recently described formalin-ethyl acetate sedimentation (FEA-SD) technique and a qPCR assay to examine fecal samples from 153 bovines (both carabao and cattle) from six barangays in Northern Samar. A high prevalence of S. japonicum was found using qPCR and FEA-SD in both cattle (87.50% and 77.08%, respectively) and carabao (80.00% and 55.24%, respectively). The average daily egg output for each bovine was calculated at 195,000. High prevalence and infection intensity of F. gigantica was also found in the bovines by qPCR and FEA-SD (95.33% and 96.00%, respectively). The identification of bovines as major reservoir hosts for S. japonicum transmission suggests that bovine treatment and/or vaccination, as one becomes available, should be included in any future control program that aims to reduce the disease burden due to schistosomiasis in the Philippines.