Salmonella spp. in Pet Reptiles in Portugal: Prevalence and Chlorhexidine Gluconate Antimicrobial Efficacy.

A fraction of human Salmonella infections is associated with direct contact with reptiles, yet the number of reptile-associated Salmonellosis cases are believed to be underestimated. Existing data on Salmonella spp. transmission by reptiles in Portugal is extremely scarce. The aim of the present work was to evaluate the prevalence of Salmonella spp. in pet reptiles (snakes, turtles, and lizards), as well as evaluate the isolates' antimicrobial resistance and virulence profiles, including their ability to form biofilm in the air-liquid interface. Additionally, the antimicrobial effect of chlorhexidine gluconate on the isolates was tested. Salmonella was isolated in 41% of the animals sampled and isolates revealed low levels of antimicrobial resistance. Hemolytic and lypolytic phenotypes were detected in all isolates. The majority (90.63%) of the Salmonella isolates were positive for the formation of pellicle in the air-liquid interface. Results indicate chlorhexidine gluconate is an effective antimicrobial agent, against the isolates in both their planktonic and biofilm forms, demonstrating a bactericidal effect in 84.37% of the Salmonella isolates. This study highlights the possible role of pet reptiles in the transmission of non-typhoidal Salmonella to humans, a serious and increasingly relevant route of exposure in the Salmonella public health framework.

Tinea corporis by Microsporum audouinii in a female chimpanzee (Pan troglodytes) from Guinea-Bissau: A case report.

We report a Microsporum audouinii infection in a female juvenile chimpanzee (Pan troglodytes) presenting generalized dermatitis compatible with dermatophytosis. Dermatophyte was identified by macro- and microscopic characterization of skin and scales cultures in Mycosel Agar. The topical treatment applied was effective, having the potential for dermatophytosis treatment in immunocompetent primates.

Phenotypic and Molecular Characterization of Salmonella 1,4,[5],12:i:- R-Type ASSuT Isolates from Humans, Animals, and Environment in Portugal, 2006-2011.

The increase in prevalence of Salmonella 1,4,[5],12:i:- related infections over the last few years has been considered a public health issue in many European countries, especially as this serovar may be associated with tetraresistance to ampicillin, streptomycin, sulfonamides, and tetracyclines (R-type ASSuT). Salmonella 1,4,[5],12:i:- isolates (n = 187) obtained by the Portuguese National Laboratory from different sources, including human clinical cases (n = 170), veterinary (n = 10), environmental (n = 6), and food samples (n = 1), were collected from 15 districts between 2006 and 2011. All isolates were serotyped using the slide agglutination method and results were confirmed by multiplex PCR for the monophasic variant. From the confirmed Salmonella 1,4,[5],12:i:-, R-type ASSuT isolates were selected by disc diffusion and minimal inhibitory concentration (MIC) determination for further characterization by pulsed-field gel electrophoresis restriction with XbaI, virulence genes determination by PCR, additional antimicrobial resistance profiling by disc diffusion, and epidemiological distribution evaluation. Out of the 187 serotyped isolates, 133 were confirmed as Salmonella 1,4,[5],12:i:- with a R-type ASSuT occurrence of 61.7%. Distribution among Portuguese districts showed a higher percentage of reported cases in coastal areas, in particular, in Porto (24.8%), Setúbal (13.5%), and Aveiro (12.8%), probably due to the higher population density. Clonality analysis revealed a high diversity of pulsotypes with the majority of human salmonellosis cases being attributed to sporadic events. All isolates harbored 14 out of the 18 virulence genes evaluated and 87.8% of the isolates showed all the resistance genes frequently associated with the European clone, blaTEM+sul2+straA-straB+tetB+. This study shows that Salmonella 1,4,[5],12:i:- resistant isolates are widely distributed in Portugal. This may be related to a selective advantage offered by R-type ASSuT profile, the presence of multiple virulent features, including the ability to form biofilms, which along with a high diversity of pulsotypes may be responsible for the dissemination through the country.

Fecal contamination of wastewater treatment plants in Portugal.

Reutilization of effluents from wastewater treatment plants (WWTP) for non-potable applications is increasing due to the reduction of sustainable water resources. These products mostly come from municipal WWTP and also from slaughterhouses effluents. The microbiological certification of these products is mandatory before their discharge into the environment. This study evaluates if the treatment applied in WWTP to municipal waters or to poultry slaughterhouse effluents distributed over the Portuguese continental territory is efficient in reducing the microbiological risk associated with the reutilization of those wastewaters and sludges. Fecal indicators Escherichia coli and enterococci were evaluated in 42 and 24 wastewater samples from 14 municipal WWTP and 8 poultry slaughterhouse treatment plants, respectively, by the conventional culture method and a rapid Fluorescent in situ hybridization (FISH) technique. Bacterial enumeration in inflow water from most WWTP was rather high (generally >10(5) cells/ml), for both E. coli and Enterococcus spp., and the bacterial quantification by FISH was generally higher than enumeration by the conventional culture method. In both types of treatment plants studied, bacterial load from effluents and sludges was not statistically different from the inflows, indicating that the treatment applied seems to be equally unable to reduce the microbiological load of the effluents. These findings may jeopardize the safe reuse of treated wastewaters in agriculture and the quality of the water environment. Therefore, products like water, sewage sludge, and biosolids originated from the municipal and slaughterhouse WWTP studied should not be reutilized, and effluents treatment should be urgently reviewed.

Antimicrobial susceptibility of Salmonella enterica isolates from healthy breeder and broiler flocks in Portugal.

Three hundred and thirty-three isolates representing 40 different serotypes of Salmonella enterica, recovered from environmental and faecal samples of breeder and broiler flocks from 2009 to 2011, were studied. Antimicrobial susceptibility was determined by measuring the minimal inhibitory concentration of 11 antimicrobials using the agar dilution method. Salmonella Havana, S. Enteritidis and S. Mbandaka were the most common serotypes isolated from broiler flocks, while S. Enteritidis was the common isolate from breeder flocks. The frequency of non-wild-type Salmonella isolates (those with decreased susceptibility to the different antimicrobials) varied according to serotype. S. Mbandaka in broilers and S. Enteritidis in both breeders and broilers showed higher frequencies of reduced susceptibility to quinolones, but clinical resistance towards ciprofloxacin was not observed. Reduced susceptibility to sulfamethoxazole, tetracycline, ampicillin and streptomycin were common in Salmonella Typhimurium isolates. Two isolates of S. Havana from broilers were resistant to cefotaxime and phenotypically categorised as extended-spectrum ß-lactamase producers. The results presented in this study provide useful data on the antimicrobial susceptibility of different Salmonella serotypes and highlight the high diversity of multi-drug resistance patterns present.

Biofilm formation by Salmonella enterica serovar 1,4,[5],12:i:- Portuguese isolates: a phenotypic, genotypic, and socio-geographic analysis.

Biofilm-forming ability is well established as an important virulence factor. However, there are no studies available regarding biofilm formation of Salmonella Typhimurium 1,4,[5],12:i:-, the new pandemic serovar in Europe. To address this problem, biofilm expression by Salmonella 1,4,[5],12:i:- was evaluated using 133 isolates from clinical, environmental and animal origins, collected in Portugal from 2006 to 2011. Biofilm detection was performed by phenotypic and genotypic methods, such growth characterization in agar and broth medium, optical density determination by microtiter assays and direct observation by fluorescent in situ hybridization. Biofilm-related genes adrA, csgD and gcpA were detected by PCR. A socio-geographic characterization of strains as biofilm producers was also performed. Results showed that biofilm formation in monophasic Salmonella is widely distributed in Portuguese isolates and could be one of the reasons for its dissemination in this country. Biofilm expression varies between locations, showing that isolates from some regions like Lisboa or Ponta Delgada have an increased ability to persist in the environment due to an enhanced biofilm production. Biofilm formation also varies between risk groups, with a higher prevalence in isolates from salmonellosis infections in women. Therefore, the analysis of the socio-geographic distribution of biofilm-forming bacteria should be considered for the establishment of more adequate regulatory measures or therapeutics regimens, especially important due to the continuous increase of infections caused by antimicrobial resistant microorganisms.

Aflatoxin B1 and zearalenone in dairy feeds in Portugal, 2009-2011.

This paper presents 3 years of data (2009-2011) on the occurrence of two mycotoxins, aflatoxin B1 (AFB1) and zearalenone (ZEA), in samples of feedstuff for dairy cows (n = 963), ewes (n = 42), and goats (n = 131) produced in Portugal. AFB1 was found in 15 samples of cow feed (1.6%), 3 samples of ewe feed (2.3%) and in 2 samples of goat feed (4.8%). All but two samples contained AFB1 at levels below the European Union maximum level (5 µg/kg). Nearly half (45%) of the samples were contaminated with ZEA, but its levels were relatively low, at 5-136.9 µg/kg, well below the European Union guidance value (500 µg/kg).

Incidencia de fumonisinas en piensos para cerdos y caballos / Occurrence of fumonisins in feed for swine and horses

Antecedentes. La contaminación por fumonisina B1 (FB1) y B2 (FB2) y por el conjunto de micotoxinas en alimentos y piensos puede causar efectos diversos sobre los productos agrícolas y la salud de los animales. La contaminación afectó de modo predominante al maíz y a los alimentos y piensos derivados. Objetivos. En este estudio, efectuada en Portugal, se examinó la incidencia de fumonisinas en muestras de pienso mixto para cerdos y para caballos. El período de la revisión abarcó de 2007 a 2010. Métodos. Mediante cromatografía líquida de alta resolución (HPLC) se analizaron 363 muestras, 258 de pienso porcino y 105 de pienso equino. El límite de detección fue de 50μg/kg para FB1 y de 100μg/kg para FB2. Resultados. Los resultados globales fueron una incidencia de contaminación por fumonisina B1 (FB1) del 13% desde 2007 a 2010. Se detectó FB1 en alrededor del 17% de las muestras de pienso porcino, siendo más frecuente en 2010 (32,9%). En dicho año, los niveles variaron entre 66,7 y 3.815,5μg/kg. En 2010 sólo se detectó contaminación por fumonisina B2 en muestras de pienso para cerdo (seis muestras, con una variación de 104,0 a 467,2μg/kg) y en muestras de pienso para caballos (una muestra). Conclusiones. Aunque los resultados de este estudio indican un aumento de la incidencia durante los años examinados, no constituye un riesgo para la salud de los animales, ya que los valores no superan los recomendados por las directrices de la Comisión Europea(AU)

Background. Fumonisin B1 (FB1), fumonisin B2 (FB2), and overall mycotoxins feed contamination may cause several effects on crops production and animal health. The contamination occurred predominantly in corn and corn-based foods and feeds. Aims. This survey intends to provide the occurrence of fumonisins in swine and equine mixed feeds in Portugal, making an overview from 2007 to 2010. Methods. A total of 363 samples were analyzed, 258 from swine feed and 105 from horse feed with HPLC method. The detection limit was 50μg/kg for FB1 and 100μg/kg for FB2. Results. The overall results were 13% of FB1 occurrence from 2007 to 2010. FB1 was detected in about 17.0% of swine feed samples, being more frequent in 2010 (32.9%). In this year (2010) levels ranged between 66.7 and 3815.5μg/kg. FB2 occurred only in 2010 in swine feed (6 samples, ranging between 104.0 to 467.2μg/kg) and in horse feed (1 sample). Conclusions. This represents an increase in occurrence through the analyzed years, but this may not be a threat to animal health, once the values were below the recommended guidance values from European Commission(AU)

Occurrence of fumonisins in feed for swine and horses.

BACKGROUND: Fumonisin B1 (FB1), fumonisin B2 (FB2), and overall mycotoxins feed contamination may cause several effects on crops production and animal health. The contamination occurred predominantly in corn and corn-based foods and feeds. AIMS: This survey intends to provide the occurrence of fumonisins in swine and equine mixed feeds in Portugal, making an overview from 2007 to 2010. METHODS: A total of 363 samples were analyzed, 258 from swine feed and 105 from horse feed with HPLC method. The detection limit was 50 µg/kg for FB1 and 100 µg/kg for FB2. RESULTS: The overall results were 13% of FB1 occurrence from 2007 to 2010. FB1 was detected in about 17.0% of swine feed samples, being more frequent in 2010 (32.9%). In this year (2010) levels ranged between 66.7 and 3815.5 µg/kg. FB2 occurred only in 2010 in swine feed (6 samples, ranging between 104.0 to 467.2 µg/kg) and in horse feed (1 sample). CONCLUSIONS: This represents an increase in occurrence through the analyzed years, but this may not be a threat to animal health, once the values were below the recommended guidance values from European Commission.

A survey on the occurrence of ochratoxin A in feeds for swine and laying hens.

Results of a 2-year (2009-2010) survey on the occurrence of ochratoxin A (OTA) in swine feed and in feed for laying hens in Portugal are reported. A total of 664 samples (478 swine feed, 186 feed for laying hens) were analyzed by a HPLC method using fluorescence detection with 2 µg kg(-1) as detection limit. In swine feed, 31 samples (6.49%) were positive for OTA. In feed for laying hens, 12 samples (6.45%) were OTA-positive. The average levels of contamination were low, with median values of positive samples at 3-4 µg kg(-1) in both years and both commodities, although a few samples contained exceptionally high levels (maximum 130 µg kg(-1)). Only the maximum level sample (swine feed) contained OTA at a concentration exceeding the European Commission guidance value. The remaining OTA concentrations found in feed samples were much lower than the guidance values.

Mycobiota and aflatoxin B1 in feed for farmed sea bass (Dicentrarchus labrax).

Thesafety characteristics of feed used in fish and crustacean aquaculture systems are an essential tool to assure the productivity of those animal exploitations. Safety of feed may be affected by different hazards, including biological and chemical groups. The aim of this preliminary study was to evaluate fungi contamination and the presence of aflatoxins in 87 samples of feed for sea bass, collected in Portugal. Molds were found in 35 samples (40.2%) in levels ranging from 1 to 3.3 log10 CFU∙g(-1). Six genera of molds were found. Aspergillus flavus was the most frequent, found in all positive samples, with a range from 2 to 3.2 log(10) CFU∙g(-1). Aspergillus niger was found in 34 samples (39.1%), ranging from 1 to 2.7 log(10) CFU∙g(-1). Aspergillus glaucus was found in 26 samples (29.9%) with levels between 1 and 2.4 log(10) CFU∙g(-1). Penicillium spp. and Cladosporium spp. were both found in 25 samples (28.7%). Fusarium spp. was found in 22 samples (25.3%), ranging from 1 to 2.3 log(10) CFU∙g(-1). All feed samples were screened for aflatoxins using a HPLC technique, with a detection limit of 1.0 µg∙kg(-1). All samples were aflatoxin negative.

Co-occurrence of mycotoxins in swine feed produced in Portugal.

A total of 404 samples of commercial swine feed from Portugal feed mills were analysed by HPLC methods for the presence of mycotoxins: 277 samples of feed for fattening pigs were analysed for ochratoxin A (OTA), zearalenone (ZEA), and deoxynivalenol (DON), and 127 samples of feed for sows were analysed for ZEA and fumonisins (FB1 + FB2). Concerning feed for fattening pigs, 21 (7.6%) samples were positive for OTA, (2-6.8 µg/kg), 69 (24.9%) were positive for ZEA (5-73 µg/kg), and 47 (16.9%) were positive for DON (100-864 µg/kg). In feed for sows, the results showed 29.9% of positive samples for ZEA (5-57.7 µg/kg) and 8.7% positive samples for FB1 and FB2 (50-391.4 µg/kg). Co-occurrence of DON/ZEA was found most frequently, but simultaneous contamination with OTA/ZEA and OTA/DON was also found.

Mycobiota and Ochratoxin A in laboratory mice feed: preliminary study.

The occurrence of mycotoxin-producing moulds in animal feed is a hazard for animals. When these undesirable substances contaminate laboratory animal feed, convey an additional problem in experimental animal assays confidence levels. The aim of this study was to evaluate fungal contamination and to determine natural occurrence of Ochratoxin A (OTA) in 31 samples. OTA is a mycotoxin produced by fungi of two genera: Penicillium and Aspergillus. OTA has been shown to be nephrotoxic, hepatotoxic, teratogenic and immunotoxic to a number of animal species and to cause kidney and liver tumors in mice and rats. In this preliminary study, feed mould counts ranged from 3 to 4.2 log(10) cfu/g (colonies forming units per gram). When these species are present, there is a significant risk of contamination with mycotoxins resulting in both acute diseases called mycotoxicoses and chronic conditions, often recognized as situations involving mycotoxins. The most frequent genus isolated was Cladosporium sp. (84%), followed by Aspergillus niger and Penicillium (81%) and Mucor sp. (77%). All rat feed samples were examined for OTA, using High Performance Liquid Chromatography (HPLC). The detection limit was 2.0 microg OTA kg(-1) and all samples revealed to be negative for this mycotoxin. These mycotoxicological researches put in evidence the importance of the use contaminant-free experimental animal feed in order to prevent any interference on the health of experimental animals and emphasizes the need for systematic control of the feed as a key issue in animal experimentation.

Changes in antimicrobial resistance among faecal enterococci isolated from growing broilers prophylactically medicated with three commercial antimicrobials.

A controlled environment poultry house (1600m(2)) was divided into two separated compartments. Three hundred twenty chick containers (each containing 100 chicks) were randomly allocated to each compartment. Antimicrobial products were provided (via drinking water) to one broiler group in three different growth phases for prophylactic purposes: lincomycin associated with spectinomycin from day 1 to day 3, trimethoprim associated with sulfamethoxazole from day 19 to day 21, and tylosin from day 26 to day 28. Feed and faecal samples from both groups were obtained on days 0, 4, 9, 14, 18, 22, 25, 29 and 33. One sample of the drinking water and samples from the broiler house environment were obtained one day before bird placement. A maximum of 16 enterococci isolated from Kanamycin Aesculin Azide Agar were tested for resistance using the disk diffusion method. A high prevalence of resistance to drugs such as tetracycline, erythromycin and nitrofurantoin was detected in enterococcal isolates from medicated and unmedicated flocks. Resistance occurrence in the non-medicated group revealed a shift in resistance profiles with the gradual change of the initial strains, isolated from the day-old chicks' cloacal swabs, by others displaying a different phenotype. Resistance phenotypes obtained from faecal strains were correlated with those isolated from the farm environment and feeds.

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.

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.

Antimicrobial resistance in Escherichia coli isolated in wastewater and sludge from poultry slaughterhouse wastewater plants.

The authors investigated the antimicrobial resistance of Escherichia coli isolates in 22 samples of crude inflow, treated effluent, and sludge collected at the wastewater treatment plants of eight poultry slaughterhouses in Portugal. A total of 549 E. coli strains were recovered and tested for resistance to 12 antimicrobial agents. Multidrug resistance was present in 55.7 percent of the isolates. Resistance to tetracycline, ampicillin, trimethoprim/sulfamethoxazole, streptomycin, and enrofloxacin was found in 80.7 percent, 56.5 percent, 47.5 percent, 39.2 percent, and 18.4 percent of the isolates, respectively. Resistance rates of E. coli to nearly all of the tested antibiotics were higher in the strains obtained from the six slaughterhouses that handled conventional broilers than in the two slaughterhouses that handled free-range broilers. Wastewater treatment resulted in an E. coli decrease of between 0.5 log and 3 log; nevertheless, an average of 5.2 x 10(5) CFUs/100 mL were present in the outflow of the plants. These data indicate that the use of antimicrobials in poultry production leads to the selection of a large pool of resistance genes and that wastewater treatment processes are unable to inactivate the bacteria and thus will result in dissemination of resistant E. coli into the environment.

A theoretical model for transdermal drug delivery from emulsions and its dependence upon formulation.

This article presents a theoretical model of transdermal drug delivery from an emulsion-type vehicle that addresses the vehicle heterogeneity and incorporates the prediction of drug transport parameters as function of the vehicle composition. The basic mass transfer model considers interfacial and diffusion resistances within the emulsion and partition/diffusion phenomena across two skin compartments in series. Drug transport parameters are predicted as follows: partition coefficients are derived from regular solutions theory, drug diffusivity in the continuous phase is computed from a free volume theory with segmental motion, and permeability of the surfactant layer around droplets is estimated based on a free surface area model. These relationships are incorporated within the basic mass transfer model, so that the overall model is able to predict temporal profiles of drug release from the vehicle and of drug concentration in plasma, as a function of vehicle composition. In this way, the proposed model provides a sound physicochemical basis to support the development of new formulations and the planning of experiments. A simulated case study regarding a nitroglycerin ointment is presented in detail, illustrating how thermodynamic and kinetic factors inherent to the emulsion vehicle can modulate drug release and subsequent systemic absorption.

Interaction of wild strains of Aspergilla with Aspergillus parasiticus ATCC15517 and aflatoxin production.

Aflatoxins are secondary metabolites produced by some competent mould strains of Aspergillus flavus, A. parasiticus and A. nomius. These compounds have been extensively studied with regards to their toxicity for animals and humans; they are able to induce liver cancer and may cause a wide range of adverse effects in living organisms. Aflatoxins are found as natural contaminants of food and feed; the main line of the strategy to control them is based on the prevention of the mould growth in raw vegetable or during its storage and monitoring of each crop batch. Mould growth is conditioned by many ecological factors, including biotic ones. Hazard characterization models for aflatoxins in crops must take into consideration biotic interactions between moulds and their potential effects on growth development. The aim of this work is to study the effect of the biotic interaction of 14 different wild strains of Aspergilla (different species), with a competent strain (Aspergillus parasiticus ATCC 15517) using an in vitro production model. The laboratory model used was a natural matrix (humidified cracked corn), on which each wild strain challenged the aflatoxin production of a producer strain. Cultures were incubated at 28 degrees C for 12 days and sampled at the 8(th) and 12(th). Aflatoxin detection and quantification was performed by HPLC using a procedure with a MRPL = 1 microg/kg. Results of those interactive cultures revealed both synergic and antagonistic effects on aflatoxin biosynthesis. Productivity increases were particularly evident on the 8(th) day of incubation with wild strains of A. flavipes (+ 70.4 %), A. versicolor (+ 54.9 %) and A. flavus 3 (+ 62.6 %). Antagonistic effects were found with A. niger (- 69.5%), A. fumigatus (- 47.6 %) and A. terreus (- 47.6 %) on the 12(th) day. The increased effects were more evident on the 8(th) of incubation and the decreases were more patent on the 12(th) day. Results show that the development of Aspergilla strains concomitantly with competent aflatoxin producing moulds has a significant influence on the natural biosynthesis pattern.

Occurrence of aflatoxin B1 in dairy cow feed over 10 years in Portugal (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 cow's 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.