ISSN 1580-4003 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK 'IbmPM V Je 1 tA»* vVft , » " c ^ • «i 9 « • V/** Wti' V.; •/♦V Volume > 43 4 Slov Vet Res • Ljubljana • 2006 • Volume 43 • Number 4 • 151-189 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Volume A 43 4 Slov Vet Res • Ljubljana • 2006 • Volume 43 • Number 4 • 151-189 The Scientific Journal of the Veterinary Faculty University of Ljubljana SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Previously: RESEARCH REPORTS OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA Prej: ZBORNIK VETERINARSKE FAKULTETE UNIVERZA V LJUBLJANI 4 issues per year / izhaja štirikrat letno Editor in Chief / glavni in odgovorni urednik: Gregor Majdič Technical Editor / tehnični urednik: Matjaž Uršič Assistant to Editor / pomočnica urednika: Malan Štrbenc Editorial Board / Uredniški Odbor: Vesna Cerkvenik Flajs, Vojteh Cestnik, Polona Juntes, Matjaž Ocepek, Zlatko Pavlica, Uroš Pestevšek, Modest Vengušt, Milka Vrecl, Olga Zorman Rojs, Veterinary Faculty University of Ljubljana / Veterinarska fakulteta Univerze v Ljubljani Editorial Advisers / svetovalca uredniškega odbora: Gita Grecs-Smole for bibliography (bibliotekarka), Leon Ščuka for statistics (za statistiko) Reviewing Editorial Board / ocenjevalni uredniški odbor: Ivor D. Bowen, Cardiff School of Biosciences, Cardiff, Wales, UK; Rudolf Cabadaj, University of Veterinary Medicine, Košice, Slovakia; Antonio Cruz, Departement of Clinical Studies, Ontario Veterinary College, Guelph, Ontario, Kanada; Gerry M. Dorrestein, Duch Research Institute for Birds and Exotic Animals, Veldhoven, The Netherlands; Wolfgang Henninger, Veterinärmedizinische Universität Wien, Austria; Simon Horvat, Biotehniška fakulteta, Univerza v Ljubljani, Slovenia; Josef Leibetseder, Veterinärmedizinische Universität Wien, Austria; Louis Lefaucheur, INRA, Saint-Gilles, France; Bela Nagy, Veterinary Medical Research Institute Budapest, Hungary; Peter O'Shaughnessy, Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Scotland, UK; Milan Pogačnik, Veterinarska fakulteta, Univerza v Ljubljani, Slovenia; Peter Popelka, University of Veterinary Medicine, Košice, Slovakia; Detlef Rath, Institut für Tierzucht, Forschungsbereicht Biotechnologie, Bundesforschungsanstalt für Landwirtschaft (FAL), Neustadt, Germany; Hans-Peter Sallmann, Tierärtzliche Hochschule Hannover, Germany; Marko Tadic, Veterinarski fakultet, Sveučilište u Zagrebu, Croatia; Frank J. M. Verstraete, University of California Davis, Davis, California, US Slovenian Language Revision / lektor za slovenski jezik: Viktor Majdič Address: Veterinary Faculty, Gerbičeva 60, 1000 Ljubljana, Slovenia Naslov: Veterinarska fakulteta, Gerbičeva 60, 1000 Ljubljana, Slovenija Tel.: +386 (0)1 47 79 100, 47 79 129, Fax: +386 (0)1 28 32 243 E-mail: slovetres@vf.uni-lj.si Sponsored by the Slovenian Research Agency Sofinancira: Agencija za raziskovalno dejavnost Republike Slovenije ISSN 1580-4003 Printed by / tisk: Birografika Bori d.o.o., Ljubljana Indexed in / indeksirano v: Agris, Biomedicina Slovenica, CAB Abstracts, IVSI Urlich's International Periodicals Directory http://www.vf.uni-lj.si/veterina/zbornik.htm SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Slov Vet Res 2006; 43 (4) Review Paper Manojkumar R. Real-time reverse transcription polymerase chain reaction technique: Usefulness as an animal viral disease diagnostic.....................................................................155 Original Research Papers Kirbiš A. Microbiological 5-plate screening method for detection of tetracyclines, aminoglycosides, cephalosporins and macrolides in milk......................................................161 Vengušt G, Bidovec A, Pogačnik M. Damage caused by large carnivores on domestic grazing animals in Slovenia. . . . 169 Kovačevič K, Budefeld T, Majdič G. Reduced seminiferous tubule diameter in mice neonatally exposed to perfume. . . . 177 Subject Index Volume 43, 2006............................................................... 185 Author Index Volume 43, 2006 ............................................................... 189 Slov Vet Res 2006; 43 (4): 155-60 UDC 619.578.2:616.9-078 Review Paper REAL-TIME REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION TECHNIQUE: USEFULNESS AS AN ANIMAL VIRAL DISEASE DIAGNOSTIC Ramanunninair Manojkumar Department of Microbiology and Immunology, New York Medical College, Valhalla, NY-10595, USA E-mail: manoj_kumar@nymc.edu Summary: Quick and early diagnosis of the causative agents is critical for countries which are enzootic to particular disease. In these diagnostics speed is paramount; Real-time Reverse Transcription PCR (rRT-PCR) is being utilized increasingly in novel clinical diagnostic assays in molecular biology. The combination of excellent sensitivity and specificity made this technique an alternative to cell culture and other laboratory testing methods for disease diagnosis. In this review, the usefulness or applications of rRT-PCR assays in the diagnosis of some of the important animal viral infections are summarized. Key words: animal diseases; virus diseases - diagnosis; reverse transcriptase polymerase chain reaction - methods; RNA, viral - genetics Introduction Real-time Reverse Transcription Polymerase Chain Reaction (rRT-PCR) is a powerful tool for quantitative analysis of nucleic acids (1, 2). rRT-PCR techniques are increasingly used to quantify RNA viruses for diagnosis (3), the standard for the detection and quantification of RNA targets (4) and is firmly established as a mainstream research technology (5). This technique is a refinement of the original PCR developed by Kary Mullis and co-workers in the mid 1980ís (2, 6). The amplification is detected by using either probe (specific) or non-probe (non-specific) and is discussed elaborately (7). In rRT-PCR the amount of product formed is monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into this reaction which is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules (2). The advantages of using rRT-PCR are as follows, 1] traditional PCR is measured at end-point, Received: 18 October 2006 Accepted for publication: 15 November 2006 while rRT-PCR collects data in the exponential growth phase, 2] an increase in reporter fluorescent signal is directly proportional to the number of am-plicons generated, 3] a permanent record amplification of an amplicon, 4] increased dynamic range of detection, 5] requirement of 1000-fold less RNA than conventional assays, 6] no-post PCR processing 7] detection ranges down to a 2-fold change 8] small amplicon size results in increased amplification efficiency and 9] less time-consuming. rRT-PCR can be applied to traditional PCR applications as well as new applications that would have been less effective with traditional PCR. With the ability to collect data in the exponential growth phase, the power of rRT-PCR has been expanded into applications such as, a] quantization of gene expression (8) including NK cell KIR gene expression (9), b] drug therapy efficacy/drug monitoring (10), c] Viral quantization (11), and d] pathogen detection (12). rRT-PCR when compared to ELISA, RT-PCR and virus Isolation in cell culture, have greater versatility, sensitivity and specificity (13, 14). The technique was and is being used in the diagnosis of a wide variety of diseases caused by RNA viruses in animals and birds and some of them are being summarized in this article. 156 R. Manojkumar rRT-PCR In diagnosis of Animal Viral Infections A. Positive-strand RNA viruses rRT-PCR assays are also useful in the diagnosis of animal viral diseases which are economically and as well as zoonotically important. These include some of the viruses listed by the Office International des Epizootics (OIE). Foot and mouth disease (FMD) is the number 1 animal viral disease of OIE list and presently the rRT-PCR technique is being widely used in the diagnosis of FMD which causes huge economic crisis world wide. The technique has proven to be more efficient and sensitive than the conventional RT-PCR and virus isolation in cell culture. rRT-PCR assay was found to be very efficient for quantitation of Foot and mouth disease virus (FMDV) in porcine tissues (15); in detecting FMDV from experimentally affected animals (16, 17) and shows 100% specific diagnosis of all the 7 serotypes of FMDV in less than 2 hrs (18, 19). The assay is also used to diagnose swine vesicular disease virus and its differential diagnosis from other vesicular disease viruses like FMD, vesicular stomatitis and ve-sivirus (20). Another 1000-fold more sensitive Taq-Man rRT-PCR was used for the specific detection of FMDV in both cell culture and clinical samples and also for the differential diagnosis of other vesicular diseases and bovine viral diarrhea (21). The two independent rRT-PCR techniques were studied and then compared to detect FMDV in clinical samples; the results suggested that the techniques could be used to enhance sensitivity of molecular methods for further FMDV detection (22). Surveillance and for field deployment another RT-loop mediated isothermal amplification technique was found to be very effective for rapid detection of FMDV (23). The worldwide occurrence and re-occurrence of trans-boundary diseases like FMD or classical swine fever (CSF) indicates that there is a high need for the development of powerful, robust and high-capacity new diagnostic methods that are able to detect the causative agents before they could spread to large populations and cause tremendous losses (24). The rapid, powerful and internationally standardized molecular diagnosis contributes to the reduction of losses caused by the trans-boundary viral diseases to a larger extent (24). Thus, rRT-PCR forms an important assay in the detection and species-specific differentiation of pestiviruses like CSF virus (25). CSF in an experimentally infected swine will produce the same extent of disease that of natural outbreaks and its detection made easier by TaqMan rRT-PCR assays in less than 2 hrs thus providing a rapid method for the diagnosis of CSF virus on herd basis (26, 27) and also for the quantitative pathogenesis study of this virus (28). Japanese encephalitis virus (JEV) is one of the most important zoonotic diseases of swine, in areas where it is endemic, such as East Asia (29). Taq-Man rRT-PCR assay using fluorogenic probes was developed to distinguish JEV with West Nile virus (WNV) and the method was tested on experimentally infected animal tissues which showed clear discrimination between WNV and JEV (29). Quantitative detection of JEV by rRT-PCR using virus specific primers showed no cross-reactions with other swine viruses and bovine viral diarrhea virus (30). TaqMan rRT-PCR assay was also used for the detection of equine arteritis virus in seminal plasma and nasal secretions of infected horses (31) and then for differentiating the avian infectious bronchitis virus isolates in clinical samples thus by identifying the serotypes involved in disease outbreak (32). B. Negative-strand RNA viruses Typical influenza is an acute respiratory herd disease and commonly observed during autumn, winter and early summer (33). Influenza viruses affect all animal and avian species. Immuno-fluorescent techniques were used initially which showed evidence of viral antigen in bronchial epithelial cells within 2 hours after infection (34). Recently, a rRT-PCR assay was used to detect swine influenza A viruses and the test was found to be 100% specific and 88-100% sensitive in screening numerous nasal swab specimens and also very efficient and specific for the respective viral genes thus able to distinguish between their viral subtypes (35) and are largely used to detect and differentiate the North American swine influenza viruses (33). rRT-PCR based on LC technology was used to detect equine influenza virus over two influenza seasons, analyzing 171 samples and they could get a positive correlation between the quantitative rRT-PCR in both cases (36) indicating the high specificity of the assay. Rapid diagnosis of H5N1 influenza A virus was performed by using multiplex rRT-PCR from 75 clinical specimens isolated from both poultry and mammals. The results highlights that the assay could be feasible and very effective for large-scale screening during times of H5N1 outbreaks (37) and also as a good tool for the rapid screening of Real-time reverse transcription polymerase chain reaction technique. 157 flocks and live bird markets (38, 39). Simultaneous detection of influenza viruses A and B was carried out using TaqMan based rRT-PCR assay which was found to be more sensitive than the combination of viral culturing and shell vial culturing of influenza viruses (40). Bovine respiratory syncytial virus (BRSV) causes respiratory disease in infected animals. Detection of BRSV was done by quantitative rRT-PCR assays based on fluorogenic probe using BioRad's iCycler iQ (41) was found to be 100 fold more sensitive than conventional RT-PCR used previously for BRSV diagnosis (42). Rabies is an enzootic fatal disease and is still a major problem in developing countries. Several RT-PCR methods have been reported for the detection of rabies and rabies-related viruses. Distinguishing the classical rabies virus and its genotypes was described in a single tube, non-nested rRT-PCR with TaqMan technology in real-time and found to be very useful in the detection and differentiation of members of the genus Lyssavirus (43). Nipah and Hendra viruses belong to the novel genus Henipavirus of the family Paramyxoviridae. Its zoonotic circulation in bats and recent emergence in Malaysia with fatal consequences for humans who were in close contact with infected pigs has made the reinforcement of epidemiological and clinical surveillance systems a priority. TaqMan rRT-PCR assay has been developed targeting the Nipah nucleoprotein so that Nipah virus RNA in field specimens or laboratory material can be characterized rapidly and specifically quantitated (44). The method was able to detect virus from hamsters infected with Nipah virus and allows a rapid detection and quantitation of Nipah RNA both from field and experimental materials used for the surveillance and specific diagnosis. Diagnosis of Newcastle disease virus (NDV) was recently carried out by using rRT-PCR from clinical samples and a positive correlation was obtained between these assays and detecting NDV by RT-PCR and virus isolation from clinical samples (45). To obtain a large diagnostic and surveillance sample workload, a high throughput rRT-PCR assay was developed during 2002-03 outbreaks of NDV occurred in California (46). And also a two-step rRT-PCR using SYBR Green I was designed for the screening large number of NDV specimens (47). C. Retro viruses Simultaneous calculation of the nucleic acid recovery rate along with the copy number of viral RNA molecules of equine infectious anemia virus in the plasma was obtained by a single tube rRT-PCR reaction using a fluorogenic probe (48). At the same time a robust, ultra-sensitive quantitative assay was developed for maedi-visna virus (MVV) RNA and DNA genomic sequences and mRNA expressed at various stages of lentiviral replication (49). The assay was designed based on PCR with real-time fluorescence resonance energy transfer measurements. The quantitative assay was found to have greater use in studying the role of genetic elements in MVV infection, pathogenesis, lentiviral vectors and packaging systems based on MVV. Quantitative rRT-PCR assay of MVV RNA in culture supernatants helped in obtaining the complete genomic sequence of a sheep lentivirus isolate that presents a slow/low phenotype (50). D. Double-stranded RNA viruses The outbreaks of bluetongue disease in sheep can be combated by extensive vaccination. In order to do so, a rapid and sensitive technique should be used to differentiate vaccine strains of bluetongue and the field strains. A new method for bluetongue virus differentiation using fluorescence resonance energy transfer probes with rRT-PCR assay was performed with LC system and described earlier (51). Infectious bursal disease virus (IBDV) causes an immunosuppressive disease in chickens and leads to severe economic losses in the poultry industry. Vaccination may not be effective if there is exposure of the vaccinated flock to a different antigenic subtype as a result of mutation in the VP2 protein upon which the major neutralizing epitopes are located (52) also they reported that the rRT-PCR assay could be a useful tool to assist in the development of more effective vaccines and control strategies of infectious diseases. Conclusion The newly established methods must be standardized to maintain high quality laboratory performance. Future challenges in the study of animal viral diseases include the application of modern techniques, such as nucleic acid chips, protein chips, proteomics and new biomarkers to avoid cross-reactivity among different samples, strains or serotypes, as well as development of internationally standardized guidelines to improve the quality of these laboratory tests. 158 R. Manojkumar References 1. Gibson UE, Heid CA, Williams PA. A novel method for real-time quantitative RT-PCR. Genome Res 1996; 6: 995-1001. 2. Kubista M, Andrade JM, Bengtsson M et al. The realtime polymerase chain reaction. Mol Aspects Med 2006; 27: 95-125. 3. Costa-Mattioli M, Monpoeho S, Nicand E, Aleman MH, Billaudel S, Ferre V. Quantification and duration of viraemia during hepatitis A infection as determined by real-time RT-PCR. J Viral Hepat 2002; 9: 101-6. 4. Bustin SA. 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Barros SC, Ramos F, Duarte M, Fagulha T, Cruz B, Fevereiro M. Genomic characterization of a slow/low maedi visna virus. Virus Genes 2004; 29: 199-210, 51. Orru G, Santis PD, Solinas F, Savini G, Piras V, Ca-porale V. Differentiation of Italian field and South African vaccine strains of bluetongue virus serotype 2 using realtime PCR. J Virol Methods 2004; 122: 37-43. 52. Mickael CS, Jackwood DJ. Real-time RT-PCR analysis of two epitope regions encoded by the VP2 gene of infectious bursal disease viruses. J Virol Methods 2005; 128: 37-46. 160 R. Manojkumar METODA OBRATNEGA PREPISOVANJA IN VERIŽNE REAKCIJE S POLIMERAZO V REALNEM ČASU: UPORABNOST V DIAGNOSTIKI ŽIVALSKIH VIRUSNIH BOLEZNI R. Manojkumar Povzetek: Hitro in pravočasno odkrivanje povzročiteljev bolezni je v državah z enzootijo za določene bolezni bistvenega pomena. Pri taki diagnostiki je hitrost odločilna in rRT-PCR (obratno prepisovanje in verižna reakcija s polimerazo v realnem času) se čedalje več uporablja kot sodoben test klinične diagnostike v molekularni biologiji. Zaradi odlične občutljivosti in specifičnosti predstavlja alternativo celičnim kulturam in drugim laboratorijskim testom za diagnostiko bolezni. V preglednem članku povzemamo uporabnost in aplikacije testov rRT-PCR v diagnostiki nekaterih pomembnih živalskih virusnih bolezni. Ključne besede: živali, bolezni; virusne bolezni - diagnostika; polimerazna verižna reakcija z reverzno transkriptazo; RNA, virusna - genetika Slov Vet Res 2006; 43 (4): 161-8 UDC 614:35-579.67:615.33:637.12 Original Research Paper MICROBIOLOGICAL 5-PLATE SCREENING METHOD FOR DETECTION OF TETRACYCLINES, AMINOGLYCOSIDES, CEPHALOSPORINS AND MACROLIDES IN MILK Andrej Kirbiš Institute for Food Hygiene and Bromatology, Veterinary Faculty, Gerbičeva 60, 1000 Ljubljana, Slovenia Corresponding author, E-mail: andrej.kirbis@vf.uni-lj.si Summary: Within the field of food hygiene and food control, the antibiotic residues in food of animal origin are analysed because their presence may have undesirable consequences. These include for example, allergic reactions in people, spread of resistance to antibiotics among microorganisms and damages in the food industry. Methods available for the detection of antibiotic residues in food are microbial, chemical and immunoassays. Microbial methods are used as screening methods and are always the first choice for this purpose. The aim of our study was to develop a microbial method for the detection of antibiotic residues from the macrolide, aminogli-coside, cephalosporine and tetracycline families. The study involved investigation of bacterial test strains and establishment of the limits of detection (LOD) of antibiotics. For cephalosporines and macrolides, the most appropriate sensitive strain proved to be Micrococcus luteus ATCC 9341, for aminoglicosides Bacillus subtilis BGA and for tetracyclines Bacillus cereus ATCC 11778. A significant component in our experiment were the so-called confirmation solutions. Magnesium sulphate inactivates aminoglicosides and can be used to confirm their presence when more than one antibiotic group can produce inhibition zones on the same plate. Cephalosporinase inactivates cephalosporines and was used to distinguish this group of antibiotics from macrolides. The LOD was at or below the allowed maximum residue level (MRL) for all tested antibiotic groups. Key words: food analysis - methods; antibiotics; drugs residues - analysis; microbial sensitivity tests - methods; milk Introduction By definition, an antibiotic is either a natural product of a micro-organism, an identical synthetic product or a similar semi synthetic product, that inhibits the growth of other microorganisms (bacteriostatic effect) or destroys other microorganisms (bactericide effect). (1). The most common cause for the presence of antibiotic residues in food of animal origin is violation of withdrawal periods (2, 3, 4, 5). Other possible causes are overdosing of antibiotics and use of antibiotics banned for treatment of food producing animals (6, 7, 8). Also, antibiotic residues can be detected in bulk milk samples from a stable where individual animals are being treated for mastitis. Received: 27 June 2006 Accepted for publication: 6 October 2006 In the field of food hygiene and food control we deal with analysis of antibiotic residues in food of animal origin due to the potential of unwanted consequences . Among them are sensitivity to antibiotics, allergic reactions and imbalance of intestinal microflora in people, spread of resistance to antibiotics in microorganisms and losses in the food industry where antibiotics can influence starter cultures used in the production of meat and milk products. Microbial methods were the first choice of systematic detection of antibiotic residues in food in the past and are still mainstream screening methods. They allow determination of the presence of antibiotics in the sample and identification of specific antibiotic groups (9). Screening methods have acceptable false-positive result rates (10, 11) and allow detection of a wide spectrum of antibiotics (9, 12). Their other 162 A. Kirbis advantages are the option to analyse a large number of samples simultaneously and the relatively short time needed for preparation of samples as no purification procedures are required (13, 14, 15, 16, 17, 18). As microbial methods can not be used to identify individual antibiotics a positive result should be confirmed with chemical or physical methods. Tetracyclines are probably the most widely used therapeutic antibiotics in food producing animals because of their broad spectrum and cost effectiveness. In the United Kingdom and the Netherlands the amount of tetracyclines used for farm animals is nearly equal to the amount of all other antibiotics. Cephalosporines are used both for humans and animals. The first and second generation are approved worldwide strictly for treatment of mastitis infections in dairy cattle. A representative of the third generation, ceftiofur, and a representative of the fourth generation, cefquinome, have been developed strictly for veterinary use and approved in several countries for treatment of respiratory disease, foot rot mastitis in dairy cattle (9, 10, 15, 16, 19). Macrolides are used in veterinary medicine for the treatment of clinical and subclinical mastitis in lactating cows and for the treatment of chronic respiratory diseases (20). The aminoglycosides are broad-spectrum antibiotics also widely used for treatment of bacterial enteritis, mastitis and other infections. Aminoglycosides most commonly used as therapeutic agents are gentamicin, neomycin and streptomycin (21). The most frequently used microbial method is based on the principle of inhibition of growth of testing strains which is known as the STAR five-plate method (22). It is used for detection of antibiotics from the macrolide, aminoglicoside, tetracycline and cephalosporine families (19, 23, 24, 25). Detailed procedures of these tests vary among laboratories and tests are not standardised for minimal detectable antibiotic concentrations, therefore comparison of results is difficult (26, 27, 28). The aim of our study was to develop a microbial method for detection of antibiotic residues from the four above mentioned families and to determine LOD for each tested antibiotic according the EU Regulation 2377/90 which prescribed maximum residue limits (29). Material and methods Microbiological methods are based on the measurement and evaluation of zones of inhibited bacterial growth on media. Two test strains are used to assess the presence of each antibiotic - one maxi- mally sensitive and the other resistant to the tested substance. With the combination of different sensitive and resistant bacterial strains, specific antibiotic groups present in the sample can be identified. In our research we used the following strains with previously established sensitivity and resistancy profiles: Bacillus cereus ATCC 11778, Micrococcus luteus ATCC 2341, Escherichia coli ATCC 10536, Staphylococcus epidermidis ATCC 12228 and E. coli ATCC 10536 (manufactured by OXOIDTM). Preparation of bacterial cultures and media Bacterial strains stored as cultures in original bacterial loops (Culti loop) were applied to a test tube containing 1ml Trypton soya broth (TSB) medium and incubated at 37 oC for one hour. The culture was then inoculated on blood agar and incubated for further 16 hours at the same temperature. Afterwards the purity of bacterial colonies was assessed with a light microscope and pure colonies were stored in a fridge at temperatures between 2 oC and 8 oC for up to one month. To compose test plates, bacterial culture was diluted with normal saline containing peptone water to produce a suspension which was then incubated at 37 oC for one hour and afterwards added to the agar medium specified below. The suspension density was standardised with the Mc Far-land method. Basic media for preparation of test plates were antibiotic agar No. 1 (MerckTM) and antibiotic agar No. 2 (MerckTM). Antibiotic agar No. 1 was prepared as follows: 1000 ml of distilled water was added to 30, 5 g of the medium, left for 15 min and then heated to boiling point so that the medium was completely dissolved. The medium was then autoclaved at 121 oC for 15 min. For antibiotic agar No. 2 1000 ml of distilled water was added to 15, 5 g of medium and then the same procedure was followed. After autoclaving, the pH of the media was set to desired values: pH 8 for Er, I BGA, Kin and AC plates and pH6 for E plates. Preparation of test plates Test plates were marked according to the bacterial strain added to the medium: AC plate - Micrococcus luteus ATCC 2341, ER plate - Staphylococcus epidermidis ATCC 12228, I-BGA plate - Bacillus subtilis BGA, Kin plate - E. coli ATCC 10536 and E plate - Bacillus cereus ATCC 11778. The pH of the medium was maintained at 8.0 for AC, E and ER plates and at 6.0 Microbiological 5-plate screening method for detection of tetracyclines, aminoglycosides. 163 for I-BGA and Kin plates. We defined the tolerance for the width of inhibition zone at (as) 8.5 mm - 0.5 mm wider than the width of the metal cylinder containing the sample. Inhibition zones between 8 mm and 8.5 mm wide were considered a non-specific reaction. To prepare a test plate 0.45 ml of suspension of bacterial culture was added to 40 ml of basic medium and heated to 40 oC. Kin plate was an exception where 0.2 ml of suspension was added to 50 ml of medium. The mixture of medium and bacterial culture was poured into a petri dish (5 ml of mixture into each petri dish). At room temperature the petri dishes with silified medium were enveloped in a parafilm and stored in a fridge. The storage period of test plates was one week. Before application of samples to test plates, plates were warmed at room temperature for 20 to 30 min. Preparation of milk samples To test the sensitivity of our method, milk samples containing known concentrations of standard antibiotics were inoculated on test plates. Prior to the addition of antibiotics, milk was always tested for the presence of inhibitory substances. As the initial step, standard antibiotic solutions were prepared using reference chemical composition and purity (Table 1). Standard antibiotics in powder were dissolved in appropriate solvents: tetracyclines in phosphate buffer with pH value 4.5, cephalosporines in phosphate buffer with pH value 6.0, aminoglicosides in phosphate buffer with pH value 8.0, and macrolides in methanol. Standard solutions were diluted to desired concentrations with UHT milk containing 1.6% fat (Ljubljanske mlekarne). These samples of milk with known concentrations of antibiotics were then poured into 10-ml test tubes and heated to 80 oC for 5 min to avoid later non-specific reaction on test plates. After heating, the samples were cooled and transferred to test plates in 8 mm wide cylinders. Test plates were incubated at 37 oC (I-BGA, AC, Er, Kin) or at 30 oC (E plate) for 18-24 hours. For each antibiotic we used milk samples containing antibiotic concentrations equal to MRL and half the MRL for that substance. If at half the MRL the result was still positive, lower concentrations of antibiotic were applied until the minimal level of detection was reached. Confirmation solutions To confirm the presence of antibiotic groups or their individual representatives we used confirmation solutions. These solutions inhibit the action of certain antibiotics and can help to distinguish between antibiotic groups which cause inhibition zones on the same test plates. Magnesium sulphate (MgSO4) was used to neutralise the aminoglicosides and cephalosporinase enzyme to neutralise the cephalosporines. 25 ^l of 20% MgSO4 solution in water was added to the sample on E, AC and I-BGA plates where inhibition zones are produced by aminoglicosides, mac-rolides or tetracyclines. 25 ^l of cephalsporinase was added to samples on AC and I-BGA plates to identify cephalosporines. Table 1: Antibiotic standards ANTIBIOTIC TRADE MARK CATALOGUE NUMBER Streptomyicin Sigma - Aldrich 46754 sGentamicin Sigma - Aldrich 46305 Neomycin Calbiochem 4801 Cephalexlne Sigma - Aldrich 33989 Cephazoline Sigma - Aldrich 22127 Cefoperazone Sigma - Aldrich 22129 Chlortetracycline Sigma - Aldrich 46133 Tetracycline Sigma - Aldrich 46935 Erythromycin Sigma - Aldrich 46256 Tylosin Sigma - Aldrich 46992 164 A. Kirbis Evaluation of results Results of microbial methods can be evaluated both qualitatively and quantitatively. Qualitative results are obtained by analysing the effect of antibiotics on a combination of sensitive and resistant bacterial strains. When required, neutralising substances (confirmation solutions) can help to differentiate between antibiotics with similar action on test bacterial strains. Quantitatively the concentration of antibiotic can be assessed with microbial methods if the sample contains a known antibiotic or an antibiotic that has previously been identified qualitatively. In each case a calibration curve is required. Results We have confirmed sensitive and resistant bacterial strains for all antibiotic groups tested in our study (Table 2). Based on our results we chose to use Bacillus cereus ATCC 11778 (E plate) as the sensitive and Micrococcus luteus ATCC 9341 (AC plate) as the resistant strain for tetracycline and chlortetracycline from the tetracyclines group. For tylosine and erythromycine from the macrolides group Micrococcus luteus ATCC 9341 (AC plate) was chosen as the sensitive and Escherichia coli ATCC 10536 (Kin plate) as the resistant strain. For gen-tamycine, sterptomycine and neomycine from the aminoglicosides group Bacillus subtilis BGA (I-BGA plate) was chosen as the susceptible and Staphylococcus epidermidis ATCC 12228 (ER plate) the resistant strain. For cephalexine, cephoperasone and cephasoline from the sensitive group Micrococcus luteus ATCC 9341 (AC plate) was chosen as the susceptible and Staphylococcus epidermidis ATCC 12228 (ER plate) as the resistant strain. We differentiated between antibiotic groups using a combination of five test plates (Table 3). To discriminate between aminoglicosides and macrolides we had to utilise used magnesium sulphate which inactivates the aminoglicosides. To discriminate between cephalosporines and macrolides we used the cephalosporinase enzyme. Table 4 shows the limit of detection for milk samples containing standardised antibiotic solutions on selected test plates. The level of detection was at or below the MRL in all tested antibiotics. Discussion Microbial methods for detection of antibiotic residues in food of animal origin are used as a screening method in the majority of laboratories in Europe that deal with analyses of drug residues in food Table 2: Sensitivity of bacterial strains: ANTIBIOTIC B.c/ E M.l.1/ AC B.s.BGA/ IBGA S.e./ ER E.c./ KIN Cephalexine - + + - - Cephasoline - + + - - Cefoperazone - + + - - Gentamicin + - + - + Neomycin + - + - + Streptomycin + - + - + Erythromycin - + + - - Tylosin - + + - - Tetracycline + - + - - Chlortetracycline + - + + sensitive strain - resistant strain B.c/E Bacillus cereus ATCC 11778/ plate E M.l.1/AC Micrococcus luteus ATCC 9341/ plate AC B.s.bga/IBGA Bacillus subtilis BGA/ plate IBGA S.e./ER Staphylococcus epidermidis ATCC 12228/ plate ER E.c./KIN Escherichia coli ATTC 10536/ plate KIN Microbiological 5-plate screening method for detection of tetracyclines, aminoglycosides. 165 Plate E Plate Ac Plate IBGA Plate Er Plate Kin Antibiotics Antimicrobial family G