Stjepan Pepeljnjak

Antimicrobial activity of Paliurus spina-christi Mill. (Christ's thorn)

Paliurus spina-christi Mill. (Rhamnaceae), also known as Christs thorn, is a perennial thorny shrub of widespread distribution in dry and rocky places in Mediterranean region and Asia. In Croatia, this plant grows along the Adriatic coast and on the islands. Christs thorn is used as a diuretic, against diarrhoea and rheumatism in traditional Croatian herbal medicine. Chemical investigations indicate the presence of the flavonoids isoquercitrin, rutin, hyperoside and quercetin-3-O-triglycoside. No literature could be found in respect of the antimicrobial activity of Paliurus spina-christi. The antibacterial effect of extracts from different plant parts was therefore investigated

Co-occurrence of aflatoxins, ochratoxin A, fumonisins, and zearalenone in cereals and feed, determined by competitive direct enzyme-linked immunosorbent assay and thin-layer chromatography.

Co-occurrence of Aflatoxins, Ochratoxin A, Fumonisins, and Zearalenone in Cereals and Feed, Determined by Competitive Direct Enzyme-Linked Immunosorbent Assay and Thin-Layer Chromatography Aspergillus, Penicillium, and Fusarium species frequently contaminate crops. For this reason mycotoxins such as aflatoxins (AFs), ochratoxin A (OTA), fumonisins (FBs), and zearalenone (ZEA) are found in food and feed in a wide range of concentrations, depending on environmental and storage conditions. Consumption of mycotoxin-contaminated food and feed has been associated with acute and chronic poisoning and carcinoma. The aim of this study was to determine the incidence and co-occurrence of AFs (B1+B2+G1+G2), OTA, FBs (B1+B2+B3), and ZEA in 37 samples of cereals and feed randomly collected in 2007 from households of an endemic nephropathy (EN) area in Croatia. The mycotoxins were determined using the competitive direct ELISA test (CD-ELISA) in combination with thin-layer chromatography (TLC). The most frequent mycotoxin was ZEA (92%, mean 318.3 μg kg-1), followed by FBs (27%, 3690 μg kg-1), AFs (24.3%, 4.6 μg kg-1), and OTA (16.2%, 9.8 μg kg-1). Levels of AFs, ZEA, and FBs detected by CD-ELISA significantly correlated with the TLC results. However, only one OTA-positive sample was confirmed by TLC due to its high limit of detection. The levels of these mycotoxins were below the permissible limit for animal feed. Twenty-nine percent of cereals were contaminated with FBs, OTA, or ZEA in mass fractions above the permissible limit for humans. Co-occurrence of two toxins varied between 4.2% and 54% and of three between 4.2% and 7.6%. Prolonged co-exposure to AFs, OTA, FBs, and ZEA might increase the risk of various chronic diseases. Određivanje aflatoksina, okratoksina A, fumonizina i zearalenona u žitaricama i krmivu primjenom kompetitivnoga direktnog imunoenzimatskog testa (CD-ELISA) i tankoslojne kromatografije (TLC) Vrste plijesni iz rodova Aspergillus, Penicillium i Fusarium česti su kontaminanti usjeva te na takvim supstratima tvore mikotoksine. Stoga su žitarice i krmiva često kontaminirana aflatoksinima (AFs), okratoksinom A (OTA), fumonizinima (FBs) i zearalenonom (ZEA) u različitim koncentracijama ovisno o mikroklimatskim uvjetima na polju i u skladištu. Konzumiranje hrane kontaminirane mikotoksinima često je povezano s akutnim ili kroničnim trovanjima, ali i s razvojem karcinoma. Cilj ovog rada bio je odrediti istodobnu pojavnost AFs (B1+B2+G1+G2), OTA, FBs (B1+B2+B3) i ZEA u uzorcima žitarica i krme (N=37) koji su nasumično skupljeni u individualnim domaćinstvima na području endemske nefropatije (EN) u Hrvatskoj (2007). Za određivanje navedenih mikotoksina korišten je kompetitivni direktni ELISA-test (CD-ELISA) u kombinaciji s tankoslojnom kromatografijom (TLC). Najzastupljeniji mikotoksin bio je ZEA (92 %, srednja koncentracija 318.3 μg kg-1), nakon čega slijede FBs (27 %, 3690 μg kg-1), AFs (24.3 %, 4.6 μg kg-1) te OTA (16.2 %, 9.8 μg kg-1). Koncentracije AFs, FBs i ZEA određene CD-ELISA-testom statistički značajno koreliraju s rezultatima dobivenim s TLC. OTA je potvrđen metodom TLC samo u jednom uzorku zbog visokog limita detekcije. Dokazane koncentracije su ispod razina dopuštenih za krmiva, dok je 29 % uzoraka žitarica sadržavalo FBs, OTA ili ZEA u koncentracijama iznad dopuštenih u hrani za ljude. Kokontaminacija s dvama odnosno trima toksinima varirala je između 4.2 % i 54 % odnosno između 4.2 % i 7.6 %. Dugotrajni unos AFs, OTA, FBs i ZEA putem hrane može povećati rizik od razvoja različitih kroničnih bolesti zbog njihova mogućega sinergističkog djelovanja.

Occurrence of fungi in air and on plants in vegetation of different climatic regions in Croatia

Research was carried out onthe presence and variation in occurrence offungal genera in air and on plant samplescollected from three different climatic regionsin Croatia in relation to temperature andrelative humidity throughout one year (1998).The sampling method employed in this study wasexposure of 150 Sabouraud agar plates withantibiotics to the air for 10 min. and byplacing 10 pieces of each plant sample (N = 150)on same agar. The spores belonging to 15 fungalgenera and sterile mycelim were identified.Cladosporium (65.3–74%),Penicillium (4.43–13.9%), Alternaria(2–4.7%), and sterile mycelium (7.4–15%) werethe most prevalent fungi in the air of all threeclimatic regions, while Fusarium(20.3–36.2%) was the most prevalent fungalgenera on plant samples especially in the modestcontinental climate. Significant differenceswere obtained for comparative fungalcontamination of air and plant samples, and bycomparing monthly occurrence in all three partsincluded. The climate factors and presence ofvegetation that produce essential oils withfungicide activity may be the reason for reducedfungal contamination of outdoor environments inthe Mediterranean climate.

Distribution and mycotoxin-producing ability of some fungal isolates from the air

Abstract Research was carried out on presence and prevalence of common fungal air spores at locations in Croatia. The sampling method employed in the study was by exposure 350 of Petri agar plates to the air for 10 min. Approximately 3400 colonies were found and mould spores belonging to 22 fungal genera were identified. Cladosporium (44.7%), Penicillium (34.4%), Alternaria (26.3%), Aspergillus (21.6%) and Absidia (12.2%) were the most prevalent fungi encountered. Investigation of toxigenic potential of airborne fungi isolates of genera Aspergillus, Fusarium and Trichoderma showed 16.9% mycotoxin-producing strains. The production of aflatoxin B 1 by A. flavus sterigmatocystin by A. versicolor zearalenon and T-2 toxin by F. graminearum and diacetoscirpenol by strains of T. viride were obtained.

Screening toxicity study in young carp (Cyprinus carpio L.) on feed amended with fumonisin B1

Fumonisin B1 (FB1) is one of several mycotoxins produced by Fusarium moniliforme, a major fungal pathogen of corn and widely spread throughout the world. FB1 produces a wide range of biological effects, some of which are specific for particular organs or species and some are common to all investigated animals. In this study we have evaluated subchronic toxicosis features in young carp (Cyprinus carpio L.) exposed to 0.5 and 5.0 mg FB1 kg−1 body weight for 42 days through nutritionally balanced diet. During the trial we observed loss of body weight in both treated groups, together with higher incidence of infective bacterial dermatological lesions erythrodermatitis cyprini (Aeromonas salmonicida subsp. nova) in the group treated with the higher FB1 dose. Several hematological parameters (erythrocyte count, platelet count) and serum chemical concentrations (creatinin, total bilirubin) and activities (aspartate aminotransferase, AST and alanine aminotransferase, ALT) were greater in the fumonisin treated groups than in the control group. Our results indicate that long-term dietary exposure to 0.5 and 5.0 mg FB1 kg−1 body weight is not lethal to young carp, but can produce adverse physiological effects. These findings also suggest that primary target organs of FB1 in the carp are kidney and liver, as it has already been observed in other animal species tested. Specifically changed red blood cell-parameters reveal that FB1 probably causes erythrocyte membrane defect or interferes with carps respiratory process.

Ochratoxinogenicity of Aspergillus ochraceus strains from nephropathic and non-nephropathic areas in Yugoslavia

Mycological analyses of 855 samples of stored grains and dried meat collected in period 1980–1987 from individual households in the nephropathic and wider non-nephropathic area in SR Croatia in Yugoslavia showed 10% of samples to be contaminated with Aspergillus ochraceus. Ability to produce ochratoxin A (OA) was tested in 70 samples (27 from nephropathic areas and 43 from non-nephropathic areas). The detection was carried out under UV-light (365 nm) (light blue fluorescence) and 6 OA-producers were found.A biosynthetic procedure on liquid nutritional substrate with sacharose and yeast extract as well as a method using wet crushed wheat revealed that 37% of the samples from a nephropathic area, and 35% of the samples from a non-nephropathic area produce OA.In the nephropathic area 1/10 strains was a strong producer of OA (concentration crushed wheat 135 mg/kg, and 240 mg/l on YES liquid substrate), 1/10 strains was a moderate producer (concentration 16.6 mg/l and 0.07–7.0 mg/l and 0.1–10.4 mg/kg).Among the strains isolated from a wider non-nephropathic area no strong producers of OA were found, but 2/15 strains were moderate producers of OA (concentration of OA 20.4–27.0 mg/l and 15.0–33.7 mg/kg). The other strains, 8/10 on the crushed wheat and 13/15 on the liquid substrate, were weak producers of OA with concentrations of OA between 0.2–9.0 mg/l and 0.2–10.0 mg/kg with the two methods respectively.

Azithromycin potency determination: Optimal conditions for microbiological diffusion method assay

The research on azithromycin was carried out to determine its potency by the microbiological diffusion method assay on an 8x8 Latin square with two standard and two sample concentrations. Results showed that Sarcina lutea ATCC 9341, Bacillus pumilus NCTC 8241 and Escherichia coli ATCC 10536 reprsented the group recommendable for the analysisof azithromycin by the diffusion method assay. These test microorganisms provided satisfactory precision and accuracy of results tested in 30 successive measurments by 8x8 Latin Square assay. Some pharmacopoeias give official methods for determination of erythromycin potency. The USP recommendation as to the diffusion method on petri dishes and relevant test microorganism is Sarcina lutea ATCC 9341. For potency determination by the diffusion method on 8x8 Latin Square and Petri dishes, PH. Eur. suggest Bacillus pumilus (NCTC 8241, CIP 765,18)and Sarcina lutea ATCC 9341. In the same instances BP suggests Bacillus pumilus NCTC 8241. All the aforesaid recommendations are in agreement with our findings, although our research includes a completly new antibiotic. As azithromycin and erythromycin belong to the same antibiotic group, our results showed that test microorganisms used were similary sensitive to both of them. The recommended group of microorganisms was also supplemented by a new member Escherichia coli ATCC 10536.

An overview of mycotoxins and toxigenic fungi in Croatia.

Mycotoxins are secondary fungal metabolites that contaminate food and feed and can affect human and animal health. Contamination occurs through the fungal growth before harvest depending on consistent environmental conditions, or after harvest as consequence of improper storage. Mycotoxins differ in their chemical characteristics and mode of action in biological systems. Between few hundred known mycotoxins aflatoxins, ochratoxins, zearalenone, trichotecens (T-2, HT-2, DON, DAS) and fumonisins have a great toxicological and economical importance.

«Suspects» in Etiology of Endemic Nephropathy: Aristolochic Acid versus Mycotoxins

Despite many hypotheses that have been challenged, the etiology of endemic nephropathy (EN) is still unknown. At present, the implications of aristolochic acid (AA) and mycotoxins (ochratoxin A—OTA and citrinin—CIT) are under debate. AA-theory is based on renal pathohistological similarities between Chinese herbs nephropathy (CHN) and EN, findings of AA-DNA adducts in EN and in patients with urinary tract tumors (UTT), as well as the domination of A:T®T:A transversions in the p53 mutational spectrum of UTT patients, which corresponds with findings of such mutations in AA-treated rats. However, exposure pathways of EN residents to AA are unclear. Experimental studies attempting to deduce whether nephrotoxins OTA and CIT appear at higher frequencies or levels (or both) in the food and blood or urine of EN residents support the mycotoxin theory. Also, some molecular studies revealed the presence of OTA-DNA adducts in the renal tissue of EN and UTT patients. In this review, data supporting or arguing against AA and mycotoxin theory are presented and discussed.

The ability of fungal isolates from human lung aspergilloma to produce mycotoxins.

This study included 11 adult patients (seven men and four women) who had been surgically treated for pulmonary aspergilloma in the Republic of Croatia within two years. Mycological analysis was positive for Aspergillus genus in five samples of surgically removed tissue. A. fumigatus was isolated in three and A. versicolor in two samples. Their mycotoxigenic potency was determined by thin layer chromatography. A. fumigatusstrains were found to produce aflatoxin B1 (AFB), and two of them aflatoxin G1. A. versicolor strains produced AFB1 and sterigmatocystin. Neither isolated Aspergillus strain produced aflatoxin G2 or ochratoxin A. Fungal growth and production of mycotoxins are the consequences of interaction of fungus, host and environment. One has also to take into consideration that the production of mycotoxins in vitro does not reflect what these fungi may produce in human organisms.