ISSN 1580-4003 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Volume 47 2 Slov Vet Res • Ljubljana • 2010 • Volume 47 • Number 2 • 35-72 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Volume 47 Slov Vet Res • Ljubljana • 2010 • Volume 47 • Number 2 • 35-72 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: Valentina Kubale Dvojmoč Editorial Board / uredniški odbor: Vojteh Cestnik, Polona Juntes, Matjaž Ocepek, Zlatko Pavlica, Modest Vengušt, Milka Vrecl, 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; 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; Nevenka Kožuh Eržen, Krka, d.d., Novo mesto, Slovenia; Louis Lefaucheur, INRA, Rennes, 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, Forschungsbericht 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, Science Citation Index Expanded, Journal Citation Reports/Science Edition http://www.slovetres.si/ SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Slov Vet Res 2010; 47 (2) Review Article Useh NM, Adamu S, Ibrahim N, Nok AJ, Esievo KAN. Outbreaks of blackleg of cattle in Northern Nigeria (1964-2003)............................................................. 39 Original Scientific Article Kusar D, Pate M, Micunovic J, Bole - Hribovsek V, Ocepek M. Detection of Salmonella in poultry faeces by molecular means in comparison to traditional bacteriological methods................................45 Skrlep M, Segula B, Zajec M, Kastelic M, Kosorok S, Fazarinc G, Candek - Potokar M. Effect of immunocastration (Improvac®) in fattening pigs I: Growth performance, reproductive organs and malodorous compounds.......57 Skrlep M, Segula B, Prevolnik M, Kirbis A, Fazarinc G, Candek - Potokar M. Effect of immunocastration (Improvac®) in fattening pigs II: Carcass traits and meat quality.....................................65 Slov Vet Res 2010; 47 (2): 39-44 UDC 636.2.09:579.852.13:616-036.2(669) Review Article OUTBREAKS OF BLACKLEG OF CATTLE IN NORTHERN NIGERIA (1964-2003) Nicodemus Maashin Useh1*, Sani Adamu, Najume Ibrahim1, Andrew Jonathan Nok2, King Akpofure Nelson Esievo1 1Department of Veterinary Pathology and Microbiology, 2Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria Corresponding author, E-mail: nmuseh@abu.edu.ng Summary: Blackleg is an endemic disease in both developed and developing countries and is a well-known cause of financial loss to cattle raisers in many parts of the world. The disease is prevalent in the United States of America, India and other parts of Asia and Europe, Latin America, and Africa. In Nigeria, the economic losses of Zebu cattle alone to the disease have been estimated at US $4. 3 million (~600 million naira) annually. A retrospective study was carried out to ascertain the current status of the disease in Northern Nigeria, and it was observed that the traditional style of livestock production by the transhumance Fulani pastoralists of rural Nigeria, who own livestock resources in the country pose a great challenge to the control of the disease. It was concluded that blackleg is still endemic in Nigeria, and its effective control can only be achieved if the traditional livestock production system of the nomads of rural Nigeria is improved. Key words: blackleg; cattle; Northern Nigeria; outbreaks; retrospective study Introduction Blackleg is a disease affecting cattle, sheep and other ruminants, caused by Clostridium chauvoei and was first reported in 1870 (1). In Nigeria, the disease was first reported in 1929 and has remained a major problem of cattle in the country (2). Although vaccination against the disease has been carried out in Nigeria since 1930, many sporadic outbreaks have been recorded annually (3). Blackleg is endemic in both developed and developing countries and is a well-known cause of financial loss to cattle raisers in many parts of the world (4). The economic losses of ruminants to the disease have not been quantified in most parts of the world, but in Nigeria, the losses of Zebu cattle alone to the disease have been estimated at US $4.3 million annually (~600 million naira) (5). In the United States of America, Latin America, India and other parts of Asia and Europe, the economic losses of ruminants to blackleg have not been estimated, but it has been reported that the disease causes major economic losses in cattle and minor losses in sheep (4,6-7). High annual rainfall has been associated with increased outbreaks of the disease in ruminants (3,8). In this study, we present outbreaks of the disease for about 40 years (1964-2003) and the possible ways of effecting efficient control of the disease in Nigeria. Materials and methods A 40 year retrospective data on annual outbreaks of blackleg (1964-2003) was collected from the Federal Ministry of Agriculture and Natural Resources Archives at Kaduna and Abuja, Nigeria and the National Veterinary Research Institute (NVRI), Vom, Nigeria. Similar data of annual outbreaks of the disease in 12 states of Northern Nigeria was also collected from the Veterinary Services Division of these states. The data was recorded according to the number of outbreaks (N. O.), number of cattle in herds infected (N. C. I.) and mortality (M). Received: 30 January 2010 Accepted for publication: 17 June 2010 40 N. M. Useh, S. Adamu, N. Ibrahim, A. J. Nok, K. A. N. Esievo Results low in some years and states and high in others. During many of the years investigated, there were The outbreaks of blackleg of cattle in Northern few records of outbreaks of all diseases including Nigeria are presented in Tables 1-3. Outbreaks were blackleg. Table 1: Blackleg outbreaks in the provinces of Northern Nigeria (1964-1970) Year Provinces Kano Sokoto Katsina Borno Sardauna Bauchi Zaria Adamawa Plateau Niger Benue Kabba florin 1964 N.O. 4 14 24 19 2 22 6 4 31 1 N.C.I. 230 2409 - 1452 132 3353 1020 741 12 M 65 81 50 39 2 164 15 13 1 1965 N.O. 1 17 12 38 1 12 9 3 3 N.C.I. 70 3298 1087 7126 144 2611 1632 698 573 M 6 198 38 262 1 105 33 3 33 1966 N.O. 8 3 1 38 1 49 10 16 11 15 1 N.C.I. 945 414 3444 30 5502 1785 4300 1923 5127 300 M 44 18 181 241 73 159 37 194 10 1967 N.O. 4 3 12 7 36 2 17 9 9 3 N.C.I. 523 186 1824 2430 3995 390 2888 1140 1558 484 M 20 5 50 39 109 14 105 23 49 24 1968 N.O. 12 1 5 11 12 20 12 3 25 8 24 N.C.I. 1230 502 315 1445 2031 1214 2734 716 11284 817 M 66 35 22 43 72 62 53 26 124 39 6 1969 N.O. 13 2 1 8 28 17 2 16 8 9 N.C.I. 26 8 7655 3254 3878 314 456 2554 498 M 35 7 1 60 98 240 14 100 109 50 1970 N.O. 9 13 28 56 10 14 2 N.C.I. 1722 - 2756 11388 1138 1391 M 79 42 107 46 77 85 11 N.O. ^ Number of outbreaks N.C.I. ^ Number of cattle in the herds infected M ^ Mortality (Number of dead animals) Table 2: Blackleg outbreaks in 5 states of Northern Nigeria (1971-1975) Year North Eastern state North Western state Benue Plateau state North Central state Kwara state 1971 N.O. 11 21 26 80 1 N.C.I. 2460 4180 468 1375 280 M 9 216 17 187 12 1972 N.O. 18 10 12 24 N.C.I. 3490 5419 620 4089 M 120 42 15 120 1973 N.O. 8 1 3 16 1 N.C.I. 214 200 418 2184 412 M 22 11 19 34 1 1974 N.O. 22 12 10 20 N.C.I. 5180 4112 384 4010 M 18 216 11 218 1975 N.O. 1 12 6 27 N.C.I. 211 4020 2116 4124 M 84 118 25 296 Outbreaks of blackleg of cattle in Northern Nigeria (1964-2003) 41 Table 3: Outbreaks of blackleg in 11 states of Northern Nigeria (1976-2003) Year Gongola Benue Plateau Bauchi Niger Kaduna Kano Sokoto Borno Gombe Taraba 1976 N.O. 2 N.C.I. 149 M 4 1977 N.O. N.C.I. M 1978 N.O. 1 1 1 N.C.I. 170 63 1 M 2 - 1979 N.O. 4 1 2 7 1 N.C.I. 168 40 144 206 144 M 3 2 8 23 1 1980 N.O. 2 5 N.C.I. 1080 153 M 75 11 1981 N.O. 1 1 N.C.I. 1000 74 M 20 13 1982 N.O. N.C.I. M 1983 N.O. 1 4 N.C.I. 66 231 M 2 17 1984 N.O. 1 1 1 N.C.I. 44 132 96 M 1 6 4 1985 N.O. 2 1 5 4 N.C.I. 58 20 320 506 M 2 1 22 34 1986 N.O. 2 3 2 N.C.I. 60 154 76 M 2 3 4 1987 N.O. 2 1 N.C.I. 78 1 M 3 1 1988 N.O. 3 4 3 N.C.I. 98 211 259 M 2 5 17 1989 N.O. 4 4 3 2 N.C.I. 100 667 210 63 M 3 5 13 10 1990 N.O. 1 1 2 N.C.I. 36 31 80 M 2 2 13 1991 N.O. 3 1 1 2 N.C.I. 1092 46 75 176 M 48 3 4 15 42 N. M. Useh, S. Adamu, N. Ibrahim, A. J. Nok, K. A. N. Esievo (Tab. 3, continuation) 1992 N.O. 1 3 N.C.I. 20 59 M 5 2 1993 N.O. 1 1 1 N.C.I. 32 70 27 M 3 3 5 1994 N.O. 1 4 18 56 N.C.I. 38 127 450 1580 M 2 5 29 85 1995 N.O. 1 3 3 3 151 N.C.I. 44 376 83 1999 742 M 1 12 4 18 55 1996 N.O. 1 2 2 2 1 N.C.I. 40 55 50 205 162 M 2 5 3 25 42 1997 N.O. 1 2 4 N.C.I. 56 88 102 M 2 4 2 1998 N.O. 19 1 4 7 N.C.I. 842 68 456 1179 M 12 2 26 33 1999 N.O. 48 1 3 2 N.C.I. 680 51 1840 11 M 23 1 64 5 2000 N.O. 10 1 1 1 N.C.I. 728 28 130 55 M 20 3 4 3 2001 N.O. 1 6 18 N.C.I. 34 108 652 M 1 5 20 2002 N. O. 1 1 N.C.I. 56 180 M 2 3 2003 N. O. 50 N.C.I. 1870 M 58 Discussion The results of this study suggest that there are still pockets of annual outbreaks of blackleg of cattle in Northern Nigeria. The Fulani pastoralists of rural Nigeria are the custodians of livestock resources in Nigeria, with about 70-80% ownership of livestock production in the country (9). Some states investigated in the study had few blackleg outbreaks, because of poor record keeping. It is therefore possible to insinuate that outbreaks of the disease may have occurred in those states during the period investigated, but were not recorded. In Nigeria, the control of blackleg has been difficult because of ineffective vaccination policy, lack of adequate logistics such as vehicles to carry out vaccination activities, lack of facilities to maintain the cold chain for vaccine storage (hence inadequate potent vaccines) and lack of disease reporting by the nomads (3). Livestock disease control in Nigeria is the responsibility of government and therefore, if reports of outbreaks are not made to the authorities, Outbreaks of blackleg of cattle in Northern Nigeria (1964-2003) 43 it is difficult for government to know the status of blackleg in a given locality. Unfortunately, in the face of outbreak or rumour of one, the nomads move away in mass, from the so called danger areas, and even those of them whose cattle are infected move away, thereby serving as a source of infection to other supposedly healthy herds (3). The nomadic Fulani pas-toralists prefer to use herbal remedies to treat diseases of their livestock, including blackleg (10), and only report disease if their herbal preparations do not provide the desired therapeutic results. In most cases, disease outbreaks are reported only if there is uncontrollable cattle mortality. The present study on the outbreaks of blackleg of cattle in Northern Nigeria is important because, in Africa, there is migration of cattle between neighbouring countries, and a disease that is endemic in one country is a potential danger to neighbouring countries. Other authors (3) suggested that to achieve effective control of blackleg in Nigeria and the rest of Africa, governments of the sister African states must intensify awareness campaigns among the nomads, through the Nomadic Education Commissions of the respective countries. It is concluded that blackleg is still endemic in Nigeria, and its effective control can only be achieved if the traditional style of livestock keeping by Nomadic Fulani pastoralists who own most of Nigeria's livestock resources is improved. This is possible if they are settled and encouraged by government to reduce transhumance, with adequate support and provision of modern livestock rearing facilities. Acknowledgements The authors of this manuscript are grateful to the Director, Federal Livestock Department and Pest Control Services, Abuja, Nigeria for approving the use of data collated by his staff to conduct the present research. We also thank the Executive Director, National Veterinary Research Institute (NVRI), Vom, Nigeria and the Directors of Veterinary Services of the 12 Northern states investigated for assisting with the data on blackleg outbreaks in their respective states. References 1. Armstrong HL, McNamee JK. Blackleg in deer. J Am Vet Med Assoc 1950; 117: 212-4. 2. Osiyemi TIO. The aetiology and data on seasonal incidence of clinical blackleg in Nigerian cattle. Bull Anim Health Prod Afr 1975; 23: 367-70. 3. Useh N M, Ibrahim NDG, Nok AJ, Esievo KAN. The relationship between annual rainfall and outbreaks of blackleg of cattle in Zaria, Nigeria. Vet Rec 2006; 158: 100-1. 4. Adams LG. Animal health issues in South Texas cattle. In: Workshop on beef cattle production systems and natural resources conservation in semi-arid lands of South Texas and North Mexico, Mexico, 1998. 5. Useh NM, Nok AJ, Esievo, KAN. Blackleg in ruminants. CAB Rev Perspect Agric Vet Sci Nutr Natur Resour 2006; 1: 1-8. 6. Ramarao D, Rao BU. Studies on the incidence of blackquarter in Karnataka during 1979-1985. Indian Vet J 1990; 67: 795-801. 7. Troxel TR Burke GL, Wallace WT, et al. Clostrid-ial vaccination efficiency on stimulating and maintaining an immune response in beef cows and calves. J Anim Sci 1997; 75: 19-25. 8. Uzal FA, Paramidani M, Assis R Morris W, Mi-yakawa MF. Outbreak of clostridial myocarditis in calves. Vet Rec 2003; 152: 134-6. 9. Awogbade MO. The nature of pastoral development: Nigeria's case in perspective. In: Proceedings of national conference on pastoralism in Nigeria. Za-ira, 1988. 10. Abdu PA, Jagun, AG, Gefu JO, Mohammed AK, Alawa CBI, Omokanye AT. Ethnoveterinary practices, research and development. In: Proceedings of National Animal Production Research Institute (NAPRI), Ahmadu Bello University Zaria, Nigeria, 2000: 163. 44 N. M. Useh, S. Adamu, N. Ibrahim, A. J. Nok, K. A. N. Esievo IZBRUHI šUMEčEGA pRISADA (GANGRENA EMpHYSEMATOSA) pRI GOVEDU V SEVERNI NIGERIJI (1964 - 2003) N. M. Useh, S. Adamu, N. Ibrahim, A. J. Nok, K. A. N. Esievo povzetek: Šumeči prisad je endemična bolezen v razvitih ter državah v razvoju in je dobro poznan vzrok finančnih izgub pri rejcih goveda v več delih sveta. Bolezen je prevladujoča v Združenih državah Amerike, Indiji in drugih delih Azije, v Evropi, Latinski Ameriki in Afriki. V Nigeriji so bile ekonomske izgube pri govedu Zebu ocenjene na 4,3 miljona dolarjev (~600 milijonov nigerijskih niarov). Z retrospektivno študijo smo želeli potrditi trenuten status bolezni v severni Nigeriji in ugotovili, da je tradicionalni način govedoreje s sezonskim potovanjem čred pastirjev Fulani v ruralnih delih Nigerije, ki premorejo večino živine v državi, velik izziv za kontrolo bolezni. Šumeči prisad je v Nigeriji še vedno endemična bolezen. Učinkovito kontrolo pa bo mogoče doseči le z izboljšanjem tradicionalne reje govedi nomadov na podeželskih področjih Nigerije. Ključne beside: šumeči prisad; Severna Nigerija; izbruhi; retrospektivna študija Slov Vet Res 2010; 47 (2): 45-56 UDC 579.62:576.851.49:616-078:577.2:631.854 Original Scientific Article DETECTION OF SALMONELLA IN pOULTRY FAECES BY MOLECULAR MEANS IN COMpARISON TO TRADITIONAL BACTERIOLOGICAL METHODS Darja Kušar, Mateja Pate, Jasna Micunovic, Vojka Bole - Hribovšek, Matjaž Ocepek* Institute of Microbiology and Parasitology, Veterinary Faculty, Gerbičeva 60, 1115 Ljubljana, Slovenia Corresponding author, E-mail: matjaz.ocepek@vf.uni-lj.si Summary: Comparison of traditional (cultivation-dependent) and molecular (nucleic acid-based) bacteriological methods was performed to detect Salmonella in reference capsules containing quantified amounts of Salmonella enterica subsp. enterica serovars Panama, Typhimurium or Enteritidis and in poultry faeces that was naturally contaminated with Salmonella or Salmonella-negative but spiked with reference materials. Traditional techniques were performed according to ISO 6579 using different enrichment (MSRV, MKTTn and RVS, respectively) and isolation plating media (XLD, BGA and Rambach agar, respectively). Molecular detection was preceded by the pre-enrichment step. Detection efficiency of two DNA isolation kits, namely High Pure foodproof I Kit (Roche Diagnostics, Germany) and QIAamp DNA Stool Mini Kit (Qiagen, Germany), in combination with classical and real-time PCR assay was compared. Results showed that traditional and molecular detection of Salmonella was unambiguous for reference control capsules, but was hindered for faecal samples. RVS medium was less appropriate than MSRV and MKTTn. Combination of MKTTn with Rambach agar plates generated the highest number of positive results with traditional approach. However, recommendation of using the semisolid MSRV medium was confirmed as it enabled detection of Salmonella in high proportion of samples, which was the least variable depending on the selection of isolation plating media. In contrast to culture-based methods, the molecular approach, especially a combination of High Pure foodproof I Kit and real-time PCR assay, enabled successful detection in all Salmonella-positive samples and should therefore be considered an important supplement to traditional protocol for Salmonella detection in foodstuffs. Key words: Salmonella; foodstuffs; faeces; detection; cultivation; molecular; PCR Introduction The routine microbiology laboratories for detection of different bacterial pathogens are complementing traditional diagnostic assays with continually evolving molecular techniques as they are not negatively affected by the presence of growth inhibitory compounds and enable rapid detection (1-3). Surveillance of alimentary zoonoses, diseases that are transmitted from animals to humans through food, and early detection of their causative agents in food producing animals and their environment are very important for the assurance of safe food. Food safety is a growing public health issue, since it was Received: 27 January 2010 Accepted for publication: 23 June 2010 estimated that up to 30 % of the population in industrialized countries is suffering from foodborne illnesses (4). Salmonelloses are the second most frequently reported human zoonoses in the European Union and can cause relatively vast economic damage due to chronic effects of the infections (5). The common reservoir of Salmonella is the intestinal tract of animals, however they can be detected in a wide variety of foodstuffs and food ingredients (5). Animal-to-human transmission occurs when bacteria are introduced into the food preparation process or through direct contact with infected animals and faecally contaminated environments. In-country laboratory-based monitoring of food-borne pathogens is being promoted (4). Traditional microbiological methods offer standardized proce- 46 D. Kusar, M. Pate, J. Micunovic, V. Bole - Hribovsek, M. Ocepek dures for their detection (e.g. ISO standards), but are time consuming and not always compatible with short-time-to-result demand. Therefore, food microbiology aims for supplementation of classical methods with molecular techniques based on detection of mi-crobial nucleic acids in foodstuffs, which shorten the analysis time and lower the limit of detection. It was shown previously that the polymerase chain reaction (PCR) has a great potential to speed-up the detection of Salmonella in food (6) and can be performed in a manner equivalent to the standard ISO 6579 culture method, which is set as the golden standard for Salmonella detection in food and feedstuffs (7,8). The main objective of our study was to evaluate the detection efficiency for different contamination levels of Salmonella spp. in the presence of competitive microorganisms. As a complex matrix, poultry faeces was selected for the starting material. The use of molecular methods polymerase chain reaction (PCR) and real-time PCR was compared to traditional, cultivation-dependent bacteriological methods. Materials and methods Reference materials (RMs) and poultry faeces were used. The RMs consisted of gelatin capsules containing a quantified amount of sub-lethally injured Salmonella strains of serovars Panama (SPan), Typhimu-rium (STM) or Enteritidis (SE) as spiked spray dried milk prepared by the Community Reference Laboratory (CRL) for Salmonella (9). The levels of contamination were SPan 5 (5 colony forming particles per capsule [cfp/caps]), STM 10 (10 cfp/caps), STM 100 (100 cfp/caps), SE 100 (100 cfp/caps) and SE 500 (500 cfp/ caps). Faeces, negative or positive for Salmonella spp., and reference capsules were stored at -20° C till use. We examined (i) 24 poultry faecal samples (numbered FC-1 to FC-24; 10 g each, negative for Salmonella spp.) in combination with a blank capsule (five samples) or a capsule containing STM (five samples STM 10 and four samples STM 100) or SE (five samples SE 100 and five samples SE 500), (ii) 20 faecal samples which were naturally contaminated with Salmonella and not spiked with capsules (numbered F-1 to F-20; 10 g each), and (iii) 10 control samples (numbered C-1 to C-10; no faeces added) consisting of two blank, two SPan 5, three STM 10 and three SE 100 capsules, respectively. In addition, two negative control samples were examined: procedure control (i.e. C-11; no faeces or capsule added) and negative faeces control (i.e. C-12; 10 g of faeces negative for Salmonella spp.). The sample outline is summarized in Table 1. Traditional bacteriological methods Cultivation-dependent detection of Salmonella was performed according to ISO 6579:2002 (10), including Amendment 1:2007 (11), and the instructions provided by CRL for Salmonella (9). In brief, detection involved the following stages: (i) overnight sample pre-enrichment in a nonselective broth medium BPW (Buffered Peptone Water), (ii) 24- (for the first isolation) and 48-hour (for the second isolation) enrichment in selective broth media MKTTn (Muller Kauffmann TetraThionate-novobiocin broth), RVS (Rappaport Vassiliadis medium with Soya) and MSRV (Modified Semi-solid Rappaport Vassiliadis medium; 11), (iii) isolation of colonies presumed to be Salmonellae on solid selective and differential plating media BGA (phenol red/Brilliant Green Agar), XLD (Xylose-Lysine-Deoxycholate agar) and R (Rambach agar; 12), and (iv) biochemical screening of Salmonella isolates on the confirmation media TSI (Triple Sugar/Iron agar), UA (Urea Agar) and LDC (1-Lysine decarboxylation medium). If colonies grown on the isolation media were not well separated, single colony isolation was performed on NA (Nutrient Agar) plates after 24-hour incubation at 37° C and followed by the aforementioned confirmation. For each of the samples from the three selective enrichment media, at least one individual colony, considered to be typical or suspect for Salmonella, was examined biochemically. If the selected colonies were not confirmed as Salmonella, maximum of five additional typical colonies were tested from the original isolation medium stored at 5° C. Sample was denoted with positive result if growth of Salmonella spp. was present at least on one of the isolation media. If not stated otherwise, media and reagents were prepared according to Annex B of ISO 6579:2002. Molecular methods Molecular detection of Salmonella involved the isolation of microbial DNA that was followed by Salmonella-specific PCR and real-time PCR assays. DNA was extracted from 1 mL of the pre-enrichment broths using two different commercial kits, namely the High Pure foodproof I Kit (Roche Diagnostics, Germany) and QIAamp DNA Stool Mini Kit (Qiagen, Germany) according to the manufacturers' instructions. The latter was not applied for samples devoid of faeces. Microbial DNA was subjected to PCR amplification using Salmonella genus-specific primers ST11 and ST15 (13) that were proved as appropriate Detection of Salmonella in poultry faeces by molecular means in comparison to traditional bacteriological methods 47 Table 1: Outline of the samples used for the study: control samples (C), naturally contaminated faecal samples (F), and faecal samples supplemented with capsules (FC). Sample name Faeces Capsule Sample name Faeces Capsule Sample name Faeces Capsule C-1 / blank F-1 pos / FC-1 neg SE 100 C-2 / SPan 5 F-2 pos / FC-2 neg blank C-3 / blank F-3 pos / FC-3 neg STM 100 C-4 / SE 100 F-4 pos / FC-4 neg SE 100 C-5 / STM 10 F-5 pos / FC-5 neg STM 100 C-6 / STM 10 F-6 pos / FC-6 neg SE 500 C-7 / STM 10 F-7 pos / FC-7 neg SE 500 C-8 / SE 100 F-8 pos / FC-8 neg blank C-9 / SE 100 F-9 pos / FC-9 neg SE 500 C-10 / SPan 5 F-10 pos / FC-10 neg SE 100 C-11 / / F-11 pos / FC-11 neg STM 10 C-12 neg / F-12 pos / FC-12 neg blank F-13 pos / FC-13 neg SE 100 F-14 pos / FC-14 neg STM 10 F-15 pos / FC-15 neg STM 100 F-16 pos / FC-16 neg blank F-17 pos / FC-17 neg STM 10 F-18 pos / FC-18 neg SE 500 F-19 pos / FC-19 neg STM 10 F-20 pos / FC-20 neg blank FC-21 neg STM 10 FC-22 neg SE 100 FC-23 neg SE 500 FC-24 neg STM 100 Legend: neg: faeces negative for Salmonella, pos: faeces positive for Salmonella, SPan: Salmonella Panama (SPan 5: 5 cfp/caps), STM: Salmonella Typhimurium (STM 10 and STM 100: 10 and 100 cfp/caps, respectively), SE: Salmonella Enteritidis (SE 100 and SE 500: 100 and 500 cfp/caps, respectively), blank: no cfp/caps for the confirmation of Salmonella-colonies obtained by the standard ISO 6579 culture method (14). Amplification was performed according to the optimized touchdown protocol as described previously (15). Real-time PCR was performed using the Light-Cycler foodproof Salmonella Detection Kit (Roche Diagnostics, Germany) according to the manufacturer's instructions. Briefly, a 20-^l reaction mixture was composed of foodproof Salmonella enzyme solution containing FastStart Taq DNA polymerase, internal amplification control (IC), master mix containing primers and hybridization probes specific for Salmonella DNA and Salmonella-specific IC, and 5 ^l of sample DNA, foodproof Salmonella positive control template or PCR-grade water as negative control. Amplification was performed by LightCy-cler 1.2 Real-Time PCR System (Roche Diagnostics, Germany). The inclusivity of foodproof Salmonella master mix for the Salmonella genus and exclusiv- ity for other genera was extensively tested by the manufacturer. Results Reference materials vs. faecal samples Detection of Salmonella in reference materials was unambiguous with no false negative or positive results regardless of the employed method (samples C in Tables 2 and 3). Both the traditional and the molecular methods in all the tested combinations were equally appropriate with detection limit of 5 cfp/ sample or lower. On the other hand, detection of Salmonella in samples containing poultry faeces was limited as it depended on the method type and the level of Salmonella contamination. Detection limit was impaired for traditional methods (above 10 cfp/ sample) in comparison to molecular methods (10 48 D. Kusar, M. Pate, J. Micunovic, V. Bole - Hribovsek, M. Ocepek cfp/sample or lower), since one sample of naturally contaminated faeces (F-2) and eight faecal samples containing reference capsules (all five samples supplemented with STM 10 [FC-11, FC-14, FC-17, FC-19 and FC-21], two samples with STM 100 [FC-3 and FC-5], and one sample with SE 100 [FC-1]) were denoted falsely negative but tested positive when molecular detection was performed (Table 3). To detail, Salmonellae from capsules STM 10 were detected by traditional methods only from the control samples, but not in samples containing faecal material. However, they were detected in all samples FC (100 %) when applying the molecular methods (particularly, real-time PCR in combination with High Pure food-proof I Kit) (Table 4). Salmonellae from samples FC supplied with higher cfp number of Salmonella Ty-phimurium (STM 100) or with Salmonella Enteri-tidis in equivalent cfp number (SE 100) were detected in marked proportions of samples (50 % or 80 %, respectively) by traditional bacteriological methods, but in all cases (100 %) when molecular approach was employed (Table 4). Salmonellae from SE 500 capsules with the highest Salmonella-contamination level were detected in all samples FC by both the traditional (with MSRV enrichment only) and the molecular approach (Table 4). Salmonellae from faecal samples F were detected in 95 % and in 100 % of cases applying traditional (particularly, MKTTn in combination with Rambach agar) and molecular (all combinations) methods, respectively (Table 4). To summarize, detection of Salmonella spp. in reference materials succeeded over the entire experimental range of contamination levels and did not depend on the method type, but was impaired in faecal samples when traditional approach was employed, enabling detection in 30 out of 39 Salmonella-positive faecal samples (19/20 for samples F and 11/19 for samples FC, respectively) (Table 3). No samples supplied with blank capsule and negative control samples tested falsely positive. Traditional bacteriological methods vs. molecular methods Results of cultivation-dependent detection of Salmonella in faecal samples after 24 and/or 48-hour incubation showed that MKTTn and MSRV selective enrichment media generated less falsely negative results than RVS (Tables 2 and 3). MKTTn enabled detection in 19 out of 20 Salmonella-positive samples F (19/20) and 10 out of 19 Salmonella-positive samples FC (10/19), MSRV in 17/20 and 9/19, and RVS in 14/20 and 3/19 samples, respectively. By traditional approach, 13 of 20 Salmonella-positive samples F and only 2 of 19 Salmonella-positive FC samples (FC-15 and FC-23) tested positive from all three enrichment media. All the rest tested positive from two (5/20 for samples F and 7/19 for samples FC) or only one (1/20 and 2/19, respectively) selective enrichment medium (Table 3). In the majority of faecal samples, detection failed from RVS enrichment regardless of the isolation medium, in particular with faecal samples FC that were contaminated with serovar Enteritidis (Tables 2 and 3). Although serovar Typhimurium was detected after RVS enrichment in 50 % of FC samples supplied with STM 100, detection of serovar Enteritidis of the same cfp number (SE 100) was completely absent (0 %) or markedly impaired (detection in 20 %) when supplied in higher cfp number SE 500 (Table 4). The highest number of true positive results (19/20 samples F and 9/19 samples FC, Table 2) was attributed to MKTTn when in combination with Rambach agar. However, the lowest number of positive results was also attributed to MKTTn, namely when it was combined with XLD or BGA isolation plating medium (6/20 or 0/20, respectively for faecal samples F; Table 2). Isolation of Salmonella colonies originating from different selective enrichment media was not affected by the selection of plating media with the above mentioned exception of MKTTn, which was likewise the only selective medium that generated higher number of positive cases when results obtained from all the three isolation plating media were combined in comparison to results obtained from individual isolation media (4 positive out of 5 samples FC supplied with SE 100 [4/5] for combined results vs. 3/5 for individual combinations of isolation media with MKTTn, respectively; Table 2). MKTTn and MSRV enrichments were comparably effective regarding the detection level, however only MSRV enabled detection of Salmonella in all samples FC supplied with SE 500 (Tables 2 and 3). That was the only case for faecal samples where detection by traditional approach was not limited. Results of molecular detection of Salmonella showed that it was successful, since the presence of Salmonella was confirmed not only in control samples but also in all faecal samples (detection level of 100 %; Table 4). However, differences were observed for samples FC regarding the procedure for DNA isolation (using the High Pure foodproof I Kit [protocol HP] or QIAamp DNA Stool Mini Kit [protocol S]) and Detection of Salmonella in poultry faeces by molecular means in comparison to traditional bacteriological methods 49 the type of PCR reaction (classical PCR or real-time PCR) (Tables 3 and 4). Protocol S enabled detection in more FC samples in comparison to protocol HP when using classical PCR detection (15/19 for S vs. 10/19 for HP), but protocol HP in more samples when performing real-time PCR (19/19 for HP vs. 15/19 for S) (Table 3). Protocol HP enabled detection of Salmonella in all samples FC, while protocol S generated false negative result for one sample supplemented with STM 10 capsule (FC-19) and for one with SE 100 (FC-1) (Table 3). Real-time PCR enabled detection in all samples FC, while classical PCR failed for three samples (FC-1, FC-19 and FC-22) (Table 3). The highest number of positive cases using the molecular approach for samples FC, where detection efficiency depended on the method type in con- trast to samples F, was obtained by protocol HP in combination with real-time PCR (19/19), followed by protocol S in combination with either classical or real-time PCR (15/15, however different samples were denoted as positive in four cases [FC-10, FC-11, FC-13 and FC-22] depending on PCR reaction type) (Table 3). The highest number of false negative results was obtained by protocol HP in combination with classical PCR that generated 10 positive cases out of 19 Salmonella-positive samples FC (10/19) (Table 3). When classical PCR reaction tested negative, in all but two such cases (samples FC-1 and FC-19 that failed to test positive when applying protocol S) real-time PCR tested positive (Table 3). When applying protocol S, two samples (FC-11 and FC-13) tested positive solely by classical PCR (Table 3). Table 2: Results of traditional detection of Salmonella: number of positive samples after 24 and/or 48-hour incubation in selective enrichment media MKTTn, RVS and MSRV as detected on individual isolation media BGA, XLD and R respectively MKTTn RVS MSRV s e ° ^ BGA XLD R t» poses ° ^ BGA XLD R t» poses BGA XLD R t» poses o 5 Z m o 5 Z m o 5 Z m o 5 Z m 2 0 0 0 0 0 0 0 0 0 0 0 0 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 20 0 6 19 19 14 14 14 14 17 17 17 17 5 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 0 0 0 0 0 0 4 1 1 2 2 2 2 2 2 1 1 1 1 5 3 3 3 4 0 0 0 0 3 3 3 3 5 2 4 4 4 1 1 1 1 5 5 5 5