Fernando Bernardo

Co-occurrence of patulin and citrinin in Portuguese apples with rotten spots

Patulin and citrinin are mycotoxins produced by certain fungi mainly belonging toPenicilliumandAspergillusand may be detected in mouldy fruits and fruit products. The data presented here refer to the simultaneous occurrence of patulin and citrinin in 351 samples of seven different varieties of apples with small rotten areas (Casanova, Golden Delicious, Red Delicious, Reineta, Richared, Rome Beauty, Starking). A rapid multidetection thin layer chromatography (TLC) method was used. The minimum detectable concentrations of patulin and citrinin were 120–130 and 15–20 µg kg-1 respectively. The percentage contamination with patulin only was higher (68.6%) than that with citrinin only (3.9%). Patulin and citrinin (19.6%) were also detected simultaneously. The highest mean patulin content was 80.50 mg kg-1 for the Richared variety, but the mean level of citrinin was lower. The lowest mean contaminations of patulin were found in Rome Beauty, Red Delicious and Reineta, ranging from 3.06 to 5.37 mg kg-1. All analysed apples varieties had low citrinin contamination, ranging from 0.32 to 0.92 mg kg-1. These findings indicate that there may be a risk of human exposure to patulin through the consumption of juices and jams manufactured with apples with small rotten areas.

Aflatoxins in spices marketed in Portugal

Seventy-nine prepackaged samples of 12 different types of spice powders (five cardamom, five cayenne pepper, eight chilli, five cloves, seven cumin, five curry powder, five ginger, five mustard, 10 nutmeg, 12 paprika, five saffron and seven white pepper) were selected from supermarkets and ethnic shops in Lisbon (Portugal) for estimation of aflatoxins by immmunoaffinity column clean-up followed by HPLC. Aflatoxin B (AFB1) was detected in 34 samples of prepackaged spices (43.0%). All of the cayenne pepper samples were contaminated with levels ranging from 2 to 32 µg AFB1/ kg. Three nutmeg samples contained levels ranging from 1 to 5 µg/kg, three samples had levels ranging from 6 to 20 µ g/kg, and there were two with 54 µg/kg and 58 µg/ kg. Paprika contained levels of aflatoxin B1 ranging from 1 to 20 µg/kg. Chilli, cumin, curry powder, saffron and white pepper samples had levels ranging from 1 to 5 µg/kg. Aflatoxins were not detected in cardamom, cloves, ginger and mustard. None of the samples analysed contained aflatoxins B2, G1 and G2.

Evaluation of microbiological quality of medicinal plants used in natural infusions.

Consumption of preparations of medicinal plants has been increasing during the last decades in occidental societies. However, there are no effective sanitary controls of these products. To evaluate the nature and content of microbiological contamination, 62 samples of seven medicinal plants (chamomile, leaves of orange tree, flowers of linden, corn silk, marine alga, pennyroyal mint and garden sage) were studied, using conventional microbiological methods. Practically all samples (96.8%) were contaminated with Bacillus cereus; 19.2% of them with levels higher than 10(3) spores/g. The highest levels were found in corn silk samples (> 10(7) spores/g). Spores of Clostridium perfringens were detected in 83.9% of samples, but only 19.2% had levels greater than 10(3)/g. The mean level of fungal population was 10(5.5) cfu/g. Corn silk samples were the most contaminated, with levels above 10(6) cfu/g. Fusarium spp., Penicillium spp., Aspergillus flavus and Asp. niger were predominant in all samples with the exception of garden sage samples. Many yeasts were found in chamomile, flowers of linden, corn silk, pennyroyal mint and garden sage. Predominant species were Cryptococcus laurentii (28.1%) and Rhodotorula mucilaginosa (22.8%). The mean level of Crypt. laurentii contamination in corn silk was greater than 10(4) cfu/g.

Fumonisins B1 and B2 in black tea and medicinal plants

Fumonisins are mycotoxins produced by Fusarium moniliforme that are prevalent in cereals and other agricultural products. These mycotoxins have been pointed to as a natural cause of equine leukoencephalomalacia, porcine pulmonary edema, and human esophageal cancer. A total of 87 samples, 18 black tea samples and 69 samples of four different medicinal plants (chamomile, leaves of the orange tree, leaves and flowers of the linden tree, and corn silk), for infusions preparations were acquired from supermarkets in Lisbon, Portugal. The samples were analyzed for the incidence and levels of fumonisin B1 (FB1) and fumonisin B2 (FB2) by high-performance liquid chromatography. The detection limit was 20 microg/kg for both FB1 and FB2. FB1 was detected in 55 (65.5%) of the 87 samples. The highest number of positive samples was found in black tea (88.8%). with levels ranging from 80 to 280 microg/kg. Relative to the medicinal plants, the leaves of the orange tree had higher concentrations of FB1 (range, 350 to 700 microg/kg) followed by leaves and flowers of the linden tree (range, 20 to 200 microg/kg). The samples of corn silk and chamomile had less contamination of FB1, with concentrations ranging from 50 to 150 microg/kg and 20 to 70 microg/kg, respectively. None of the samples tested had contamination of FB2. This is the first report of the natural occurrence of fumonisins in black tea and medicinal plants in Portugal. We reinforce the necessity to implement risk management measures for safety control of this kind of product.

Presencia de aflatoxina B1 en piensos para ganado lechero en Portugal durante el periodo 1995-2004

In Portugal, there is rather little information about the natural occurrence of aflatoxin in feedstuffs. The aim of this work was to report the results of screening the natural incidence of aflatoxin B1 (AFB1) in samples of cattle feed collected from seven dairy cows farms from Portugal distributed in several locations of the country. One thousand and one samples were taken from 1995 to 2004. High performance liquid chromatography (HPLC) was used for separation, identification and quantification of the compound. Detection limit was 1 microg/kg. Aflatoxin B1 was detected in 374 (37.4%) of the samples. The incidence and mean content of AFB1 was generally low. Levels of aflatoxin B1 above the maximum limit established in Portugal (5 microg/kg) for dairy cattle feed samples were observed in 62 samples (6.2%) with levels ranging from 5.1 to 74 microg/kg. Out of those 62 samples, 3.7% had levels between 5.1 to 10 (mean 7.8); 1.8% had a contamination level of 10.1 to 20 (mean 12.0), and 0.7% exceeded 20.1 microg/kg (mean 50.4). On the last two years (2003-04) none of the samples exceeded the maximum permissible level of the toxin.

Effects of antimicrobial treatment on selection of resistant Escherichia coli in broiler fecal flora.

Under field conditions, three commercial antimicrobials were sequentially prescribed to 16,000 broiler chickens during their rearing period, via drinking water using subtherapeutic levels for 3 days. A control group of 16,000 broilers was placed in the same controlled environment poultry house. Feed diet and fecal samples from both groups were collected periodically. One sample of the drinking water along with samples from the broiler house environment was also collected 1 day before bird placement. Samples were plated onto Tergitol BCIG Agar media; a maximum of 26 Escherichia coli were isolated per sample, and their susceptibility was tested to 12 antimicrobials by disk diffusion agar method. We have observed that day-old chicks were rapidly colonized by new antibiotic-resistant patterns shortly after treatment with lincomycin associated with spectinomycin. After medication with the second (sulfadiazine with trimethoprim) and third (tylosin) antimicrobials, a more radical displacement was observed, and, concurrently, antimicrobial resistance phenotypes have become more complex. In contrast, more than 70% of the strains isolated in control group during the experiment displayed exactly the same resistance pattern found in the day-old chicks. This study provides clear evidence that a sequential medication of a broiler flock, with different antimicrobial classes during short periods of time for prophylactic objectives, was accompanied by a dramatic increase in both antimicrobial resistance rates and phenotype diversity of E. coli strains.

Antibiotic Resistance of Enterococcus spp. Isolated from Wastewater and Sludge of Poultry Slaughterhouses

Antibiotimicrobial resistance was investigated in 537 Enterococcus spp. isolates recovered from 22 samples of crude inflow, treated effluent and sludge collected in wastewater treatment plants of eight poultry slaughterhouses of Portugal. No significant differences (P > 0.05) were found in the resistance to each antimicrobial agent with regards to the origin of the sample (inflow, sludge and effluent). Many of the isolates displayed resistance to tetracycline (85.7%), erythromycin (45.7%), nitrofurantoin (34.0%) and rifampicin (17.8%). Resistance was also observed, but to a lesser extent, to ciprofloxacin (10.2%), ampicillin (8.0%), chloramphenicol (4.6%), vancomycin (0.9%) and gentamicin (0.4%). Resistance to three or more antimicrobial classes was present in 37.1% of the isolates. Wastewater treatment resulted in viable enterococci decrease between less than 1 log and 4 log; nevertheless, more than 4.4 × 105 colony forming units (CFU)/100 mL were present in the outflow of the plants and thus resistant enterococci are not prevented from reaching the general environment.

Field trial evaluating changes in prevalence and patterns of antimicrobial resistance among Escherichia coli and Enterococcus spp. isolated from growing broilers medicated with enrofloxacin, apramycin and amoxicillin.

The present study investigates, under field conditions, the influence of antimicrobial administration on prevalence and patterns of antimicrobial resistance among Escherichia coli and Enterococcus spp. isolated from growing broilers. For this purpose, a group of 16,000 commercial broiler chickens was treated with enrofloxacin from day 1 to day 3, gentamicin from day 19 to day 21, and ampicillin from day 26 to day 28. A control group of 16,000 broilers was placed in the same controlled environment poultry house. Fecal (from both groups) and feed samples were collected at regular intervals. Few E. coli isolates were obtained from either farm environment or poultry feed samples, while enterococci were found to be ubiquitous among these samples. The frequency of resistance against most antimicrobials tested was significantly higher (P<0.05) in E. coli isolated from broilers receiving intermittent antimicrobial pressure than that from non-medicated broilers, whereas in enterococci these differences were only observed among structurally related antimicrobial drugs and over a short period of time. By the time the broilers reached market age (33 days), several multi-resistant E. coli and enterococci were detected in the feces of the medicated group. Results suggest that antimicrobial resistance in E. coli was mainly medication-dependent, whereas among enterococci, changes observed over time were apparently influenced by factors apart from antimicrobial exposure, namely the resistance organisms previously present in farm environment and those present in feedstuffs.

Mycotoxins in feedstuffs in Portugal: an overview

Mycotoxins are secondary metabolites produced by many genera of fungi in many commodities, under certain conditions. Mycotoxicological control of feed is a procedure that aims to protect human and animal health, avoiding the adverse effects of these undesirable substances. This component of the sanitary control of feed and food is essential to prevent the presence of those substances which can seriously affect the health of the animals. In Portugal, there is relatively few information related to the natural occurrence of mycotoxins in feed. In this context, the authors present results and data compilation concerning the occurrence of mycotoxins in raw materials and also feed for dairy cattle, swine, poultry, horses, fish, laboratory rats and pet; making a generic qualitative appreciation of the risks associated to the presence of mycotoxins in these feedstuffs. The mycotoxins studied: aflatoxins (AFs), ochratoxin A (OTA), fumonisin B1 and B2 (FB1, FB2) were analysed by High Performance Liquid Chromatoghraphy (HPLC). Deoxynivalenol (DON) and zearalenone (ZEN) were determined by Thin-Layer Chromatography (TLC). The results suggest that contaminations with these mycotoxins in feed are quite common, revealing the need for surveillance and monitoring programs for the prevention of the sanitary impacts of these “non desirable substances”.

A six year survey (1999-2004) of the ocurrence of aflatoxin M1 in daily products produced in Portugal.

Aflatoxin M1, a mutagenic and carcinogenic metabolite of aflatoxin B1, occurs in milk from animals fed on food contaminated with some species ofAspergillus. Aiming to investigate the occurrence of AFM in dairy products produced in Portugal. 598 samples of raw milk were analysed during six years (1999–2004). 25 samples of powder milk and 42 traditional fresh cheeses were also analysed. The toxin was extracted using an immunoaffinity column method and quantified by HPLC. AFM1 was detected in 394 (65.8%) of the raw milk samples. Along the analysed period AFM: was detected at a low level (0.005–0.05μg/l) in 54.8% of the samples and at a level ranging from 0.041–0.05 in 2.8% of the samples. From 2001 to 2004, 49 samples (8.2%) were contaminated with levels above the maximum permitted level (>0.051 to 0.08). None of the samples of powder milk and traditional fresh cheese revealed to be contaminated with AFM1.