ISSN 15804003 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK ■ Volume a 50 4 Slov Vet Res • Ljubljana • 2013 • Volume 50 • Number 4 • 145-203 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Volume 50 Slov Vet Res • Ljubljana • 2013 • Volume 50 • Number 4 • 145-203 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 Assistants to Editor / pomočnici urednika: Valentina Kubale Dvojmoč, Klementina Fon Tacer Editorial Board / uredniški odbor: Robert Frangež, Polona Juntes, Matjaž Ocepek, Alenka Seliškar, Modest Vengušt, Milka Vrecl, Veterinary Faculty University of Ljubljana / Veterinarska fakulteta Univerze v Ljubljani; Vesna Cerkvenik, Reziduum s.p. 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, Paton and Martin Veterinary Services, Adegrove, British Columbia; Gerry M. Dorrestein, Dutch Research Institute for Birds and Exotic Animals, Veldhoven, The Netherlands; Sara Galac, Utrecht University, 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; Henry Stämpfli, Large Animal Medicine, Department of Clinical Studies, Ontario Veterinary College, Guelph, Ontario, Canada; Frank J. M. Verstraete, University of California Davis, Davis, California, US; Thomas Wittek, Veterinärmedizinische Universität, Wien, Austria 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 Book Agency Sofinancira: Javna agencija za knjigo Republike Slovenije ISSN 1580-4003 Printed by / tisk: DZS, d.d., 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 2013; 50 (4) Original Scientific Articles Kastelic M, Špehar M, Barac Z. Productivity of milk and milk composition of Istrian sheep in Croatia and Slovenia...............149 Premrov Bajuk B, Drobnič Košorok M, Malovrh T. The efect of a peptide inhibitor of cysteine peptidases produced by the dermatophyte Trichophyton mentagrophytes on a mouse immune system..................................................157 Biasizzo M, Vadnjal S, Mičunovic J, Henigman U, Kirbiš A. Estimation of Yersinia enterocolitica prevalence in slaughtered pig tonsils in Slovenia by using three cultural isolation procedures...........................................167 Dobeic M, Ocepek M, Kožuh-Eržen N, Pintarič Š, Cerkvenik-Flajs V, Gobec I. Doramectin degradation and bacterial community during sheep manure composting...................................................................................173 Žan Lotrič M, Gorjanc G, Kompan D. Geographical distribution of sheep and goat breeds in Slovenia............................183 Štukelj M, Toplak I, Valenčak Z. An attempt to eliminate porcine reproductive and respiratory syndrome (PRRS) by serum inoculation on small pig farm............................................................................................193 Author Index Volume 50, 2013...............................................................................................................201 Slov Vet Res 2013; 50 (4): 149-56 UDC 636.32/.38.08: 637.12(497.12/.13) Original Scientific Article PRODUCTIVITY OF MILK AND MILK COMPOSITION OF ISTRIAN SHEEP IN CROATIA AND SLOVENIA Miran Kastelic1*, Marija Špehar2, Zdravko Barač2 University of Ljubljana, Biotechnical Faculty, Animal Science Department, Groblje 3, 1230 Domžale, Slovenia, 2Croatian Agricultural Agency, Ilica 101, 10000 Zagreb, Croatia Corresponding author, E-mail:miran.kastelic@bf.uni-lj.si Summary: The effects of country, flock nested within country, litter size, parity, lambing season, interval between lambing and the first milk recording, and year of lambing on production of Istrian sheep in Slovenia and Croatia was studied in period between 2005 and 2009. Data consisted of 3,347 lactations in Croatia and 1,788 in Slovenia that were collected as a part of separate breeding programs. The Croatian animals produced 124.87 kg total milked milk (TMM) with 7.31 % of fat (FC) and 6.05 % of protein content (PC). Slovenian ewes produced only 79.33 kg TMM with 7.25 % FC and 5.87 % PC. The increase of TMM was 5.40 kg per year (P<0.05). Ewes that lambed between October and the end of January had the highest TMM, fat yield (FY), and protein yield (PY). The productivity did not differ between countries. Flock effect nested within country caused differences (P<0.05) in TMM, FC, and PC. The effect considered genetic, management and climatic conditions (average temperature, above mean sea level (AMSL), and precipitation). Productivity was not affected by the litter size. Higher productivity was observed in ewes reared in the Mediterranean area. The exception was one flock coming from harsher continental conditions. The productivity in such conditions can be improved with earlier lambing, high quality forage before the grazing season and with an extra feeding on pasture after the dry weather begins. Key words: Istrian sheep; dairy production; Croatia; Slovenia; environmental effects Introduction Istrian sheep (Istarska ovca, Istrska pramenka or Istriana) is an autochthonous dairy sheep breed originating from Istrian Peninsula and its hinterland. In the year 2009, estimated population size was between 2,600 and 3,100 animals in Croatia, 1,150 animals in Slovenia, and 920 animals in Italy (1). The population of Istrian sheep was divided after the Second World War to Yugoslav and Italian subpopulations. At the beginning of 1990s, the Yugoslav subpopulation was further split between Croatia and Slovenia. Migration of animals between the two countries has nearly stopped. Geographic and climatic characteristics are changing very rapidly considering a rather small area. The altitude of breeding ranges from the sea to 1,000 meters above mean sea level (AMSL). The border between Slovenia and Croatia is also geographical. The Croatian part of breeding area consists mostly of the Istrian peninsula, therefore under influence of the sea. The temperature and the yearly rainfall levels are changing with the AMSL increase and with the distance from the sea. Pula (AMSL 30 m) on the southern coast of Istria has an average temperature of 14.7oC [v "nogo" na ustreuno stran : The climatic data for period 1990 - 2010 were provided by Croatian Meteorological and Hydrological Service and Slovenian Environment Agency]. The coldest months are January and February with an average temperature of 6.0oC and 6.3oC respectively. Pazin (AMSL 242 m) is located in the middle of Istrian Peninsula with the average temperature of 11.5oC. Slovenian breeding area is placed around Ilirska Bistrica (AMSL 421 m), which is situated outside the Istrian peninsula in the typical Karstic valley. It is not influenced by Mediterranean climate. The average temperature is 9.9° C. The annual sum of precipitation for all three areas reaches the peak in winter months: 828 mm in Pula, 1,065 mm in Pazin and 1,356 mm in Ilirska Bistrica. In Istria, the pasture is available during the winter and especially in early spring period. However, in Ilirska Bistrica and its surrounding mountains, snowing is common and pasture season starts in April. The dry period lasts in Istria (Pula and Pazin) from spring to middle autumn, and from the middle July to the end of August in Slovenia (Ilirska Bistrica). Poor summer vegetation is a consequence of the low summer precipitation and porous Karstic pastureland on limestone bedrock with a very thin layer of soil. Observed differences in climatic conditions between Croatian and Slovenian breeding area were not very important in the past. The larger flocks from Slovenian hinterland moved to the winter transhumance pasture (2) to the seaside of Trieste and Istria. At the beginning of April, the flocks returned home and prolonged good pasture to the middle of July. The exploitation of Mediterranean and continental vegetation was a comparative advantage of flocks from Slovenian territory. This practice was abandoned during the Second World War and since then animals are kept in the stables during the winter period. Under such conditions, early lambings are possible only with hayof highquality and a supplement of concenfrates. Latelambings causedshorter lactation end honssquentlylowea paodettion ie lactation.Most of aioaenrwiefrianshesgflocks remained on Seen sraductivesgricnnurtl Inane, opposite fs whai wne oboecved in Italo, Spvin mod France. The aim of this study was to evaluate the effects of country, flock, lambing season, parity, litter size, interval between lambing and first milk recording, and year of lambing on the productivity of Istrian sheep. Material and methods Data were collected as part of separate breeding programs in Croatia and Slovenia for the period from 2005 to 2009. Breeding programs were established in the 1990-ties, 20 to 30 years later as in France, Italy or Spain (3). Daily milk yield and milk components were recorded according to the ICAR guidelines (4) using the AT4 method. The total number of recorded lactations was 3,347 in Croatia and 1,788 in Slovenia. The number of lactations per year did not change in the studied period in Slovenia. However, the number of recorded lactations per year increased more than six times (from 195 in 2005 to 1,266 in 2009) in Croatia. The total milked milk (TMM) was calculated from recorded daily milk yield using test interval method (4). Fat (FY) and protein yields (PY) in the milking period were calculated with remodelled formula for TMM where daily yields of fat and protein were used instead of daily milk yield. Fat (FC) and protein (PC) contents in milking period were calculated from TMM, FY, and PY. The exact weaning date was not recorded. Therefore, the start of the milking period was set 15 days before the first milk record. Lambing season (S) starts on the 1st October. The season was expressed as continuous variable and calculated as the number of days after October 1st. Data were analysed using the following statistical model: yse = 0.98) and the mean recovery of the analytical method > 80% were assured for the analysis. To approach the real values, the measured concentrations were corrected for mean recoveries of the respective series and used as final results. Bacterial count, PCR amplification and T-RFLP analysis for identification of microbial communitie. Bacterial cell counts were determined by quantitative estimations of direct counting using fluorescence microscopy. For this purpose LIVE/ DEAD® BacLight™ (Invitrogen, USA) dye was used, which selectively stained live and dead bacteria. Microbial communities were identified by means of a polymerase chain reaction (PCR) with subsequent restriction analysis (15). Prior to PCR, microbial DNA was extracted using the PowerSoil™ DNA Isolation Kit (Mo Bio Laboratories, USA). Fluorescence-labelled eubacterial primers 27F (16) and 1392R (17, 18) were used in PCR, targeting the 16S rRNA gene. PCR amplification was performed using the GeneAmp PCR System 2700 (Applied Biosystems, USA). The obtained PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Germany) according to the manufacturer's instructions and were subjected to HhaI, MspI and RsaI (Promega, USA) restriction. Restriction fragments were cleaned using the QIAquick Nucleotide Removal Kit (Qiagen, Germany). The length of fluorescence-labelled terminal restriction fragments was determined using the ABI PRISM 310 genetic analyser (Applied Biosystems, USA), employing standard size marker 2500 ROX, and the GENEMAPER 2.0 analysis software programme (Applied Biosystems, USA). Results were compared with restriction patterns from the public database using the web based tool MiCA (Microbial Community Analysis, http://mica. ibest.uidaho.edu/trflp.php) (MiCA) (19). Temperature, moisture, dry matter, pH value, nitrogen and carbon determination Temperature and air flow in the composted substrate were monitored using PT 100 and hot wire probe (Testo 450, Germany) directly in the compost and analysed using the computer software VisiDAQ™ runtime (Advantech®, Sunnyvale, USA). The moisture content and dry matter of the compost were analysed by drying (for 24 hours at 105°C) and weighing. The content of nitrogen and carbon were determined by a Vario MAX CNS analyzer (Elementar, Hanau, Germany) after incineration of the sample at 900°C. The pH values of a liquid phase of the material were measured by a pH meter (Hanna HI 221, Germany). Data evaluation Statistical evaluation of results was carried out by ANOVA, t-test and correlation analysis using the SPSS (Statistical Package for the Social Sciences) 17.0 statistics software (Rainbow Technologies, USA). The Pearson product-moment correlation and linear regressions ABS versus time were accepted for r >0.95, and values of P <0.05 of the slopes were considered statistically significant. Results Temperature profile, moisture content, carbon, nitrogen, and pH value characteristics Temperatures of the compost in bioreactors varied from 24 to 69.3°C. (Table 2). The mean moisture content of the compost was 612g/kg before composting (day 0) and decreased to 461g/ kg at the end of composting (day 21) (Table 2). The ratio of carbon (C) : nitrogen (N) in the compost mixtures decreased significantly (P<0.05) from the start to the end of composting: day 0 = 27:1, day 21 = 21:1. The pH value in the compost mixtures within 21 days of composting ranged from 7.4 to 6.4. Temperature / moisture levels in the compost samples with additional water were constant for 21 days (21 ± 0.5°C/ 0,34, 0,53, 0,71 and 0,96 ± 0,05 ml/g). The C : N ratio and the pH value were the same as in the compost in bioreactors. Temperature in the sterilised compost samples was constant for 21 days (68 ± 0.2°C), The mean moisture level in the sterilised compost was 670.9g/kg before composting (day 0) and decreased to 653.0g/kg by the end of composting (day 21). The C : N ratio in the sterilised compost mixtures was the same as in the compost in bioreactors. The pH in the sterilised compost mixtures during 21 days of composting ranged from 6.8 to 7.7. Degradation of doramectin in compost mixtures The results of tests showed that the mean concentration of doramectin in all samples (K1-K3) declined significantly (P<0.05) by 31.841.8% during 21 days of composting. The mean concentrations of doramectin in samples with initial concentration K1 and K2 declined significantly (P<0.001) with the time of composting, thus from day 0 to day 21 the mean concentration of doramectin declined by 37.0-37.8%. The most pronounced decrease in doramectin (P<0.001) in the samples K1 and K2 was observed between day 0 and day 14 of composting (41.9-42.6%). Table 2: Temperature profile and moisture content of the compost mixtures (K0-K3) in three experimental batches (P1-P3). Temperature (°C) Moisture content (g/kg) Batch n Lowest Highest Average Standard deviation day 0 7 14 21 P1 21 24.0 69.3 55.0 12.9 634.9 511.6 545.6 486.8 P2 21 25.7 68.3 56.3 11.5 632.8 466.7 459.1 555.4 P3 21 29.2 65.1 48.9 12.8 572.0 517.6 517.3 342.1 Table 3: Doramectin concentrations in the compost and sterilised compost mixture samples (n) with initial concentrations K1-K3, according to the sampling time (day 0 - day 21) Doramectin concentration day n Lowest Highest Average Standard deviation Average doramectin reduction from day 0 (%) (pg/kg dry matter) K1 0 6 962 1232 1108.3 100.5 / 7 6 777 1202 1022.3 161.1 7.7 14 6 606 714 643.2 41.2 41.9 21 6 595 845 698.7 90.4 30.8 K2 0 6 1709 2134 1878 152.3 / 7 6 1147 1969 1470 272.6 21.8 14 6 936 1352 1078 171.6 42.6 21 6 1014 1330 1167 127.1 37.8 K3 0 6 3896 6056 4625 998 / 7 6 2772 3146 2980 154 35.5 14 6 2245 3444 2976 486 35.6 21 6 1112 3584 2688 1102 41.8 Sterilised compost mixture 0 6 906 1986 1428 / / 7 6 748 1932 1316 / 7.8 14 6 660 1667 1312 / 8.2 21 6 697 1863 1398 / 2.1 The highest decrease of doramectin (41.8%) was observed in the compost samples with initial concentration K3 but this reduction was insignificant. Nevertheless, doramectin decrease in samples K3 was significant (P<0.05) (35.6%) from day 0 to day 14 of composting (Table 3). Degradation of doramectin in sterilised compost mixtures The degradation of doramectin in sterilised compost mixtures was 2.1% and within the accuracy of the respective analytical method. Degradation of doramectin in relation to compost moisture Declination of doramectin and reduction of moisture in compost mixture samples (K1 -K3) during the composting (0 - 21 day) were in significant correlation (P<0.001; r=0.512), thus higher concentrations of doramectin were found in compost with higher concentrations of water. The same was asceratained in the laboratory test since mean values of doramectin were significantly (P<0.05) lower in samples with the addition of water. Number of viable bacteria in compost mixture samples Irrespective of doramectin concentration (K1-K3) in all groups of samples, the mean count of viable bacteria was lowest prior to composting (day 0) and highest on day 7 of composting. The difference was significant (P<0.05). The number of viable bacteria gradually decreased in the period between days 7 and 21. The mean total count (n = 7.65 x 108) of viable bacteria in the samples without doramectin (K0) was higher in comparison to the samples with doramectin (K1-K3) and steadily grew from the day 0 to day 21 (Fig. 1). Phylogenetic analysis of bacterial community in compost mixture samples Most taxonomic groups of bacteria in the samples (K0-K3) belonged to the phyla Fibrobacteres. Other bacteria belonged mostly to the rumen bacteria from the phylum Bacteriodetes and its genera Porphyromonas, Tannerella, Prevotella, Cytophaga, Bacteroides, Alistipes, Microscilla and Rikenella and to the unidentified bacteria as presented in Figure. 2. Regarding the T-RFLP analysis among all thedoramectin supplements (K0-K3), 2.7 to 11.6 times higher number of molecular fragments were found using the MspI enzyme compared to the number of fragments acquired by the enzyme HhaI, and 53.6 to 128.8 times higher than that acquired by the enzyme RsaI. Identification of fragments using the enzyme MspI indicates the presence of numerous potential bacterial taxa belonging to the phylum Proteobacteria, genus Pseudomonas, phylum Actinobacteria, genus Rhodococcus and to the phylum Firmicuites, genus Streptococcus, Enterococcus and Lactobacillus. The potential presence of bacteria belonging to the phylum Spirochaees was also determined. Discussion The results of the experiment indicate a gradual reduction of doramectin in thermophilic phase of composting and suggest an impact on the number of live bacteria, but not on their taxonomic diversity. Namely the average concentration of doramectin in all the samples (K1-K3) significantly (P < 0.05) decreased during 21 days of composting (mainly between days 7 and 14) meanwhile doramectin reduction did not differ significantly among samples K1-K3. Doramectin degradation was obviously associated with the biological processes of composting, since the test results of doramectin degradation in sterilised compost mixtures demonstrated that in the absence of viable bacteria the degradation of doramectin was Figure 1 (K0-K3) sampling days Count of viable bacteria (n) in the compost mixture samples depending on doramectin concentration uncultured Deltaproteobacteriaceae pig manure bacteria uncultured Spirohaeta uncultured Porphyromonas uncultured Cytophagales Persicobacter diffluens Rikenella microfusus Persicobacter diffluens Microsdikl sp. Flexibacter aggregans. Cyclobacterium marinum Bacteroidetes bacterium bacteria like Bacteroides Alkaliflexus imshenetskii Alistipes putredinis Algoriphagus ratkowskyi uncultured bacteria Treponema unidentified bacteria bacteria uncultured soil bacteria uncultured compost bacteria Spirochaeta uncultured Cytophaga rumen bacteria uncultured Prevotella Bacteroidetes uncultured Bacteroidetes uncultured anaerobic bacteria Tannerella Porphyromonas unidentified rumen bacterid uncultured Bacteroides Prevotella Bacteroides uncultured bacteria uncultured rumen bacteria Fibrobacteres =H —I 0 50 100 Records of bacterial taxa 150 □ Day 21 □ Day 14 ■ Day 7 ■ Day 0 Figure 2: Records of bacterial taxa (taxonomic units) in the compost mixture samples according to the time of composting very low. The highest counts of viable bacteria in bioreactors were observed in the samples without doramectin (K0) compared to the samples with doramectin (K1-K3). Namely the viable bacterial population did not alter significantly in the samples with doramectin addition (K1-K3) while the number of viable bacteria in the samples without addition of doramectin (K0) slightly increased with the time of the composting process (day 0 to the 21st day). Beside biological degradation of doramectin it can be hypothetically concluded that declination of doramectin was related to the moisture content of the compost. It is assumed that the reason for this relationship is based on the physicochemical properties of the compost, even though doramectin has low water solubility and low vapour pressure (20). Therefore it was assumed that the mean doramectin concentrations were reduced in accordance with decreasing water content in the compost. This hypothesis was confirmed by significant (P<0.05) correlation between doramectin content and moisture content in the samples K3 (r=0.969) K1 and K2, although in insignificant positive correlation (r>0.80). This was also proved in the laboratory test in which the mean values of doramectin correlated significantly (P<0.05) with the amount of added water, since doramectin concentrations in the samples with no addition of water were lower by 63-118% than those in the samples to which some water was added. On the other hand, concentrations of doramectin in sterilised compost mixtures remained almost the same where the mean moisture level remained almost constant throughout the investigation. These results are comparable to the previous studies (21, 7) which demonstrated a parallel increase in the concentrations of avermectins and the moisture content in sheep faeces. However, those samples were not treated as in the composting process. Results of this study showed that among all the taxonomic groups of bacteria determined in test samples of compost mixtures (K0-K3), most of them belonged to the phyla Fibrobacteres, Bacteroidetes and to the unidentified bacteria. Very similar results were reported by Green et al. in the research on cattle manure composting, which found that as many as 19 (out of 31) groups belonged to the phylum Bacteroidetes. However, regarding the potential degradation of the environmental pollutants, bacteria from genera Pseudomonas and Rhodococcus were described as extremely important decomposers (3). The experiment showed that the highest number of molecular fragments were found using the enzyme MspI indicating the potential presence of bacterial taxa belonging to the phylum Actinobacteria, genus Rhodococcus. This is an important indicator to the assumption of biological degradation of doramectin in the experiment. For this reason, reliable future research is essential to confirm the presence of bacteria from the genus Rhodococcus in sheep manure composts. To conclude in this study the degradation of doramectin in relation to biophysical composting factors was demonstrated, and reciprocally the impact of doramectin on the count of viable bacteria was proved. In addition, the correlation between doramectin degradation and compost moisture was proved. Due to the complexity of the composting process, a multi-variable analysis should be performed in the future to elucidate the mutual impacts of avermectin drugs and composting processes. Moreover, the development of specific micro-organisms for avermectin decomposition in the composting process should be examined in the future. Acknowledgements We would like to thank the Ministry of Higher Education, Science and Technology, and the Ministry of Agriculture, Forestry and Food of Slovenia, for funding this research. The main source of data for this paper was the doctoral thesis of Ivan Gobec, who was awarded a Doctoral Fellowship from the Slovenian Research Agency (ARRS no. 33104-831611). We would also like to thank Mr. Andrej Škibin, DVM from the Centre for Sustainable Recultivation Vremščica of the Veterinary Faculty, for helping, permitting and facilitating this study. References 1. Haruta S, Nakayama T, Nakamura K, et al. Microbial diversity in biodegradation and reutilization processes of garbage. J Biosci Bioeng 2005; 99: 1-11. 2. Martinez J, Dabert P, Barrington S, Burton C. Livestock waste treatment systems for environmental quality, food safety, and sustainability. Bioresour Technol 2009; 100: 5527-36. 3. Fritsche W, Hofrichter M. Aerobic degradation of recalcitrant organic compounds by microorganisms. In: Jordening HJ, Winter J, eds. Environmental biotechnology: concepts and applications. Weinheim: Wiley-VCH, 2005: 203-27. 4. McKellar QA, Benchaoui HA. Avermectins and milbemycins. J Vet Pharmacol Ther 1996; 19: 331-51. 5. Kolar L, Cerkvenik Flajs V, Kužner J, et al. Time profile of abamectin and doramectin excretion and degradation in sheep faeces. Environ Pollut 2006; 14: 197-202. 6. Montforts MHMM, Kalf DF, Vlaardingen PLA, Linders JBHJ. The exposure assessment for veterinary medicinal products. Sci Total Environ 1999; 225: 119-33. 7. Virant Celestina T, Kolar L, Gobec I, et al. Factors influencing dissipation of avermectins in sheep faeces. Ecotoxicol Environ Saf 2010; 73:18-23. 8. Dolliver H, Gupta S, Noll S. Antibiotic degradation during manure composting. J Environ Qual 2008; 37:1245-53. 9. Kim KR, Owens G, Kwon SI, So KH, Lee DB, Ok YS. Occurence and environmental fate of veterinary antibiotics in the terrestrial environment. Water Air Soil Pollut 2011; 214: 163-74. 10. Kožuh Eržen N, Kolar L, Gobec I, Pogačnik M. Degradation of avermectins in contaminated sheep faeces under various conditions. J Vet Pharmacol Ther 2006; 29: 327-8. 11. Ramaswamy J, Prasher SO, Patel RM, Hussain SA, Barrington SF. The effect of composting on the degradation of a veterinary pharmaceutical. Bioresour Technol 2010; 101: 2294-9. 12. Cessna AJ, Larney FJ, Kuchta SL, et al. Veterinary antimicrobials in feedlot manure: dissipation during composting and effects on composting processes. J Environ Qual 2011; 40: 188-98. 13. Boxall A, Fogg LA, Kay P, Paul A, Pemberton EJ, Croxford A. Prioritisation of veterinary medicines in the UK environment. Toxicol Lett 2003; 142: 207-18. 14. Shoop WL, Mrozik H, Fisher MH. Structure and activity of avermectins and milbemycins in animal health. Vet Parasitol 1995; 59: 139-56. 15. Ogram A, Feng X. Methods of soil microbial community analysis. In: Knudsen GR, Thomashow L, eds. Manual of environmental microbiology. Washington: American Society for Microbiology, 1997: 422-30. 16. Stahl DA, Flesher B, Mansfield HR, Montgomery L. Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Appl Environ Microbiol 1988; 54: 1079-84. 17. Kutzner HJ. Microbiology of composting. In: Klein J, Winter J, eds. Biotechnology. 2nd ed. New York: Wiley-VCH, 2000: 35-100. 18. Lane DJ. 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M, eds Nucleic acid techniques in bacterial systematic. New York: John Wiley & Sons, 1991: 115-75. 19. MiCA. Microbial Community Analysis III. University of Idaho, 2007. (last update 8. April 2011) http://mica.ibest.uidaho.edu/trflp.php 20. Bloom RA, Matheson JC. Environmental assessment of avermectins by the U.S. Food and Drug Administration. Vet Parasitol 1993; 48: 281-94. 21. Kolar L, Kožuh Eržen N. Degradation of abamectin and doramectin in sheep faeces under different experimental conditions. Int J Environ Pollut 2007; 31: 22-33. DEGRADACIJA DORAMEKTINA IN BAKTERIJSKE ZDRUŽBE MED KOMPOSTIRANJEM OVČJEGA GNOJA M. Dobeic, M. Ocepek, N. Kožuh-Eržen, Š. Pintarič, V. Cerkvenik-Flajs, I. Gobec Povzetek: Raziskana je bila razgradnja doramektina in bakterijskih združb v ovčjem gnoju med najbolj bioaktivno-termofilno fazo kompostiranja v poskusnih bioreaktorjih. V vzorce komposta, ki smo ga kompostirali 21 dni,so bile dodane različne koncentracije doramektina. V povprečju so se koncentracije doramektina v vseh vzorcih (K1-K3), analiziranega po postopkih kromatografske analize (HPLC), s časom kompostiranja (21 dni) znižale za 31,8 - 41,8 %. Ugotovljena je bila značilna korelacija (P<0,001; r=0,512) med koncentracijo doramektina in znižanjem vlage v vzorcih (K1-K3). Domnevamo, da se povprečne koncentracije doramektina v vzorcih znižujejo skladno z zniževanjem vsebnosti vode. Iz tega razloga je bil vzporedno izveden laboratorijski preskus, v katerem so bile ugotovljene značilno (P<0,05) nižje povprečne vrednosti za doramektin v vzorcih z nižjo vsebnostjo vode. Doramektin je vplival na številčnost bakterij, ki je bila najvišja v vzorcih brez doramektina v primerjavi z vzorci, obogatenimi z doramektinom. PCR in filogenetska analiza (T-RFLP) sta pokazali prisotnost bakterij iz rodov Fibrobacteres in Bacteroidetes, medtem ko je analiza z restrikcijskim encimom MspI pokazala morebitno prisotnost bakterij iz rodu Rhodococcus, ki so odgovorne za potencialno razgradnjo doramektina. Študija je pokazala učinkovanje biofizikalnih dejavnikov na degradacijo doramektina in njegov vpliv na številčnost živih bakterij, ne pa tudi na raznolikost bakterijske združbe. Ključne besede: kompostiranje; degradacija doramektina; bakterije Slov Vet Res 2013; 50 (4): 183-91 UDC 636.3.08:574.9(497.12) Original Scientific Article GEOGRAPHICAL DISTRIBUTION OF SHEEP AND GOAT BREEDS IN SLOVENIA Metka Žan Lotrič*, Gregor Gorjanc, Drago Kompan Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domžale, Slovenia Corresponding author: E-mail: metka.zan@bf.uni-lj.si Summary: Geographical distribution of small ruminant breeds kept in Slovenia and included into the National selection program was studied. Analyses of the population size and its structure were made together with the geographical distribution (geographical coordinates of flocks locations upon Gauss-Krueger coordinate system; the values of x and y). For individual flocks the radial distance from the geographic centre of gravity was calculated, and a distribution graph was made where cumulative distribution of animals depending on theirdistance from the geographiccentre of gravity is presented.The calculated geographical centre for the autochthonous breeds is in the area of their origin, while traditional and foreign breeds gravitate towards the central part of Slovenia. It is characteristic of autochthonous breeds that the majority of their population is located within a small radius, compared to foreign and traditional breeds. So, the autochthonous breeds are mostly concentrated just to a smaller geographical area. Three Slovenian autochthonous breeds of sheep (Bela Krajina Pramenka, Istrian Pramenka and Bovec sheep) have 90 % of their population within a distance of less than 25 km, while the Slovenian autochthonous breed of Drežnica goat has 90 % of the total population within a radius of less than 30 km. Traditional and foreign breeds are not confined to only one region or area. Due to the occurrence of natural disasters or sudden outbreak of diseases the scarce Slovenian autochthonous sheep and goat breeds are considered as most endangered population. Key words: sheep; goats; breeds; geographical distribution; endangered breeds; endemic area Introduction In the report entitled "The State of the World's Animal Genetic Resources for Food and Agriculture" prepared by the International Organisation of the United Nations for Food and Agriculture [1] an urgent need is stressed to improve the overview of each animal breed population (size, endangerment, etc.). However, other important factors such as geographical concentration should also be taken into account [2]. This involves the study of the distribution of individual species, breeds and flocks of farm animals which is particularly important for the "in situ" conservation in an environment where the farm animal genetic resources originate from and where they developed their distinctive traits [3]. An assessment of the risk status of livestock breeds or populations is an important element in the planning of animal genetic resources management. The risk status of each breed informs stakeholders whether, and how urgently, actions need to be taken [1]. Gandini et al. [4] define "degree of endangerment" as a measure of the likelihood that, under current circumstances and expectations, the breed will become extinct. Accurately estimating degrees of risk is a difficult undertaking and incorporates both demographic and genetic factors. Clearly, current population size is important factor in determining risk status. A small population concentrated in one area is at greater risk of being wiped out by natural disasters, disease, or inappropriate management [1]. An essential aspect of risk is the geographical distribution of flocks because potential threat for farm animal genetic resources is positively associated with high geographical concentration. Carson et al. [5] stresses that in determining the level of risk status of certain breed besides the number of population, the number of flocks within particular breed etc., special attention should be given also to other factors such as geographical concentration of an individual breed. The example of a large impact of the geographical concentration of animals on one area was clearly visible in the UK during the outbreak of foot-and-mouth disease in 2001 [5]. Small numerous autochthonous breeds are often kept in a well-defined (original) geographical area where they were selected to be maximally adapted to the local conditions. However, some breeds can be present in very small areas, while other breeds are more dispersed. The species, which is tied to a specific geographical area, can be considered as endemic [6]. Breed diversity in small ruminants in Slovenia is relatively large. Slovenian autochthonous (Jezersko-Solčava sheep, Bovec sheep, Istrian Pramenka, Bela Krajina Pramenka, Drežnica goat) and traditional (Improved Jezersko-Solčava sheep, Slovenian Saanen goat and Slovenian Alpine goat) breeds of sheep and goats and the foreign Boer goat are included into the National selection program according to the breeding program of each individual breed. The goal of the breeding program are selected purebred females and males that are suited to meet the individual needs and requirements of the sheep and goat breeders. This goal also helps in the recovery of endangered species by preserving the existing gene pool and prevents inbreeding especially in endangered breeds. The aim of this work was to evaluate the potential threat for the spread of infection in purebred flocks due to geographical distribution in case the infection would arise in the vicinity. Based on these results the conservation policy could be modified or improved by considering the geographical component. The risk of losing an endangered breed due to infectious diseases could therefore be reduced. Material and methods Population data of sheep and goat breeds in Slovenia for 2011 were obtained from the database of National selection program for small ruminants. The database includes selection aspects for each individual animal (e.g. identification number, birth date, pedigree information and production recording) and is being updated daily. The proportion of sheep and goat breeds covered by the database of National selection program differs among breeds. For traditional and foreign breeds the number of animals included in the database is less than half of the whole population kept in Slovenia, while the percentage of animals by autochthonous breeds included in the database is as follows: Bovec sheep = 76.4 %; Istrian Pramenka = 83.74 % Bela Krajina Pramenka = 88.86 %; Drežnica Goat = 83.12 % and Jezersko-Solčava sheep - the only non-endangered autochthonous breed = 29.22 %. All purebred males of individual breeds are used as breeding males. We analysed the population size and structure together with the geographical distribution (geographical coordinates of flocks according to the Gauss-Krueger coordinate system, values x and y). Gauss-Krueger coordinates system is commonly used in the Republic of Slovenia (http: / /www. spatialreference.org/ref/sr-org/7011/), where meter is used as a unit for x and y. A potential danger of endemic diseases for a particular breed is best show on the geographical map, where it is clearly seen how individual breed flocks are dispersed or concentrated in a particular geographic area. Data processing and presentation of the results was performed with the software package R [7]. By using coordinates (pairs of values x and y) we indicated the locations as well as flock size on the map of the Republic of Slovenia. Flock size is represented by a point size [8]. Due to the proximity of certain flocks we sketched a partially transparent point. In the case of multiple overlapping of nearby points it is plotted as a darker colour. Like Carson et al. [5] we calculated the geographical centre of gravity for flock locations and marked it on the map. The centre of gravity was calculated as the weighted median of coordinate values: X = median (x, w) and Yt = median (y, w), where x and y are vectors of coordinates of individual flocks, while w is the vector of relative weights calculated in relation to the size of an individual flock (population number of a particular breed per farm). Compared to Carson et al. [5] we used the median instead of the mean. It is clearly seen that a single strong isolated point can greatly affect the location of the centre of gravity, which is determined by the arithmetic mean. The median is insensitive to an individual remote flock and therefore more suitable for the evaluation of the centre of gravity, mostly for concentrated flocks. For individual flocks the distance from the centre of gravity was calculated, and a distribution graph was made where cumulative proportion (distribution) of animals depending on their distance from the centre is shown. Apart from the points on the map which represent location and flock size, there are also circles drown to show the distance from the calculated geographic centre of gravity, where 50, 90 and 95 % of all animals included into the National selection program for the individual breed are located. Results Geographical distribution of flocks and flock size for individual breeds of sheep and goats kept in Slovenia is presented in Figures 1-4. Individual flocks are presented by circle, while the calculated geographic centre of gravity is marked by an x. From the images it is clear that the geographical distribution differs among breeds. Bela Krafina Pramenka In 2011, there were 41 purebred males, 652 purebred females and 115 purebred young females included in the National selection program. Bela Krajina Pramenka is characterized by the concentration of a large part (95 %) of the population at a distance calculated from the geographic centre of gravity of less than 30 km while 90 % of the population is located within the radius of ~ 18 km (Figures 1a and 2 a). Jezersko-Solčava sheep Jezersko-Solčava sheep is the only non-endangered Slovenian autochthonous small ruminant breed. In 2011, there were 126 flocks of 5202 purebred animals included in the National selection program which means 203 purebred males, 4247 purebred females and 752 purebred young females. The average flock size was relatively small, amounting only to 36.6 animals. Jezersko- Solčava sheep is otherwise scattered throughout the entire territory of Slovenia. However, most of the flocks are in the area of two Slovenian statistical regions (Gorenjska and Savinjska). The breed is therefore in its native region, as well as outside and it is not geographically confined to only one statistical region or area (Figure 1b). A relatively uniform distribution of flocks depending on the distance from the calculated geographic centre of gravity is shown in Figure 2b. It is necessary to take into account the fact that the percentage of animals included into the National selection program according to the number of animals in the entire Jezersko-Solčava population is different in comparison to other Slovenian autochthonous breeds of sheep and goats. In less numerous breeds there are more than 80 % of the population included into the National selection program, while less than 30 % belong to Jezersko-Solčava sheep. Bovec sheep In 2011, there were 148 purebred males, 1886 purebred females and 640 purebred young females included in the National selection program. The average flock size was 64.6 animals. There are only a few flocks of bigger size. Most farmers keep a small number of animals, therefore the median for the flock size is 36.0 animals. Istrian Pramenka In 2011 there were 43 purebred males, 650 purebred females and 270 purebred young females included in the National selection program. The average flock size was 151 animals. Figure 1d shows the small size of Istrian Pramenka population and a local distribution in the Karst and Istria region. A little less than a half of the total population of Istrian Pramenka were bred in a single flock. For this reason a median for flock size is only 41.5 animals. Improved Jezersko-Solčava sheep Improved Jezersko-Solčava sheep is the most numerous sheep breed in Slovenia. In 2011, there were 151 purebred males, 4052 purebred females and 670 purebred young females included into the National selection program. Improved Jezersko-Solčava sheep is the most geographically dispersed breed in Slovenia (Figure 1e). e) 50 10CK 150' 200 • • -_____-4 Figure 1: Location and flock size of a) Bela Krajina Pramenka, b) Jezersko-Solčava sheep, c) Bovec sheep, d) Istrian Pramenka and e) Improved Jezersko-Solčava sheep with calculated geographic centre of gravity (x) and radius where 50 % (inner circle), 90 % (middle circle) and 95 % (outer circle) of animals included into the National selection program are located Table 1: Flock dispersion of sheep breeds from the calculated geographic centre of gravity Breed Purebred females (n) in NSP* in 2011 Distance (km) from the calculated geographic centre of gravity Calculated geographic centre of gravity by Gauss-Krueger coordinate system Percentage of animals (%) 50 90 95 Bela Krajina Pramenka 767 ~ 6 ~18 ~ 28 x=523488, y=41375 Mala sela close to Adlešiči Jezersko-Solčava sheep 4999 ~ 35 ~ 95 ~ 103 x=135715,y=477431 Zg. savinjska dolina and Luče Bovec sheep 2526 ~ 1 ~ 12 ~ 55 x=133610,y=389289 Bovec Istrian Pramenka 920 0 ~ 25 ~ 100 x=62814,y=423325 Gabrče close to Divača Improved Jezersko-Solčava sheep 4722 ~ 49 ~90 ~105 x=121970,y=502424 Gomilsko *National selection program Distance (km) Distance (km) c) d) Distance (km) 0 50 100 150 Distance (km) e) Figure 2: Percentage of a) Bela Krajina Pramenka, b) Jezersko-Solčava sheep, c) Bovec sheep, d) Istrian Pramenka and e) Improved Jezersko-Solčava sheep included into the National selection program according to the distance from the calculated geographic centre of gravity Distance (km) Based on the results for individual sheep breeds the distance (km) from the calculated geographic centre of gravity and the calculated geographic centre following Gauss-Krueger coordinate system is presented in Table 1. Differences in flock dispersion among various sheep breeds occur mainly due to the group of breeds (autochthonous, traditional) (Table 1). Thus, the Slovenian autochthonous sheep breeds are mostly concentrated in their own calculated geographic centre of gravity. At the same time these centres are also their area of origin according to the historical sources on development of the individual breed. For example, in Istrian Pramenka a half of the population is located in its own geographic centre of gravity, while a larger share of animals is located within a distance of about 25 km. The most numerous Slovenian autochthonous sheep breed (Jezersko-Solčava sheep) has a half of its population in a distance of about 35 km from the calculated geographic centre of gravity, whereas practically all animals are located within a distance of about 103 km from the calculated geographic centre of gravity. Traditional sheep breed (Improved Jezersko-Solčava sheep) is fairly evenly dispersed throughout the territory of Slovenia. a) b) Herd size: 50 100 150 200 • • • • 50 1:00 150 200 • • • • c) d) 50 100 150 200 • • • • Herd size: j 50 100 1^0 200 • • m • Figure 3: Location and flock size of a) Drežnica goat, b) Slovenian Alpine goat, c) Slovenian Saanen goat and d) Boer goat with calculated geographic centre of gravity (x) and radius where 50 % (inner circle), 90 % (middle circle) and 95 % (outer circle) of animals included in the National selection program are located Drežnica goat In 2011, there were 39 purebred males, 355 purebred females and 105 purebred young females included in the National selection program. Almost all flocks of Drežnica goat breed are located in the area of breed origin (Figure 3a). Within a distance less than 30 km there are 90 % of all population of Drežnica breed included in the National selection program (Figure 4 a). Slovenian Alpine goat In 2011, there were 1241 animals; 37 purebred males, 976 purebred females and 228 purebred young females included in the National selection program. Based on the distance from the calculated geographic centre of gravity, flocks of Slovenian Alpine goat breed have a relatively even distribution throughout the country (Figures 3b and 4b). Slovenian Saanen goat In 2011, there were 526 animals; 26 purebred males, 324 purebred females and 176 young purebred females included in the National selection program. Slovenian Saanen goat breed is small in number, therefore it is difficult to speak of greater or lesser dispersion. Depending on the distance from the focus the flocks of Slovenian Saanen goat breed are relatively evenly distributed (Figures 3c and 4c). Boer goat Boer goat is the most numerous goat breed in Slovenia and it is spread throughout the country (Figures 3d and 4d). In 2011, there were 297 purebred males, 1465 purebred females and 640 young purebred females included in the National selection program. a) b) Distance (km) Distance (km) 0 50 100 150 0 50 100 150 Figure 4: Percentage of a) Drežnica goat, b) Slovenian Alpine goat, c) Slovenian Saanen goat and d) Boer goat according to the distance from the calculated geographic centre of gravity Table 2: Flock dispersion of goat breeds from the calculated geographic centre of gravity Breed Purebred females (n) in NSP* in 2011 Distance (km) from the calculated geographic centre of gravity Calculated geographic centre of gravity by Gauss-Krueger coordinate system Percentage of animals (%) 50 90 95 Drežnica goat 460 ~ 5 ~ 29 ~ 48 x= 126070,y= 392751 Drežniške Ravne Slovenian Alpine goat 1204 ~ 38 ~ 96 ~ 110 x = 87495,y = 437278 Žibrše close to Logatec Slovenian Saanen goat 500 ~ 10 ~ 60 ~ 90 x= 102600,y= 434683 Žirovski vrh Boer goat 2105 ~38 ~ 95 ~ 102 x= 115563,y= 505911 Marija Reka close to Trbovlje *National selection program The smallest distance from the calculated geographic centre of gravity has Slovenian autochthonous Drežnica goat breed (Table 2). A half of the entire population of this breed included in the National selection program is located within a distance of about 5 km. The calculated geographic centre of gravity of Drežnica goat is the area of its origin. Other goat breeds included in the analysis have a similar distance from the geographic centre of gravity calculated for 50, 90 and 95 % of the animals. Their calculated geographical focus gravitates away from the central part of Slovenia. Discussion Slovenian autochthonous breeds of sheep and goats are mostly concentrated in the area of their origin. The small numerous breeds of sheep such as Istrian Pramenka, Bela Krajina Pramenka and also Bovec sheep have 90 % of their population within a distance less than 25 km. Drežnica goat, the only Slovenian autochthonous goat breed has 90 % of their population within a distance less than 30 km. The calculated geographic centre reflects a single rearing centre of all Slovenian autochthonous sheep and goat breeds. In more dispersed traditional breeds like Improved Jezersko - Solčava sheep, Slovenian Saanen goat, Slovenian Alpine goat and the foreign Boer goat the calculated geographic centre is not a single rearing centre. Istrian Pramenka sheep is characterized by a small geographical dispersion (Figure 1d) and it is small in flock number (Figure 2d). In the event of any natural disaster or outbreak of infectious and communicable diseases this breed could suffer serious consequences, so we must emphasise the risk in this respect. It may be noted that due to the high concentration of flocks in smaller geographical areas in Slovenia a small population number is particularly vulnerable and dangerous for autochthonous breeds with the exception of Jezersko-Solčava sheep. It has its calculated geographic centre range in the area of its origin, but as a relatively productive meat type it is extended to other parts of Slovenia. In case of transfer of infectious diseases within a narrow range, the concentration of animals of certain species within the area is also important because in high concentrations animal diseases spread quickly. Geographical concentration of breeds indicates that the animals are very well adapted to their specific environment and to the traditional uses and farming management, as well as the attachment of breeders to them. Increased geographical dispersion of breeds to larger area of Slovenia means lesser possibility for spread of infection, while increased geographical concentration of breeds means higher possibility for spread of infectious diseases. The traditional Improved Jezersko - Solčava sheep breed dispersed in a large area of Slovenia and not confined to only one region or area, is therefore not highly at risk. Density of the same and different species located in the specific area is a risk factor for disease transmission, therefore further investigations on density would be necessary. Acknowledgment We are grateful to prof. dr. Martina Klinkon for her critical review of the manuscript and her constructive comments. References 1. Pilling D, Rischkowsky B, eds. The state of the world's animal genetic resources for food and agriculture. Rome: Commission on Genetic Resources for Food and Agriculture, Food and Agriculture Organization of the United Nations, 2007: 511 str. 2. Alderson L. Breeds at risk: definition and measurement of the factors which determine endangerment. Livest Sci 2009; 123: 23-7. 3. Šalehar A, Čepon M, Žan Lotrič M, et al. Geografija živinoreje v Sloveniji v letu 2000. Razširjenost in gostota reje kmetijskih živali ter raba kmetijskih zemljišč po statističnih regijah in v Sloveniji. In: Ohranjanje biotske raznovrstnosti živinoreji. Poročilo za leto 2004. Domžale: Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za zootehniko, 2005: 152-77. 4. Gandini GC, Ollivier L, Danell B, et al. Criteria to assess the degree of endangerment of livestock breeds in Europe. Livest Prod Sci 2004; 91: 173-82. 5. Carson A, Elliott M, Groom J, Winter A, et al. Geographical isolation of native sheep breeds in the UK: evidence of endemism as a risk factor to genetic resources. Livest Sci 2009; 123: 288-99. 6. Kryštufek B. Osnove varstvene biologije. Ljubljana: Tehniška založba Slo venije, 1999: 155 str. 7. The R development core team. R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing, 2011: 1651 str. 8. Tanimura S, Kuroiwa C, Mizota T. Proporional symbol mapping in R. J Stat Software 2006; 15: 5. GEOGRAFIJA RAZŠIRJENOSTI PASEM OVC IN KOZ V SLOVENIJI M. Žan Lotrič, G. Gorjanc, D. Kompan Povzetek: Proučevali smo geografijo razširjenosti pasem drobnice v Sloveniji, ki so vključene v Rejski program. Analizirali smo velikost populacije, njeno strukturo in geografijo razširjenosti (geografske koordinate rej po Gauss-Kruegerjevem sistemu; vrednosti x in y). Za vsako rejo smo izračunali zračno razdaljo oddaljenosti od geografskega težišča ter prikazali porazdelitveno sliko, ki prikazuje delež živali glede na oddaljenost od geografskega težišča pasme. Izračunano geog rafsko težišče za avtohtone pasme je njihovo izvorno območje, za tradicionalne in tujerodne pasme pa je usmerjeno proti osrednjemu delu Slovenije. Za avtohtone pasme je značilno, da večji del populacije obstaja znotraj manjšega radija, kot je le-ta v primerjavi s tujerodnimi in tradicionalnimi pasmami in da so reje večinoma skoncentrirane v manjšem geografskem območju. Tri slovenske avtohtone pasme ovc (belokranjska pramenka, istrska pramenka in bovška ovca) imajo 90 % populacije znotraj razdalje, ki je manjša od 25 km, slovenska avtohtona pasma koz drežniška koza pa znotraj radija, ki je okoli 30 km. Tradicionalne in tujerodne pasme niso omejene le na eno regijo ali območje. Zaradi ogroženosti in tveganj ob naravnih nesrečah ali nenadnih izbruhih bolezni so najbolj izpostavljene maloštevilne slovenske avtohtone pasme ovc in koz. Ključne besede: ovce; koze; pasme; geografija razširjenosti; ogroženost; endemično področje Slov Vet Res 2013; 50 (4): 193-200 UDC 636.4.09:616.9-097 Original Scientific Article AN ATTEMPT TO ELIMINATE PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME (PRRS) BY SERUM INOCULATION ON SMALL PIG FARM Marina Štukelj1*, Ivan Toplak2, Zdravko Valenčak1 1Institute for Health Care of Pigs, institute for Microbiology and Parasitology Veterinary faculty, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia Corresponding author, E-mail: marina.stukelj@vf.uni-lj.si Summary: The great heterogeneity among porcine reproductive and respiratory syndrome virus (PRRSV) isolates is probably the main obstacle to its effective control using current commercial vaccines, since the induced immunity by one strain is specific onlytothisstrain.ExposureofallbreedingpigstothePRRSVcirculatingonthefarmisanoptionforeliminationofPRRSinbreeding herd. Adoption of strict biosecurity measures is essential. The objective of this study was to eliminate the PRRS from a farrow-to-finish small pig farm (130 breeding pigs) by serum inoculation. The owner was acquainted with the biosecurity measures (strict biosecurity protocols and herd closure for at least 200 days). Breeding pigs were immunized with serum obtained from weaners. The number of high positive breeding pigs decreased from six months after the II. serum inoculation till the end of the study, but the prevalence of antibody were almost the same comparing the sampling before serum inoculation to last sampling 13 months after the II. serum inoculation. The breeding herd were free of virus during all testing, but PRRSV circulated in the two-month old weaners. The owner did not implement herd closure and other required biosecurity measures and a new strain of PRRSV was introduced. Hence, serum inoculation proved to be unsuccessful for the elimination of PRSS from the farrow-to-finish farm. Implementation of biosecurity measures in field conditions is a much more difficult challenge than what was expected at the beginning. Key words: control; immunization; pig; PRRS; serum inoculation Introduction Porcine reproductive and respiratory syndrome (PRRS) is a viral infection endemic in most swine-producing countries and leads to major economic losses (1). The disease is characterized by reproductive failure, including late-term abortions, early farrowing, stillbirths, weak born piglets and increased mortality in neonates, nursery and growing pigs, and respiratory tract illness that can be especially severe in neonatal and nursery- age pigs (2). PRRSV infection is difficult to control due to large heterogeneity among the isolates. A variety of strategies have been described for PRRS elimination, including total depopulation/ repopulation, partial depopulation (3), isowean (4) or segregated early weaning (5), test and removal (6), mass vaccination with unidirectional pig flow and herd closure (7). Elimination of a disease is disappearance of all clinical cases of a specific disease (8) which is the consequence of desistance of virus replication and circulation in the population of pigs. No single strategy for elimination will work on infected farms; therefore, the program must be individually designed based on the unit's pig flow and facility design as well as serological results (9). PRRS elimination is a long term goal, and the first step is stabilization of the breeding herd. Herd stability is defined as a herd which lacks clinical signs and in which a virus is not actively circulating and transmitting between pigs (9). Stabilization can be achieved with simultaneous immunization of the breeding herd (10). Immunization can be achieved with commercial vaccines, serum inoculations and natural exposure. It appears that currently available vaccines may not be effective in protecting against infections with genetically different strains of PRRSV (11). Numerous studies have shown some cross-protection against different strains which are reflected only in the reduction of clinical signs and lesions but not in elimination of the virus (12). Moreover, inoculation with a homologous strain provides a high level of protection against the same or nearly the same virus strain (13). It is, however, readily accepted that homologous immunity is more protective than heterologous immunity. In fact serum inoculation is the intentional immunization of pigs with the strain of PRRS virus originating from the same, infected farm (homologous herd strain). This method consists of intramuscular injection of complete breeding herds with serum derived from acutely infected pigs that contain the particular farm-specific PRRSV (14). Shibata et al. (15) showed that, after exposure to a homologous PRRSV strain, pigs subsequently challenged with that strain did not develop clinical signs, and virus replication was reduced in both the titer and the length of infection. In addition, herd closure is also required to achieve herd stability. In the period of herd closure new pigs cannot be introduced to the farm. This applies also to internal replacements of gilts to the breeding herd (7). The success of PRRS elimination depends on biosecurity practices and cooperative work (9). Consequently, one very important measure is to follow strictly biosecurity protocol, which includes preventing direct routes of spread as well as indirect and miscellaneous routes, as authored by Pitkin et al. in the American Association of Swine Veterinarians Foundation (AASV) website (www.aasv.org/aasv/PRRSV_BiosecurityManual.pdf). The objective of this study was to eliminate PRRS from a small farrow-to-finish pig farm with herd closure, improved biosecurity and serum inoculation. Materials and methods Farm The study was carried out from June 2010 until March 2012 on one farrow-to-finish farm consisting of four boars and 130 breeding sows. Six months after the second round of serum inoculations, the owner reduced the number of breeding sows to 88 due to the lower price of pigs on the Slovenian market and not due to our request as a measure for the elimination of PRRSV. Semen originated from their four boars. Serum inoculation was performed twice on the farm: the first being after the conformation of PRRS and the second three months after I. serum inoculation. Herd closure The introduction of new pigs to the farm was prohibited for 200 days. Also in this period gilts from the farm could not enter the breeding herd. Biosecurity measures The owner was acquainted with obligatory measures: strict biosecurity protocols (entering the farm after changing clothes; having personnel aid in the changing of coveralls and boots; the washing of hands; using footbaths; maintaining individual responsibility for each pig category; use of the all in/all out system; one age category of pigs in one room; one way pig flow; the cleaning and disinfection of pens, pig equipment kept on the farm; deratization and disinsection). Preparation of inoculum for serum inoculation of breeding pigs The weaners at age 8 to 14 weeks of age were bleeding and tested by RT-PCR. Inoculum was prepared from positive serum samples. The PRRSV positive serum samples were pooled. To one part of each pool four parts of RPMI-1640 medium (Gibco, Germany) were added and mixed with 1% of Antibiotic-Antimyco tic (100x), (Invitrogen, Germany). The inoculums contained 102 to 104 TCID50 PRRSV/ml. All breeding pigs were inoculated intramuscularly with 2 ml/pig on the same day. Samplings procedure All together 704 blood samples were collected for serology and 456 for molecular testing. The sequencing of PRRSV positive samples were performed 6 times. Enzyme-linked immunosorbent assay (ELISA) The HerdChek, IDEXX Laboratories, PRRS X3 ELISA test was used for detecting antibodies in serum samples. The ELISA was performed according to the manufacturer's instructions. Sample results were divided in four groups: samples with S/P less than 0.4 (negative), samples with S/P between 0.4-1 (low positive), samples with S/P between 1 and 2 (positive) and samples with S/P more than 2 (high positive). Detection of PRRSV with gel-based RT-PCR and direct sequencing of PRRSV positive samples Total RNA was extracted from 140 ^l of serum samples using the QIAamp® viral RNA mini kit (Qiagen, Germany) according to the manufacturer's instructions. 456 samples were tested individually or as pools (maximum 5 samples in pool) by one-step RT-PCR (One-Step RT-PCR Kit, Qiagen, Germany) using sequences based on the open reading frame 7 (ORF7), which detect Type 1 and Type 2 PRRSV strains respectively (16, 7). The PRRS strain VR-2332 (Type 2) and the Lelystad viruses (Type 1) were used as positive controls. Reaction mixtures without RNA served as negative controls. Fifteen PRRSV positive samples were directly sequenced in both directions using the Macrogen sequencing service (Macrogen, South Korea) and the RT-PCR amplification primers. For each sample, 258 nucleotide long sequences were aligned with the published data using BLAST (available at http://www.ncbi.nlm.nih.gov/) at the National Centre for Biotechnology Information (NCBI), and PRRSV sequences obtained were compared using the sequence analysis software Lasergene® (DNASTAR Inc., Madison, WI, USA). Results PRRS was confirmed on the farm via testing of 10 animals which showed positive or high positive results in ELISA. Before I. serum inoculation 13 (9.7%) samples were negative, 24 (17.9%) were low positive, 43 (32.1%) were positive and 54 (40.3%) Table 1: Number of tested sera for serology (ELISA for detection of PRRS antibodies), PRRSV detection (RT-PCR method for PRRSV genome detection) and times of sequencing Sampling No. of tested sera by ELISA No. of tested sera with RT-PCR Sequencing of PRRSV positive samples Breeding pigs Pigs Breeding pigs Pigs Breeding pigs Pigs Confirmation of PRRS 10 - - - - - Before I. serum inoculation 134 - - 15 - yes 3 months after I. serum inoculation 134 - - 15 - yes 3 months after II. serum inoculation 133 - 133 - - - 6 months after II. serum inoculation 88 20 88 20 - yes 10 months after II. serum inoculation 20 30 20 30 - yes 13 months after II. serum inoculation 97 13 97 13 - yes 17 months after II. serum inoculation - 25 - 25 - yes Footnote: In the "Pigs" column, all categories from weaning pigs to fatteners are included Table 2: Results of PRRSV Detection By RT-PCR and Sequencing of PRRSV Sampling Results of RT-PCR Age of positive pigs (weeks) Identification number of sequence Nucleotide identity to 08066t/2010 Breeding Pigs Pigs Before I. serum inoculation - positive 10 08066t/2010 06088t/2010 100% 100% 3 months after I. serum inoculation - positive 8-12 Meol/2010 98.4% 3 months after II: serum inoculation negative - - - - 6 months after II: serum inoculation negative positive 10 2768-81/2011 2768-83/2011 2768-84/2011 2768-86/2011 2768-87/2011 276889/2011 99.6% 97.7% 99.6% 98.1% 97.7% 98.1% 10 months after II. serum inoculation negative positive 10 Meol15/2011 Meol19/2011 97.3% 96.9% 13 months after II: serum inoculation negative positive 10 Meol20/2011 Meol21/2011 99.2% 99.2% 17 months after II. serum inoculation - positive 10 0803-1/2012 08032/2012 99.2% 99.2% Type 1 Type 2 28.6 ■ VR-2332 (U87329) Meol20/2011 Meol21/2011 0803-2/2012 0803-1/2012 2768-81/2011 2768-84/2011 08066t/2010 08068t/2010 r Meol 15/2011 H- Meol 19/2011 2768-86/2011 2768-89/2011 Meol/2010 -2768-83/2011 L 2768-87/2011 PRRSV LV4.2.1 (AY588319) 25 20 15 10 Nucleotide Substitution (x100) 0 Figure 1: The Phylogenetic Tree of 15 Sequenced PRRSV Samples. The strains 08066t/2012 and Meol/2010 which were used for serum inoculation are presented in bold 70 60 50 40 30 20 10 Hi before .serum 3 months after . 3 months elfter 6monthsafter 1C months after I . 13 months after II Sampling I Negative I Low positive Positive I High positive Figure 1: Serological Results of the Breeding Herd Table 3: Results of PRRSV Detection By RT-PCR and Sequencing of PRRSV Biosecurity measures Required Implemented Herd closure (introducing replacement gilts into the breeding herd) yes no Herd closure (introducing new pigs to the farm) yes yes Entering the farm after changing clothes yes no One age category of pigs in one room yes yes One way pig flow yes yes Changing of coveralls between pig category yes no Changing of boots between pig category yes no Washing hands between pig category yes no All in/ all out in farrowing and fattening units yes no Individual responsibility for each pig category yes no Cleaning and disinfection of pens yes no Footbath yes yes Pig equipment kept on the farm yes no Deratization and disinsection yes yes were high positive (figure 1). Three months after the I. serum inoculation 6 (4.5%) breeding pigs were still negative and 21 (15.7%) breeding pigs were low positive therefore the second serum inoculation was performed. Three months after II. serum inoculation 2 (1,5%) samples of breeding pigs were negative; 18 (13.5%) were low positive; 30 (22.5%) positive and 83 (62%) high positive, which points to improved immune response of breeding herd. Six months after II. serum inoculation the number of high positive pigs decreased to 35 breeding pigs (39.8%); the number of positive breeding pigs increased to 39 (44.3%); and the percentage of low positive breeding pigs remain at the same level (13.6%) compared to prior sampling. Six months after II. serum inoculation we also checked the status of weaners. Two were negative; three, low positive; seven, positive; and six were high positive which indicate a persistent circulation of the wild type of PRRSV. Samplings at 10 and 13 months after II. serum inoculation still present a trend of decreasing high positive and positive pigs in the breeding herd while the low positive increased. One possible reason is that the breeding herd developed protective immunity; and, in spite of the new introduction of the homologous PRRSV, the titers of antibodies (high positive and positive) decreased. The last sampling—17 months after II. serum inoculations— was performed only in weaners and fatteners to check if PRRS was eliminated from the herd. Results showed the following: 2 negative, 4 low positive, 7 positive and 12 high positive which indicated persistent circulation of the PRRSV. During the whole period of the study, the breeding herd was negative for PRRSV by the RT-PCR method in all samplings. Sample 08066t /2010 (Fig 1) was used for the I. serum inoculation of the entire breeding herd (134 breeding pigs). A second serum inoculation of the entire breeding herd (134 breeding pigs) was carried out using the detected PRRSV strain Meol/2010. Observed sequence homology between detected strains in herd and topology from phylogenetic tree obtained from fifteen sequenced positive samples in the period of study suggested that the second strain was introduced into the farm between I. serum inoculation and II. serum inoculation. The owner implemented only 5 of the required biosecurity measures, and the rest of the measures were neglected. Discussion To be successful, any PRRS herd elimination strategy must stop replication of the virus within a population of breeding pigs and this will prevent infection of neonates (18). PRRSV circulates in endemically infected herds because, at any given time, animals are in various stages of infection and immunity (19). Considering the heterogeneity of PRRS serotypes and the importance immunity of homologues, serum inoculation can be a successful measure to eliminate PRRS from the farm (20). With simultaneous serum inoculation of homologues PRRSV strain (strain 08066t/2010) of the breeding herd, we tried to stop the circulation of PRRSV in the breeding herd which would lead to stabilization of breeding herd and which resulted in, as much as possible, uniform S/P ratios (between 1 to 2) and production of negative fatteners. Three months after I. serum inoculation, 4.5% breeding pigs were still negative and 15.7% positive, which was too high for the number of breeding pigs to ensure good protection. We decided to use the II. serum inoculation (strain Meol/2010) in order to ensure the stoppage of shedding the PRRSV. From the beginning of the study we implemented one additional method, herd closure. PRRSV elimination through herd closure is based on the fact that naturally developed immunity eliminates virus infection from the farm (7, 21). With serum inoculation we try to enhance the development of homologous immunity which did not prove as good a protection, according to our serological results. One good candidate for the elimination of the PRRSV is the three-site farm (7); moreover, the success rate is above 85% for farms with segregate production (22), keeping in mind that our study was performed on farrow-to-finish farm. According to the results of RT-PCR method and sequencing, we confirmed the introduction of a new closely-related strain PRRSV after I. serum inoculation. The observed sequence homology between 08066t/2010 strain (used for I. serum inoculation) and Meol/2010 strain which we used for II. serum inoculation was 98.4% (table 3, figure 1). Both strains were detected also 6, 10 and 13 months after II. serum inoculation in weaners age of 10 weeks, confirming long period of circulation of strains in farm, although the breeding herd was negative in all testing during the study. The results of serology 6 months after II. serum inoculation show a trend of decreasing high positive breeding pigs which continued until the end of the study. On the other hand, the prevalence of antibody is almost the same comparing the sampling before serum inoculation (90.3%) to sampling 13 months after II. serum inoculation (96.9%). Merely the percentage of high positive breeding pigs decreased from 40.3 to 25.8. We expected the number of high positive breeding pigs to be much lower or non-existent. The results 17 months after serum inoculation indicated by the periodical introduction of PRRSV into the breeding herd resulted in persistently high S/P ratios. The results of RT-PCR of weaners aged 8 weeks from testing 6, 10, 13 and 17 months after II. serum inoculation were negative, but the virus constantly persisted in group of 10 week old weaners. Pigs born from PRRSV infected dams maintain maternal antibody until 4 to 8 weeks of age using the indirect ELISA (23). Thus it is obvious that after decreasing maternal immunity, the weaners got infected. Shortly, 25% of breeding pigs were high positive 13 months after II. serum inoculation and at the age of 10 weeks, the virus was persistently circulating among the weaners. In this category of weaners, they were in various stages of infection and immunity. While some developed antibodies, the virus replicated in others due to not following the all-in/ all-out protocol. Newly incoming weaners were infected from prior weaners that remained in the room. In every visit to the farm we checked if the biosecurity measures were implemented according to our written guidelines. On the basis of owner assurance, the owner followed all required measures. But the facts presented a completely different picture. He equivocated on lack of time and personnel. We established that only 5 of the 14 required biosecurity measures were followed. Probably, it is very difficult to change the daily routine, which takes one more time and energy. The owner introduced his own replacement gilts into the breeding herd. In the case of introducing the negative replacement gilt, that animal can be a source of virus replication and transmission. The herd closure was not implemented as proposed at the beginning of the study and consequently this can be one of the reasons that the breeding herd could not reach stabilization. So the first goal in the process for achieving the elimination of PRRSV was not accomplished. Although some breeding pigs were identified as negative during the study, when we analysed the individual data the same animals did not remain negative. Hence this suggested that we did not stop the circulation of the virus in breeding herd despite all of the negative results of RT-PCR. One very important measure was the all-in/all-out protocol which was not followed and thus resulted in pore pen hygiene due to non-vacant pens being thoroughly cleaned and disinfected. It follows that both pigs and pens were the source of PRRSV. Moreover, additional staff were not appointed to a single pig category and did not change coveralls or boots between pig categories, nor wash hands between pig categories. Hence these factors were the reason as well as the route of transmission of PRRSV between categories and between facilities. Pig equipment was not kept on the farm but rather brought to the farm without prior sterilisation (tattooing pliers). From the results of serology, molecular testing and biosecurity measures, we can conclude that the owner did not follow the required biosecurity measures nor carry out strict herd closure which proved to be the reasons for the unsuccessful elimination of PRRSV from the farm. In order to eliminate PRRSV from the farm, the proposed measures should be strictly followed and additional measures, immunization of fatteners and partial depopulation should be implemented since we are dealing with a one-site farm. Dee et al. (3) reported that partial depopulation and strict biosecurity measures can stop the circulation of PRRSV in weaners. Thus it can be concluded that the serum inoculation did not prove itself as a successful measure for elimination of PRRSV from the farrow-to-finish farm and implementation of herd closure and biosecurity measures in field conditions is a much more difficult challenge than expected. Nonetheless, further study focusing on the education of farmers must be undertaken. References 1. Stadejek T, Stankiewicz I, Pesjak Z. Concurrent circulation of PRRSV-EU and PRRS-US within swine herd in Poland. In: 4th International Symposium on Emerging and Re-emerging Pig Diseases. Rome, 2003: 67-8. 2. Wensvoort G. Lelystad virus and the porcine epidemic abortion and respiratory syndrome. Vet Res 1993; 24, 117-24. 3. Dee SA, Morrison RB, Joo HS. Eradicating porcine reproductive and respiratory syndrome (PRRS) virus using two-site production and nursery depopulation. J Swine Health Prod 1993; 1(5): 20-3. 4. Gramer ML, Christianson WT, Harris DL. Producing PRRS negative pigs from PRRS positive sows. In: Proceedings of the Annual Meeting of the American Association of the Swine Practitioner. Louisiana, 1999: 413-16. 5. Rejic A, Dewey CE, Deckert AE, Friendship RM, Martin SW, Yaoo D. Production of PRRSV-negative pigs commingled from multiple, vaccinated, serologically stable, PRRSV-positive breading herds. J Swine Health Prod 2001; 9: 179-84. 6. Dee SA. A protocol for defining breeding herd stability and classifying farms according to PRRS status to identify potential intervention strategies: a summary of 200 farms. In: 15th International Pig Veterinary Society Congress. Birmingham: IPVS, 1998: 2. 7. Torremorell M, Christianson WT. PRRS eradication by herd closure. Adv Pork Prod 2002; 13: 169-76. 8. Toma B, Vaillancourt JP, Dufour B, et al. Dictionary of veterinary epidemiology. Ames: Iowa State University Press, 1991: 83. 9. Gillespie TG, Caroll AL. Techniques for PRRSV elimination utilizing modified live virus vaccines on single-site swine farms. In: Proceedings of the 4th International Symposium on Emerging and Re-emerging Pig Diseases. Rome, 1999: 127-8. 10. Menard J. Canadian PRRS eradication: a dream or a future reality? Adv Pork Prod 2008; 19: 77-82. 11. Meng XJ. Heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development. Vet Microbiol 2000; 74: 309-29. 12. Murtaugh MP, Gezow M. Immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (PRRS). Vaccine 2011; 29: 8192-204. 13. Batista L, Pijoan C, Torremorell M. Experimental injection of gilts with porcine reproductive and respiratory syndrome virus (PRRSV) during acclimatization. J Swine Health Prod 2002; 10: 147-50. 14. Fano E, Olea L, Pijoan C. Eradication of porcine reproductive and respiratory syndrome virus by serum inoculation of naive gilts. Canad J Vet Res 2005; 69: 71-4. 15. Shibata I, Mori M, Yazawa S. Experimental reinfection with homologous porcine reproductive and respiratory syndrome virus in SPF pigs. J Vet Med Sci 2000; 62: 105-8. 16. Donadeu M, Arias M, Gomez-Tejedor C, et al. Using polymerase chain reaction to obtain PRRSV-free piglets from epidemically infected herds. J Swine Health Prod 1999; 7: 255-61. 17. Toplak I, Rihtarič D, Hostnik P, Grom J, Štukelj M, Valenčak Z. Identification of genetically diverse sequence of porcine reproductive and respiratory syndrome virus in Slovenia and the impact on the sensitivity of four molecular tests. J Virol Meth 2012; 179: 51-6. 18. Zimmerman J, Benefield DA, Murtaugh MP, Osorio F, Stevenson GW, Torremorell M. Porcine reproductive and respiratory syndrome virus (Porcine arterivirus). In: Zimmerman JJ, Karriker LA, Ramirez KJ, Stevenson GW. Diseases of swine. 10th ed. Ames: Blackwell Publishing Professional, 2012: 461-86. 19. Dee SA, Joo HS, Tokach L, Park BK, Molitor TW, Pijoan C. Detecting subpopulations after PRRSV virus infection in large breeding herds using multiple serologic tests. J Swine Health Prod 1996; 4: 181-4. 20. McCawe M. Different approaches to handling PRRS. In: London Swine Conference: Thinking globally, acting locally. London, 2006: 21-33. 21. Torremorell M, Henry S, Christianson WT. Eradication using herd closure. In: Zimmerman J, Yoon KJ, eds. The 2003 PRRS Compendium. 2nd ed. Des Moines, Iowa: National Pork Board, 2003: 157-61. 22. Schaefer N, Morrison RB. Effect on total pigs weaned of herd closure for elimination of porcine reproductive and respiratory syndrome virus. J Swine Health Prod 2007; 15(3): 152-5. 23. Molitor TW, Bautista EM, Choi CS. Immunity to PRRSV: double-edged sword. Vet Microbiol 1997; 55: 265-76. POSKUS ELIMINACIJE PRAŠIČJEGA REPRODUKCIJSKEGA IN RESPIRATORNEGA SINDROMA NA MANJŠI FARMI Z INOKULACIJO SERUMA M. Štukelj, I. Toplak, Z. Valenčak Povzetek: PoglavitnirazlogzavelikogenetskoraznolikostvirusovPRRSjeverjetnoneučinkovitakontrolabolezniskomercialnimi cepivi, ki vsebujejo samo en sev virusa, saj je zaščita po preboleli okužbi homologna. Ena izmed možnosti za eliminacijo PRRS je prekužitev plemenske črede s farmskim sevom virusa. Za uspešno eliminacijo je nujno upoštevati biovarnostne zahteve. Namen študije je bil eliminirati virus PRRS iz manjše farme (130 plemenskih prašičev) z inokulacijo seruma. Rejec se je obvezal, da bo izvajal stroge biovarnostne ukrepe in zaporo reje vsaj za 200 dni. Plemensko čredo smo imunizirali s pozitivnim serumom tekačev. Šest mesecev po drugem vnosu seruma je število visoko pozitivnih prašičev padlo in trend padanja se je nadaljeval do konca študije, vendar pa je prevalenca protiteles pred serumizacijo v primerjavi s prevalenco na koncu študije (13 mesecev po vnosu seruma) ostala skoraj enaka. Plemenska čreda je bila v vseh testiranjih negativna na prisotnost virusa, virus pa smo stalno dokazovali pri kategoriji tekačev, starih 10 tednov. Rejec se ni držal zapore reje in ostalih predpisanih biovarnostnih zahtev, saj je med drugim vnesel na farmo nov sev virusa PRRS. Eliminacija PRRS z inokulacijo seruma zato ni bila učinkovita. Ugotovili smo, da je izvajanje biovarnostnih zahtev v praksi za rejca zelo velik izziv. Ključne besede: kontrola; imunizacija; prašiči; PRRS; vnos seruma Slov Vet Res 2013; 50 (4): 201-3 AUTHOR INDEX VOLUME 50, 2013 Antonov A, see Fasulkov I, Atanasov A, Antonov A............................31 Atanasov A, see Fasulkov I, Atanasov A, Antonov A............................31 Barač Z, see Kastelic M, Špehar M, Barač Z............................. 149 Biasizzo M, Vadnjal S, Mičunovič J, Henigman U, Kirbiš A. Estimation of Yersinia enterocolitica prevalence in slaughtered pig tonsils in Slovenia by using three cultural isolation procedures.......................... 167 Bilandžič N, Sedak M, Dokič M, Varenina I, Solomun Kolanovič B, Božič D, Brstilo M, Sokolič-Mihalak D, Jurkovič Z. Comparative study of iron, magnesium and zinc and daily intakes in certain meats and meat products........................... 103 Bošnjak D, see Stančič I, Bošnjak D, Radovič I, Gvozdič D, Savič B, Stančič B..........111 Božič D, see Bilandžič N, Sedak M, Dokič M, Varenina I, Solomun Kolanovič B, Božič D, Brstilo M, Sokolič-Mihalak D, Jurkovič Z. . . 103 Brstilo M, see Bilandžič N, Sedak M, Dokič M, Varenina I, Solomun Kolanovič B, Božič D, Brstilo M, Sokolič-Mihalak D, Jurkovič Z. . . 103 Cerar A, see Perše M, Cerar A.......... 127 Cerkvenik-Flajs V, see Dobeic M, Ocepek M, Kožuh-Eržen N, Pintarič Š, Cerkvenik-Flajs V, Gobec 1............................. 173 Cerovšek M, Plavec T, Zrimšek P, Pogačnik M, Zabavnik J. Clinicopathological survey of 56 canine malignant mammary tumours in Slovenia - prognostic value of clinical stage and histological grade...................93 Chen S, see Liu H, Luo P, Chen S, Shang J.............................13 Cheng M, see Gong Q, Cheng M, Niu M, Qin C................................5 Cociancich V, Gombač M, Švara T, Pogačnik M. Malignant mesenchymoma of the aortic valve in a dog.........................83 Cvetnič Ž, see Petrovič M, Špičič S, Potkonjak A, Lako B, Kostov M, Cvetnič Ž........... 117 Čandek-Potokar M, see Fazarinc G, Uršič M, Gjurčevič Kantura V, Trbojevič Vukičevič T, Škrlep M, Čandek - Potokar M............67 Dobeic M, Ocepek M, Kožuh-Eržen N, Pintarič Š, Cerkvenik-Flajs V, Gobec I. Doramectin degradation and bacterial community during sheep manure composting.............. 173 Dokič M, see Bilandžič N, Sedak M, Dokič M, Varenina I, Solomun Kolanovič B, Božič D, Brstilo M, Sokolič-Mihalak D, Jurkovič Z. . . 103 Drobnič Košorok M, see Premrov Bajuk B, Drobnič Košorok M, Malovrh T........... 157 Fasulkov I, Atanasov A, Antonov A. Anogenital cleft in a bitch - A case report. ... 31 Fazarinc G, Uršič M, Gjurčevič Kantura V, Trbojevič Vukičevič T, Škrlep M, Čandek-Potokar M. Expression of myosin heavy chain isoforms in longissimus muscle of domestic and wild pig.............................67 Gagič A, see Hadžiabdič S, Rešidbegovič E, Gruntar I, Kušar D, Pate M, Zahirovič L, Kustura A, Gagič A, Goletič T, Ocepek M.....45 Giadinis ND, see Papaioannou N, Giadinis ND, Tsaousi P............................35 Gjurčevič Kantura V, see Fazarinc G, Uršič M, Gjurčevič Kantura V, Trbojevič Vukičevič T, Škrlep M, Čandek-Potokar M.............67 Gobec I, see Dobeic M, Ocepek M, Kožuh-Eržen N, Pintarič Š, Cerkvenik-Flajs V, Gobec 1............................. 173 Goletič T, see Hadžiabdič S, Rešidbegovič E, Gruntar I, Kušar D, Pate M, Zahirovič L, Kustura A, Gagič A, Goletič T, Ocepek M.....45 Gombač M, see Cociancich V, Gombač M, Švara T, Pogačnik M....................83 Gombač M, Švara T, Paller T, Vergles Rataj A, Pogačnik M. Post-mortem findings in bottlenose dolphins (Tursiops truncatus) in the Slovene sea...........................75 Gong Q, Cheng M, Niu M, Qin C. Study on immune efficacy of genomic expression library vaccines against avian Pasteurella multocida. . . 5 Gorjanc G, see Žan Lotrič M, Gorjanc G, Kompan D.......................... 183 Gruntar I, see Hadžiabdič S, Rešidbegovič E, Gruntar I, Kušar D, Pate M, Zahirovič L, Kustura A, Gagič A, Goletič T, Ocepek M...........45 Gvozdič D, see Stančič I, Bošnjak D, Radovič I, Gvozdič D, Savič B, Stančič B..........111 Hadžiabdič S, Rešidbegovič E, Gruntar I, Kušar D, Pate M, Zahirovič L, Kustura A, Gagič A, Goletič T, Ocepek M. Campylobacters in broiler flocks in Bosnia and Herzegovina: Prevalence and genetic diversity...........45 Henigman U, see Biasizzo M, Vadnjal S, Mičunovič J, Henigman U, Kirbiš A........ 167 Jurkovič Z, see Bilandžič N, Sedak M, Dokič M, Varenina I, Solomun Kolanovič B, Božič D, Brstilo M, Sokolič-Mihalak D, Jurkovič Z. . . 103 Kastelic M, Špehar M, Barač Z. Productivity of milk and milk composition of Istrian sheep in Croatia and Slovenia................ 149 Kirbiš A, see Biasizzo M, Vadnjal S, Mičunovič J, Henigman U, Kirbiš A........ 167 Kompan D, see Žan Lotrič M, Gorjanc G, Kompan D.......................... 183 Kostov M, see Petrovič M, Špičič S, Potkonjak A, Lako B, Kostov M, Cvetnič Ž........... 117 Kožuh-Eržen N, see Dobeic M, Ocepek M, Kožuh-Eržen N, Pintarič Š, Cerkvenik-Flajs V, Gobec 1............................. 173 Kustura A, see Hadžiabdič S, Rešidbegovič E, Gruntar I, Kušar D, Pate M, Zahirovič L, Kustura A, Gagič A, Goletič T, Ocepek M.....45 Kušar D, see Hadžiabdič S, Rešidbegovič E, Gruntar I, Kušar D, Pate M, Zahirovič L, Kustura A, Gagič A, Goletič T, Ocepek M.....45 Lako B, see Petrovič M, Špičič S, Potkonjak A, Lako B, Kostov M, Cvetnič Ž............. 117 Liu H, Luo P, Chen S, Shang J. Cloning of basic fibroblast growth factor from Chinese Small Tail Han sheep and its effects on proliferation of murine C2C12 myoblasts. ... 13 Luo P, see Liu H, Luo P, Chen S, Shang J.............................13 Malovrh T, see Premrov Bajuk B, Drobnič Košorok M, Malovrh T.................. 157 Meshref AMS. Bacteriological quality and safety of raw cow's milk and fresh cream. ... 21 Mičunovič J, see Biasizzo M, Vadnjal S, Mičunovič J, Henigman U, Kirbiš A........ 167 Mrkun J, see Podpečan O, Mrkun J, Zrimšek P............................57 Niu M, see Gong Q, Cheng M, Niu M, Qin C................................5 Ocepek M, see Dobeic M, Ocepek M, Kožuh-Eržen N, Pintarič Š, Cerkvenik-Flajs V, Gobec 1............................. 173 Ocepek M, see Hadžiabdič S, Rešidbegovič E, Gruntar I, Kušar D, Pate M, Zahirovič L, Kustura A, Gagič A, Goletič T, Ocepek M.....45 Paller T, see Gombač M, Švara T, Paller T, Vergles Rataj A, Pogačnik M..............75 Papaioannou N, Giadinis ND, Tsaousi P. Concurrent scrapie and chronic coenurosis in two Chios sheep.......................35 Pate M, see Hadžiabdič S, Rešidbegovič E, Gruntar I, Kušar D, Pate M, Zahirovič L, Kustura A, Gagič A, Goletič T, Ocepek M...........45 Perše M, Cerar A. Animal models of human pathology - our experience.............. 127 Petrovič M, Špičič S, Potkonjak A, Lako B, Kostov M, Cvetnič Ž. Epizootiological, clinical and pathological characteristics of sheep flocks infected with Brucella ovis in the republic of Serbia............................ 117 Pintarič Š, see Dobeic M, Ocepek M, Kožuh-Eržen N, Pintarič Š, Cerkvenik-Flajs V, Gobec 1............................. 173 Plavec T, see Cerovšek M, Plavec T, Zrimšek P, Pogačnik M, Zabavnik J.........93 Podpečan O, Mrkun J, Zrimšek P. Associations between the fat to protein ratio in milk, health status and reproductive performance in dairy cattle...............57 Pogačnik M, see Cerovšek M, Plavec T, Zrimšek P, Pogačnik M, Zabavnik J.........93 Pogačnik M, see Cociancich V, Gombač M, Švara T, Pogačnik M....................83 Pogačnik M, see Gombač M, Švara T, Paller T, Vergles Rataj A, Pogačnik M.......75 Potkonjak A, see Petrovič M, Špičič S, Potkonjak A, Lako B, Kostov M, Cvetnič Ž.. . 117 Premrov Bajuk B, Drobnič Košorok M, Malovrh T. The efect of a peptide inhibitor of cysteine peptidases produced by the dermatophyte Trichophyton mentagrophytes on a mouse immune system............. 157 Qin C, see Gong Q, Cheng M, Niu M, Qin C................................5 Radovič I, see Stančič I, Bošnjak D, Radovič I, Gvozdič D, Savič B, Stančič B............ 111 Rešidbegovič E, see Hadžiabdič S, Rešidbegovič E, Gruntar I, Kušar D, Pate M, Zahirovič L, Kustura A, Gagič A, Goletič T, Ocepek M............................45 Savič B, see Stančič I, Bošnjak D, Radovič I, Gvozdič D, Savič B, Stančič B............ 111 Sedak M, see Bilandžič N, Sedak M, Dokič M, Varenina I, Solomun Kolanovič B, Božič D, Brstilo M, Sokolič-Mihalak D, Jurkovič Z. . . 103 Shang J, see Liu H, Luo P, Chen S, Shang J.............................13 Sokolič-Mihalak D, see Bilandžič N, Sedak M, Dokič M, Varenina I, Solomun Kolanovič B, Božič D, Brstilo M, Sokolič-Mihalak D, Jurkovič Z.......................... 103 Solomun Kolanovič B, see Bilandžič N, Sedak M, Dokič M, Varenina I, Solomun Kolanovič B, Božič D, Brstilo M, Sokolič-Mihalak D, Jurkovič Z.......................... 103 Stančič B, see Stančič I, Bošnjak D, Radovič I, Gvozdič D, Savič B, Stančič B.... 111 Stančič I, Bošnjak D, Radovič I, Gvozdič D, Savič B, Stančič B. Fertility of gilts with prolonged preinsemination anestrus after progestagen-eCG treatment.............111 Škrlep M, see Fazarinc G, Uršič M, Gjurčevič Kantura V, Trbojevič Vukičevič T, Škrlep M, Čandek-Potokar M.....................67 Špehar M, see Kastelic M, Špehar M, Barač Z............................. 149 Špičič S, see Petrovič M, Špičič S, Potkonjak A, Lako B, Kostov M, Cvetnič Ž........... 117 Štukelj M, Toplak I, Valenčak Z. An attempt to eliminate porcine reproductive and respiratory syndrome (PRRS) by serum inoculation on small pig farm............ 193 Švara T, see Cociancich V, Gombač M, Švara T, Pogačnik M.........................83 Švara T, see Gombač M, Švara T, Paller T, Vergles Rataj A, Pogačnik M..............75 Terčič D. Divergent selection experiments in poultry............................. 139 Toplak I, see Štukelj M, Toplak I, Valenčak Z.......................... 193 Trbojevič Vukičevič T, see Fazarinc G, Uršič M, Gjurčevič Kantura V, Trbojevič Vukičevič T, Škrlep M, Čandek-Potokar M.. . . 67 Tsaousi P, see Papaioannou N, Giadinis ND, Tsaousi P............................35 Uršič M, see Fazarinc G, Uršič M, Gjurčevič Kantura V, Trbojevič Vukičevič T, Škrlep M, Čandek-Potokar M.....................67 Vadnjal S, see Biasizzo M, Vadnjal S, Mičunovič J, Henigman U, Kirbiš A........ 167 Valenčak Z, see Štukelj M, Toplak I, Valenčak Z.......................... 193 Varenina I, see Bilandžič N, Sedak M, Dokič M, Varenina I, Solomun Kolanovič B, Božič D, Brstilo M, Sokolič-Mihalak D, Jurkovič Z........................... 103 Vergles Rataj A, see Gombač M, Švara T, Paller T, Vergles Rataj A, Pogačnik M.......75 Zabavnik J, see Cerovšek M, Plavec T, Zrimšek P, Pogačnik M, Zabavnik J.........93 Zahirovič L, see Hadžiabdič S, Rešidbegovič E, Gruntar I, Kušar D, Pate M, Zahirovič L, Kustura A, Gagič A, Goletič T, Ocepek M............................45 Zrimšek P, see Cerovšek M, Plavec T, Zrimšek P, Pogačnik M, Zabavnik J.........93 Zrimšek P, see Podpečan O, Mrkun J, Zrimšek P............................57 Žan Lotrič M, Gorjanc G, Kompan D. Geographical distribution of sheep and goat breeds in Slovenia.................... 183 Izterjava dolgov in upravljanje s terjatvami ^ © M D Svetovanje Finančne storitve wvuw.vasefinance.si Namen ustanovitve in delovanja podjetja MD svetovanje d.o.o. je pomagati podjetjem pri poslovanju z nudenjem produktov in storitev, ki ne spadajo v osnovno dejavnost podjetja. To dosegamo s celovito ponudbo predstavljenih produktov in storitev. Zato smo naš moto Skupaj bomo uspešnejši! nadgradili še z motom in sloganom Vse za Vas na enem mestu! Vizija Postati vodilna neodvisna družba s celotno ponudbo za podjetja in posameznike na enem mestu in na ta način prihraniti podjetjem in posameznikom čas in denar. Vse to nam bo uspelo s trdim delom in kakovostno izvedbo storitev in zaupanih nam nalog, predvsem če bomo sledili naslednjim načelom: zagotavljanje celovite ponudbe, vedno delo v dobro stranke, strokoven razvoj, organizacijsko izpopolnjevanje, zagotavljanje visoke stopnje kakovosti storitev z upoštevanjem predlogov naših strank, ustvarjanje novih delovnih mest, povečanje produktivnosti in dobičkonosnosti, visoko motiviran in usposobljen kader s primernim vodenjem, kar zagotavlja kakovost izvajanja storitev, postati vodilno podjetje, ki ponuja rešitve, ki podjetju omogočajo da si na enem mestu zagotovi vse dejavnosti, ki ne spadajo v njegovo osnovno dejavnost. Prednosti poslovanja z nami: vse svoje potrebe in vizije uresničite s klicem na eno telefonsko številko, razbremenite se ukvarjanja z obrobnimi zadevami, posvetite se svojemu strokovnemu delu, informacijska tehnologija, prilagodljivost, zanesljivost, povečanje dobičkonosnosti, zmanjšanje stroškov dela, MD svetovanje, poizvedbe in storitve d.o.o. Dunajska cesta 421, 1231 Ljubljana - Črnuče PE Ljubljana-Vič Cesta dveh cesarjev 403, 1102 Ljubljana 01 / B20-47-01 01 / B20-47-04 041 /614-090 www. mdsvetovanje. eu Zakaj M D Svetovanje visoka profesionalizacija, visoka strokovnost, visoka uspešnost, konkurenčne cene, vse na enem mestu. d.o.o. i SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Slov Vet Res 2013; 50 (4) Original Scientific Articles Kastelic M, Špehar M, Barac Z. Productivity of milk and milk composition of Istrian sheep in Croatia and Slovenia...............149 Premrov Bajuk B, Drobnič Košorok M, Malovrh T. The efect of a peptide inhibitor of cysteine peptidases produced by the dermatophyte Trichophyton mentagrophytes on a mouse immune system..................................................157 Biasizzo M, Vadnjal S, Mičunovic J, Henigman U, Kirbiš A. Estimation of Yersiniaenterocolitica prevalence in slaughtered pig tonsils in Slovenia by using three cultural isolation procedures...........................................167 Dobeic M, Ocepek M, Kožuh-Eržen N, Pintarič Š, Cerkvenik-Flajs V, Gobec I. Doramectin degradation and bacterial community during sheep manure composting...................................................................................173 Žan Lotrič M, Gorjanc G, Kompan D. Geographical distribution of sheep and goat breeds in Slovenia............................183 Štukelj M, Toplak I, Valenčak Z. An attempt to eliminate porcine reproductive and respiratory syndrome (PRRS) by serum inoculation on small pig farm............................................................................................193 Author Index Volume 50, 2013 201