ISSN 15804003 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Volume 53 Slov Vet Res • Ljubljana • 2016 • Volume 53 • Number 4 • 181-240 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Volume 53 Slov Vet Res • Ljubljana • 2016 • Volume 53 • Number 4 • 181-240 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 Co-Editor / sourednik: Modest Vengušt Technical Editor / tehnični urednik: Matjaž Uršič Assistants to Editor / pomočnici urednika: Valentina Kubale Dvojmoč, Klementina Fon Tacer Editorial Board / uredniški odbor: Vesna Cerkvenik, Robert Frangež, Polona Juntes, Tina Kotnik, Matjaž Ocepek, Milka Vrecl, Veterinary Faculty University of Ljubljana / Veterinarska fakulteta Univerze v Ljubljani Editorial Advisers / svetovalca uredniškega odbora: Gita Grecs-Smole for Bibliography (bibliotekarka), Leon Ščuka for Statistics (za statistiko) Reviewing Editorial Board / ocenjevalni uredniški odbor: 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; 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 79100, 47 79129, Fax: +386 (0)1 28 32 243 E-mail: slovetres@vf.uni-lj.si Sponsored by the Slovenian Research Agency Sofinancira: Javna agencija za raziskovalno dejavnost 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 2016; 53 (4) Review Article Novosel D, Petrovic T, Acinger - Rogic Z, Stukelj M. Epidemiology and status of porcine reproductive and respiratory syndrome in the Western Balkan region: Challenges and prospects.......................................................................185 Original Research Articles Ambrozic M, Henigman U, Jevsnik M, Kirbis A, Raspor P. Viral contamination in mussel production chain on the Slovenian coastline..............................................................................................................................195 Pugliese M, Spadola F, Morici M, Piazza A, Caracappa G, Persichetti MF, Lommi A. Evaluation of the conjunctival bacterial flora in 140 rabbits (Oryctolagus cuniculus) farmed in Sicily Island.............................................................205 Mutluay D. Distribution of primitive endoderm and epiblast lineage specific factors in late stage blastocysts.....................211 Pogorevc E, Lukanc B, Seliskar A, Pelc R, Zorko B. Radiological comparison of lumbosacral anatomy between German and Belgian Shepherd (Malinois) working dogs.................................................................................219 Case Report Brloznik M, Faraguna S, Slavec B, Kostanjsek R, Vergles Rataj A, Gruntar I. Pentatrichomonas hominis coinfection in a puppy from a Slovenian animal shelter....................................................................................................229 Author Index Volume 53, 2016...............................................................................................................237 Slov Vet Res 2016; 53 (4): 185-93 UDC 636.4.09:616.98-036.22:578 Review Article EPIDEMIOLOGY AND STATUS OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME IN THE WESTERN BALKAN REGION: CHALLENGES AND PROSPECTS Dinko Novosel1*, Tamaš Petrovič2, Žaklin Acinger - Rogič3, Marina Štukelj4 1Department of Pathology, Croatian Veterinary Institute, Savska cesta 143, 10000 Zagreb, Croatia; 2Scientific Veterinary Institute "Novi Sad", Rumenacki put 20, 21000 Novi Sad, Serbia; 3Veterinary and Food Safety Directorate, Ministry of Agriculture, Planinska 2a, 10000 Zagreb, Croatia; institute for Health Care of Pigs, University of Ljubljana, Veterinary Faculty, Grbiceva 60, 10000 Ljubljana, Slovenia Corresponding author, E-mail: dinko.novosel@gmail.com Summary: Two decades after its emergence, porcine reproductive and respiratory syndrome (PRRS) remains a challenge to the sustainability of the porcine industry worldwide. In the Western Balkan region in particular, control of the disease is hampered by fragmentation of pig production; lack of farmer knowledge regarding health care; the fact that most farms are small, single-site pig operations with low biosecurity standards; and intensive trading and import of pigs from different countries without known health status and without quarantine. All these factors contribute to rapid disease transmission among pig operations. PRRS entered the Western Balkan region in 1995, when it appeared in Croatia and slightly later in Serbia, and again after 2004 when it entered Slovenia and potentially other countries. All PRRS cases originally described in the Western Balkans appear to have been caused by infection with type 1 subtype 1 PRRSV; more recently, infection with type 2 virus has also been reported. Veterinary services have an important role to play in monitoring and controlling spread of PRRS, but control programs in the region are either inconsistent or non-existent. Available epidemiological data suggest that new PRRSV introduction into Western Balkan countries is less likely to occur via animal transfers within the region and more likely to occur via arrivals from elsewhere in the EU. Strong efforts are needed to develop and implement guidelines for pig movement, implement biosecurity measures, establish consistent diagnostic testing for PRRS virus, and classified pig herds according to health status and farmer education. Key words: PRRSV; Slovenia; Croatia; Serbia Introduction Two decades after its emergence, porcine reproductive and respiratory syndrome (PRRS) remains a challenge to the sustainability of the global porcine industry. As a result, many countries in North America and the European Union (EU) have implemented continuous monitoring programs. The picture is quite different among countries in the Western Balkans, where Received: 26 February 2015 Accepted for publication: 435 April 2016 pig production and veterinary services are highly fragmented, and PRRS control measures are inconsistent or non-existent. This troubling state of affairs reflects a lack of harmonized diagnostic methods and sparse available data on PRRS prevalence, nature of virus and farm management practices. This poses a problem not only for control and reduction of PRRS virus (PRRSV) already present in the Western Balkans, but also for prevention of new virus introduction due to extensive trade with other EU countries. The problem of PRRS monitoring and control in this region is even more challenging because 186 D. Novosel, T. Petrovic, Z. Acinger - Rogic, M. Stukelj Croatia, Slovenia and Serbia contain large numbers of sustainable farms where small numbers of animals are kept, mostly for household needs. For example, of approximately 168,000 breeding sows in Croatia, only 25,000 are in large pig production units; the remainder are on semiintensive or -extensive farms, according to the Croatian Agriculture Agency (http://www.hpa. hr/, accessed 15 May 2016). Most pig farms in Slovenia are small, single-site production farms: according to a census of 4162 farms in 2013, 3909 farms had 1-20 breeding pigs; 206 farms, 21-50; 31 farms, 51-100; 12 farms, 101-200 breeding pigs; 2 farms, 501-1000 and 2 farms, 1001 or more (data from 1.2.2013 in the VOLOS database, Ministry for Agriculture, Forestry and Food, UVHVVR). Though the first PRRS outbreak was reported in lower Saxony in 1992 (1), PRRSV appears to have emerged before that in Eastern European countries behind the "Iron Curtain" (2), when the present-day countries of the Western Balkans formed part of the single federation of Yugoslavia. This federation literally served as a bridge for PRRS: it was the only Communist country with soft borders, and it traded extensively with Eastern and Western Europe. The paths of live animal movements potentially carrying PRRSV changed often in the region as a result of complex political changes that molded and remolded trade routes. The Berlin Wall fell in 1989, Yugoslavia broke up into several republics in 1990, and war raged in Croatia, Bosnia and Herzegovina, Serbia and Montenegro in 1991-1995. Sloveniajoined the Central European Free Trade Association (CEFTA) in 1996, followed by Croatia in 2003. The following year, Slovenia joined the EU and left CEFTA, while in 2007 most other former Yugoslav republics joined CEFTA. In 2013 Croatia joined the EU and left CEFTA (Figure 1). All these changes probably modified live animal movements in complex ways, making it difficult to track PRRSV epidemiology and predict prevalence. Figure 1. Evolution of political alliances affecting trade routes and therefore live animal movements in the Western Balkan region. EU member states are shown in blue; CEFTA member states, in yellow. Membership status is shown (A) before 2003 and (B) in 2015 Epidemiology and status of porcine reproductive and respiratory syndrome in the Western Balkan Region: Challenges and prospects 187 Pig production and trade in the Western Balkans Western Balkan countries import large amounts of pork and pigs from Western Europe. The biggest exporter to the Western Balkans in 2012 was Hungary, with the traded animals originating outside Hungary [Croatian Agriculture Agency (www.hpa.hr), accessed 15 May 2016]. This reflects the fact that large pig-exporting firms in Hungary had distribution centres for the Western Balkans but were not producing their own animals. In the same year, Croatia imported more pig and pork from the EU27 bloc than all other Western Balkan countries (Table 1). Croatia also exported large amounts to Bosnia and Herzegovina and Albania. In fact, membership in CEFTA facilitated pig and pork trading with Bosnia and Serbia (Table 2). Evidence also strongly suggests illegal trading among Western Balkan countries that bypassed veterinary inspections (3). Before joining the EU in 2004, Slovenia was free of PRRS. The disease was introduced after entry as a result of pig import from other EU members; quarantine is not required for animals moving between EU members. Epidemiology of PRRSV in selected Western Balkan countries and attempts at control Epidemiological studies suggest that most pig pathogens in the Western Balkans came from outside the region, and that some local enzootic strains have emerged. Most strains of porcine circovirus type 2 (PCV2) in the Western Balkan region are highly homologous to Dutch strains (4), as are most strains of PRRSV (5, 6). One exception is porcine parvovirus genotype 3 (PPV3), which appears to have originated in Croatia (5). A different PCV2 genotype was recently discovered in wild boars (4). Some local enzootic strains appear to have spread from the Western Balkans to other regions, such as PPV3, while others have not, such as PCV2d (8). Relatively good data exist about PRRSV seroprevalence in Slovenia and Serbia, but little is known about the situation in Croatia, and the few data available are sometimes contradictory. For example, only 11 ELISA and PCR tests were performed in 2009 (9) and only 5 in 2010 (10). Seroprevalence in Croatia is thought to be much higher than 25%, based on analyses of neighboring Slovenia and Serbia, suggesting that nearly all farms suffer economic losses due to PRRSV. Table 1: Key data on Western Balkan countries. Source: FAO (www.fao.org, accessed 15 May 2016) Country Total area (103 Ha) Agriculture area (103Ha) Population (1000s) GDP (mil €) HDI Export (%) Import (MT) Albania 2875 1201 3238 11781 0.19 8.6 Bosnia and Herzegovina 5121 2151 3736 16578 0.71 Croatia 5659 1326 4379 60852 0.76 26.36 Montenegro 1381 512 633 411 0.769 7.3 Romania 23839 13982 21339 161624 0.767 65.8 Bulgaria 11100 5088 7349 47714 0.7343 37.9 Serbia 8836 5061 9835 38423 0.701 FYROM 2571 1118 2069 9189 0.701 7.7 Slovenia 2027 458 2045 46908 0.828 18 Western Balkans 63409 30897 54623 393480 18 153.66 EU 4381376 507890 17577000 0.876 Abbreviation: 1 Area of country in 103 Ha; 2 Total agriculture area in 103 Ha; 3 Country population; 4 Gross domestic product; 5 Human development index; 6 Percentage of production that was exported to other countries; 7 Import in millions of tons 188 D. Novosel, T. Petrovic, Z. Acinger - Rogic, M. Stukelj Table 2: Pig imports and exports to and from Western Balkan countries. Source: UN Comtrade database (http:// comtrade.un.org/, accessed 15 May 2016) Country Pig type Exports (MT) Destination countries Imports (MT) Countries of origin Albania pure breed 7066 GR, H, HR, I < 50 kg 135 GR, NL, I Austria pure breed 1363 D, SLO, CZ, H 38402 D, DK, SLO, CZ < 50 kg 1028 SLO, H, HR, D 4814 D, SLO, CZ, H Bosnia and Herzegovina pure breed 16 MNE 4932 HR, SRB, H < 50 kg 1,6 MNE 711 H, D, SRB, HR Croatia pure breed 7983 BiH, SRB, AL 142 H, CZ, SLO < 50 kg 19 BiH 13887 NL, D, H, DK Denmark pure breed 46443 D, BY, I, E < 50 kg 237313 D, PL, I, CZ Greece pure breed < 50 kg 34 AL 104 H, N, E Montenegro pure breed 833 H, SRB,BiH < 50 kg 651 SRB, BiH Romania pure breed 20686 H, BG, NL < 50 kg 11 MD 18709 NL, H ,D Bulgaria pure breed 89 GE, AL 18 DK, D, GR < 50 kg 574 NL, GR, RO Serbia pure breed < 50 kg FYROM pure breed 355 SRB < 50 kg 43 SRB Slovenia pure breed 2655 A, MNE, AL 306 A, H, I < 50 kg 19 A 1844 A, D, NL Hungary pure breed 55 AL 13 F, E < 50 kg 7948 RO, HR, NL 9042 NL, D, SK EU27 pure breed 23443 RU, AL, SRB 1,8 CH, US < 50 kg 24622 HR, UA 20 CH Western Balkans pure breed 11098 HR, BiH 2691 < 50 kg 127,6 36615 Abreviation: 1 Pure breed - reproductive gilts and boars, <50 kg - imported weaners for fattening; 2 Exports in million tons of body weight; 3 Imports in million tons of body weight Epidemiology and status of porcine reproductive and respiratory syndrome in the Western Balkan Region: Challenges and prospects 189 Croatia The first outbreak of a severe reproductive disorder in breeding animals in Croatia occurred in 1995 (11), most probably resulting from sow insemination with imported Duroc semen contaminated with PRRSV. At that time, Croatia had not yet developed methods to diagnose PRRS, so serum samples were sent to the Veterinary Diagnostic Institute in Lelystad in the Netherlands; these samples tested positive for PRRSV by the enzyme-linked immunosorbent assay (ELISA). Subsequently the Croatian Veterinary Institute became a major testing centre for PRRSV, testing approximately 60,000 serum samples between 1996 and 2010 (12). During that time, the disease spread to nearly all major pig breeding herds, and seroprevalence in domestic swine was estimated to be over 90% (12). The situation appeared to have improved in 2009, though only 709 serum samples were tested, followed by only 955 in 2010. Testing of sera from boars, sows, gilts and fatteners for the presence of anti-PRRSV antibodies using three commercial ELISA kits revealed no positives in 2009, compared to positive rates of 1.84% (5 of 272) in sows and 2.59% (5 of 193) in fatteners in 2010. These results likely underestimated the prevalence of PRRSV, since only 1.1% of sows were tested in 2009 and only 1.6% in 2010 (13). Indeed, testing of samples using nested reverse transcription (RT)-PCR, immunohistochemistry and pathology indicated the presence of PRRSV in nearly all large pig production units in Croatia. This lack of progress in controlling or eradicating PRRS contrasts with the fact that PRRS is a notifiable disease in Croatia: every positive PRRS case must be notified to the Croatian veterinary directorate. The problem may be that the national authority has not mandated any specific control measures to combat PRRS. As a result, control measures are usually applied at the farm level by the local veterinary service assigned to that particular farm. Meanwhile, large pig production farms and units usually apply only nonspecific (mainly biosecurity) measures. Outbreaks of classical swine fever in 2006 and 2007-08 led the national authority to mandate biosecurity measures on all farms, with more stringent measures required on farms keeping more than 100 pigs. Efforts to control and eradicate PRRS in Croatia continue to lack any coordination at the national level. As of 2015, the veterinary authority does not require PRRSV testing of national breeding stock, such as testing of boars before transfer to artificial insemination facilities or regular testing of sows and gilts. As a result, such testing is performed at farm level by the local veterinary service assigned to that particular farm based on individual health programs. No national-level data on PRRSV vaccination are available, nor are PRRSV-positive farms required to notify the veterinary authority of depopulation-repopulation programs or other control measures. In 2004, a PRRSV emergency was declared and imported inactivated vaccine was applied. The results were ambiguous and left many farmers disappointed and unconvinced of the efficacy of coordinated intervention at the national level. An attenuated vaccine against PRRSV entered the Croatian market in 2007, but it has been little used. Epidemiological data on PRRSV-positive animals are less reliable because all animals that test positive locally are automatically recorded as PRRSV-positive, regardless of diagnostic criteria and testing method. Testing and confirmation methods have not been standardized at the national level. Available epidemiological data suggest persistence of the virus throughout Croatia: in 2009, 26 pigs on 6 farms tested positive for PRRSV; in 2010, 6 pigs on 6 farms; in 2011, 8 pigs on 3 farms; in 2012, 295 pigs on 48 farms; in 2013, 43 pigs on 7 farms; in 2014, 31 pigs on 10 farms. Positive farms were located mainly in Osijek-Ba-ranja, Vukovar-Srijem and in Medjimurje County, consistent with the higher concentration of large pig production sites in these areas. Slovenia A survey of swine sera during 1999-2004 in Slovenia showed all herds to be free of PRRSV. In the beginning of 2005, soon after Slovenia joined the EU, animals positive for anti-PRRSV antibody using ELISA we first detected among breeding pigs in a few herds (14). In 2010, a survey of 267 herds revealed that 44.8% were seropositive for antibody (15). Positive herds manifested clinical signs of disease, including reproductive and/or respiratory disorders. To reduce economic losses, farms with positive animals relied on vaccination with the same 190 D. Novosel, T. Petrovic, Z. Acinger - Rogic, M. Stukelj two live-attenuated vaccines currently available in the EU, as well as on herd closure, roll-over and serum inoculation. A volunteer project funded by the Slovenian Research Agency and the Ministry of Agriculture and Environment eliminated or eradicated PRRS from 7 of 19 farms (16). The hypothesis that EU entry led to PRRSV introduction into Slovenia through import of infected animals seems more plausible given that something similar appears to have happened with Hungary. Shortly after Hungary acceded in 1996, the first seropositive pigs were recorded in 1996. In contrast to the situation in Slovenia, between 1995 and 2002, 27925 pig sera were tested and the seroprevalence remained below 3% until 2002, despite brisk pig trade between Hungary and Western Europe (17). Although a national program to eradicate PRRS was proposed in 2011, it was never implemented. The main problem was that farmers did not want PRRS status to become publicly known. Serbia The first suspected cases of PRRS in Serbia occurred in 2001, when serious respiratory disorders associated with high mortality affected large numbers of pigs on two large industrial farms located in the northern region close to the borders with Croatia and Hungary. The suspected cause of the cases was boar semen illegally imported from neighbouring countries. Subsequently in 200102, respiratory syndrome with high morbidity and moderate mortality, which was diagnosed as PRRS, occurred on several large industrial farms in the northern Serbian province of Vojvodina, where pig production is intensive. This syndrome subsequently spread to parts of central Serbia. Severe health problems and high economic losses led the veterinary directorate to perform PRRS serology screening in 2002, 2004-2005 and 20062007 (Table 3). PRRSV monitoring using RT-PCR and immunofluorescence in 2002 detected the virus in 2 of 16 piglets who died on infected farms. Monitoring in 2006-2007 revealed PRRSV-positive herds in all Serbian regions at prevalences of 1.56-60.86%; the disease was most prevalent in northern, western and central parts of the country, where prevalence was 17.30-60.86%. In contrast, prevalence was only 1.56-8.98% among herds in the eastern and southern parts of the country (18). These screening results suggest that two major PRRS introductions occurred in the Western Balkan region. The first one was in 1996 when the disease moved from Croatia into Hungary and shortly thereafter into Serbia. The second introduction occurred in Slovenia and Hungary after they joined the EU, when quarantine of imported animals from EU countries was no longer required. As a result of the first virus introduction and resulting outbreak, an emergency-vaccination campaign to control PRRSV was carried out on large industrial farms in northern Serbia in 20022003. Other than this limited intervention, no monitoring or control program against PRRS has ever been proposed at the national level. Genetic diversity of PRRSV in the Western Balkan region Extensive studies have identified only one PRRSV type (EU type 1 subtype 1) circulating in the Western Balkan region, with no evidence of other Eastern European subtypes (5, 6, 19, 20). ORF5 sequences from field samples in Croatia show 95.2-99.7% homology with the Lelystad vaccine strain used in the country (5). In Serbia, phylogenetics indicate that all 18 genetically typed isolates belong to EU subtype 1 or Lelystad-type viruses that are distributed across Europe as well as other parts of the world (19). In Slovenia, six genetically different PRRSV strains were found in circulation, all belonging to type 1 subtype 1. Table 3: Results of PRRSV screening in Serbia Period Farms/animals, n Positive animals, n / % Positive farms, n / % 2002 32 / 880 511 / 58.07 20 / 62.50 2004-2005 43 / 1135 540 / 47.58 28 / 65.12 2006-2007 562 / 3069 No data 11 / 20.46 Epidemiology and status of porcine reproductive and respiratory syndrome in the Western Balkan Region: Challenges and prospects 191 Median-joining (MJ) phylogenetic networks can illustrate microevolutionary process in viruses (21), particulary for viruses in which recombination frequently occurs (22). Therefore we generated an MJ phylogenetic network based on partial 281-bp ORF7 sequences from Slovenia (n = 22), Serbia (n = 17) and Croatia (n = 7) (6, 19, 20), as well as on seven additional ORF7 sequences from Croatia (5). Sequences were aligned using the ClustalW program in MEGA 5 software (23); sequence polymorphism and haplotype diversity were computed using DnaSP 5 (2). Finally, MJ networks of haplotypes were constructed using Network 4.6.0.0. (Fluxus Technology). The MJ network clearly shows that all Serbian and Slovenian and most Croatian sequences belong to EU type 1, subtype 1. Eastern EU subtypes 2-4, previously identified in Belarus, Lithuania, Russia and Kazakhstan, were not present among the Western Balkan samples. The phylogenetic relationships suggest that the PRRSV genotype 1 was introduced six times into the Western Balkans, and these events involved the appearance of the following six strains: a strain with an ORF5 sequence similar to that of the Porcillis PRRSV strain used in vaccines in Serbia and Croatia (5), a strain that has been circulating for a long time in Slovenia and that has mutated several times, a strain recently introduced into Slovenia, a strain in Slovenia originating from an Amervac vaccine strain, a strain present in Croatia and Slovenia, and a strain circulating for a long time in Serbia that shows homology to Eastern European subtype 3. Two Croatian sequences clustered with American virus type - Genotype 2, one of which appears to be derived from a vaccine strain, while ResPRRS vaccine was never used and registered in Croatia. These sequences came from farms that imported breeding animals from the US. These insights from MJ phylogenetic networks should be interpreted with caution when reconstructing the evolution of PRRSV strains in the Western Balkan region. The MJ analysis is based only on partial ORF7 sequences. Definitive determination of the origins of PRRSV strains in Croatia, Serbia and Slovenia requires more comprehensive analysis based on both ORF5 and ORF7 sequences. If the preliminary results in Figure 2 are verified, they may indicate that PRRSV movement among Western Balkan countries poses a much smaller threat than import of infected animals from Western Europe. Figure 2: MJ network of PRRSV isolates from Croatia, Serbia and Slovenia based on partial ORF7 sequences, showing phylogenetic relationships among PRRSV sequences and related sequences in GenBank. Red dots indicate Croatian haplotypes; blue, Serbian; and green, Slovenian. Yellow dots indicate haplotypes isolated in other countries and deposited in GenBank. Haplotypes are labelled by geographic origin. Dot size is proportional to the frequencies of isolates belonging to the given haplotype 192 D. Novosel, T. Petrovic, Z. Acinger - Rogic, M. Stukelj Conclusions Complex trade relations and a poor understanding of the true economic costs of PRRSV have dampened political will to create control and eradication programs. Strong leadership from government agencies is needed throughout the Western Balkan region to develop guidelines for pig movements, develop diagnostic methods and screening approaches and implement requirements for clear division status at the herd and animal levels. Farmers and veterinarians need to be educated about diagnosis and biosecurity measures; uncertainties about these issues lead many to be unconcerned about PRRS as a threat. Systematic and rigorous epidemiological studies of PRRS are needed in Croatia in order to guide future monitoring, control and eradication efforts. Collaborative epidemiological studies involving the various Western Balkan countries may generate much-needed hard data on the economic impact of PRRSV, its prevalence, herd risk and effectiveness of diagnostic tools. Acknowledgments This work was conducted in the framework of COST Action FA0902 ("Understanding and combating porcine reproductive and respiratory syndrome in Europe - EuroPRRSnet"), the Ministry of Science, Education and Sport of the Republic of Croatia (project 0481153) as well as the Slovenian Research Agency (ARRS) and Ministry of Agriculture and Environment (CRP V4-1111). 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In: EuroPRRS2011: understanding and combating PRRS in Europe. Novi Sad, Serbia, 2011: 50-6. 20. Toplak I, Lazič S, Lupulovič D, et al. Study of the genetic variability of Porcine circovirus type 2 detected in Serbia and Slovenia. Acta Vet Hung 2012; 60(3): 409-20. 21. Morrison DA. Networks in phylogenetic analysis: new tools for population biology. Int J Parasitol 2005; 35(5): 567-82. 22. Olvera A, Busquets N, Cortey M, et al. Applying phylogenetic analysis to viral livestock diseases: moving beyond molecular typing. Vet J 2010; 184(2): 130-7. 23. Tischer I, Rasch R, Tochtermann G. Characterization of papovavirus-and picornavirus-like particles in permanent pig kidney cell lines. Zen-tralbl Bakteriol Orig A 1974; 226(2): 153-67. NAČINI UKREPANJA IN STANJE V NEKATERIH DRŽAVAH ZAHODNEGA BALKANA GLEDE PRAŠIČJEGA REPRODUKCIJSKEGA IN RESPIRATORNEGA SINDROMA (PRRS) D. Novosel, T. Petrovič, Ž. Acinger - Rogič, M. Štukelj Povzetek: Prašičji reprodukcijski in respiratorni sindrom (PRRS) so dokazali že pred 20 leti in še vedno povzroča velike izgube v prašičereji. V zahodnih balkanskih državah se srečujemo s specifično situacijo; v glavnem obstajajo majhne družinske reje, v katerih navadno ne izvajajo nikakršnih ukrepov zoper PRRS. Večina omenjenih držav ima nizek odstotek samooskrbe s prašičjim mesom, zato ga uvažajo iz drugih evropskih držav. Na podlagi rezultatov lahko sklepamo, da je do vnosa bolezni prišlo najprej na Hrvaškem leta 1995, malo kasneje pa v Srbiji. Drugi pomembni vnos bolezni se je zgodil pol leta po vstopu Slovenije v Evropsko unijo leta 2004. Najpogosteje v zahodnih balkanskih državah ugotavljamo genotip 1 virusa PRRS in podtip 1, medtem ko drugih podtipov, ki se pojavljajo v vzhodnoevropskih državah, nismo dokazali. Poglaviten vzrok neukrepanja zoper PRRS je v zahodnih balkanskih državah nepoznavanje dejanskih izgub, ki jih povzroča bolezen v posamezni reji, v nekaterih državah pa tudi slaba laboratorijska diagnostika. Verjetno je treba med vzroke prišteti tudi nezainteresiranost politike za izvajanje nacionalnih programov ukrepanja zoper PRRS. Izračuni so pokazali, da je PRRS na Hrvaškem leta 2011 povzročil za 17 milijonov EUR izgub v prašičereji. Poglavitni krivec za nastali položaj je veterinarska uprava, ki ni izpeljala potrebnih ukrepov. Širjene bolezni med omenjenimi zahodnimi balkanskimi državami ne predstavlja velikega tveganja, saj praktično ni trgovanja med njimi, nasprotno pa so uvozi iz drugih držav velika nevarnost za vnos novih sevov virusa PRRS. Na podlagi omenjenih dejstev bi bilo treba pripraviti načrt ukrepov zoper PRRS, ki bi med drugim vključevali tudi testiranje na prisotnost tako protiteles kot virusa PRRS in posledično vzpostavitev statusov čred, kar bi pripomoglo k zajezitvi širjenja bolezni. Ključne besede: PRRSV; Slovenija; Hrvaška; Srbija Slov Vet Res 2016; 54 (4): 195-204 UDC 614.31:637.56:578 Original Research Article VIRAL CONTAMINATION IN MUSSEL PRODUCTION CHAIN ON THE SLOVENIAN COASTLINE Mateja Ambrožič1, Urška Henigman2, Mojca Jevšnik3, Andrej Kirbiš2, Peter Raspor1 1Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, institute for Food Hygiene, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 3Department of Sanitary Engineering, Faculty of Health Sciences, University of Ljubljana, Zdravstvena pot 5, 1000 Ljubljana, Slovenia Corresponding author, E-mail: peter.raspor@guest.arnes.si Summary: Noroviruses (NoVs) and Hepatitis A virus (HAV) are significant agents of foodborne human viral illness worldwide, both as agents of greatest concern from the consumption of shellfish. In the framework of official national monitoring shellfish samples have been collected since 2013 to determine the spread of NoVs and HAV. Contamination of shellfish samples with NoVs varied from 25% in year 2014 to 40% in year 2015. HAV was not found in any of the analyzed samples, which correlates with the low number of human infections in Slovenia. Alongside official surveillance, semi-structured interviews were carried out with local shellfish farmers regarding this subject. The qualitative analysis highlighted that Slovenian shellfish farmers are aware of food safety hazards, especially associated with hazards to primary production, but only in the context of Escherichia colias an indicator microorganism, and not in the concept of viral food safety. Despite detected foodborne viruses in shellfish on the Slovenian market, local farmers are not aware of or do not recognize foodborne viruses as distinctive food safety hazards. It can be concluded that local farmers possess knowledge and information of critical points in the mussels food supply chain connected to food safety in general. However, in particular, they are not familiar with viruses that represent barrier and consequently critical point to food safety management practices. Training and education on all important aspects of viral food safety according to the current recommendations is strongly recommended for all actors within the shellfish supply chain. Key words: mussel farmer; Slovenia; official control; food safety; foodborne viruses; semi-structured personal interviews Introduction Mariculture is a traditional activity in the Primorska region. Mediterranean mussels (Mytilus galloprovincialis) are the main harvested species and, of a smaller quantity, also warty venus (Venus verrucosa). Mussel farming takes place in a standard manner in lines of floating buoys linked together, with longline nets hung from them. In Slovenia, within 46.6 km of coastline there are three official harvesting areas of Mediterranean Received: 492 February 2015 Accepted for publication: 435 April 2016 mussels: Seča, Strunjan, and Debeli rtič, with 56 registered shellfish farmers on a total area of 46 ha. In 2013, 231 persons were involved in aquaculture activities in Slovenia, and only 39 persons were involved in marine fish and shellfish farming. The aquaculture sector in Slovenia is mainly characterized by small self-employed family farms, most of which have one employee, and some are assisted by unpaid family members. Total shellfish production in 2013 was 311 tonnes, and current production covers mainly the needs of the domestic market. The major cultured shellfish species, Mediterranean mussel, accounts 196 M. Ambrozic, U. Henigman, M. Jevsnik, A. Kirbis, P. Raspor for 83 percent of total mariculture production in Slovenia (1, 2). Next to that there were about 153 tons of imports of mussels in Slovenia but only 23 tons of export, what is a negligible quantity in alimentation compared to consumption of fresh fruit and vegetables, which are also recognized as vulnerable to viral contamination. Yet, the level of shellfish consumption by country is very different. In some countries, the mussel consumption per capita is over 3 kg per year, while it is not even part of the local diet in others (3). Per capita consumption of fresh fruit in Slovenia was in 2013 75 kg and 73 kg of fresh vegetables. However, there were no contaminated samples with NoVs or HAV of fresh produce found within Slovenian national monitoring (27). Shellfish specialties are commonly part of the culinary and gastronomic specialties only along the Slovene coast and are generally prepared and served in restaurants. The filter-feeding nature of bivalves and the traditional way of consuming them (often raw or slightly cooked) make shellfish one of the most common vehicles of viral foodborne illness. Shellfish are filter-feeding animals, which ingest and accumulate human pathogens (5). Lees (6) reported that shellfish grown in sewage-polluted waters tend to bio-accumulate environmentally stable enteric viruses. Processing interventions such as depuration do not eliminate viral particles (7), and food consumption practices increase the health risk related to shellfish consumption. The increasing amount of data on virus detection in shellfish (8, 9, 10) and shellfish-borne viral outbreaks (11) indicates the necessity of a constant surveillance system in European countries, including Slovenia (12). The management of the harvesting areas continues to rely uniquely on bacterial standards, such as Escherichia coli, despite the proven fact of being unreliable tools to indicate the viral presence in harvesting areas or to control the efficiency of the process, such as depuration (13, 14). From a virological point of view, shellfish safety continues to be a sanitation challenge and to protect consumers the EU strives towards establishing legislation on this matter. With better diagnostic technology and investigative epidemiology, it is now accepted that enteric viruses are major contributors to foodborne disease as well. Enteric viruses are transmitted through contaminated food, but also in combination with person-to-person contact or through environmental contamination. They have been increasingly recognized a significant cause of foodborne disease, despite the measures already in place, mainly targeted at reducing bacterial contamination, because of the increasing consumption of ready-to-eat foods, raw and/ or minimally processed shellfish, fruits, and vegetables. This is because products are often imported from areas lacking strict hygienic measures, they are often eaten uncooked, and they often come into contact with potentially contaminated animal manure, water, ice, human hands and surfaces from the "farm-to-table" continuum (10). Most foodborne viruses are more resistant (15) than bacteria to commonly used control measures, (e.g. refrigeration, freezing, pH, drying, UV radiation, heat, pressure, disinfection, etc.). There are currently no effective, realistic and validated risk management options to eliminate viral contamination prior to consumption without changing the normally desired characteristics ofthe food. Because of concerns about virus persistence during food processing, effective control strategies need to focus on the prevention of contamination. From the limited available information, foodborne viruses have a low infectious dose and are dispersed in stool or emesis in high numbers. Only a few viral/infectious particles are needed to cause an infection that may lead to illness (10, 15, 17). Shellfish aquaculture is a marine-based industry that is affected by other land users such as tourism, recreation, forestry, agriculture, and urban development. In many cases, the public is unaware of the detrimental impact their activities have on the aquaculture sector and, consequently, also on shellfish food safety. Food safety embraces the absence or acceptable and safe levels of contaminants, adulterants, naturally occurring toxins or any other substances that can make food dangerous to human health. Microbial food safety is considered a significant public health issue but historically has focused mostly on the control of bacterial contamination; however, enteric viruses have been increasingly recognized as an important cause of foodborne disease, and control measures are being developed (16, 17). The food supply chain from stable to table includes activities such as production, processing, distribution, retail, packaging and labeling of foodstuffs, which are governed by a mass of laws, regulations, codes of practice and guidance. Nowadays, the distance that food travels from producer to consumer has increased as a result of globalization in the food Viral contamination in mussel production chain on the Slovenian coastline 197 trade. Moving these food products safely and efficiently from farm to fork requires a highly coordinated series of links in a long chain of trading partners. Food miles, as a term that refers to the distance food is transported from where it is grown or raised to where it is purchased by a consumer, is part of the broader issue of sustainability that deals with a large range of environmental, social and economic issues. Therefore, keeping safety and quality along the food supply chain has become a significant challenge, whereas good traceability systems, defined as the ability to trace and follow a food, feed, food-producing animal or substance intended to be, or expected to be incorporated into a food or feed, through all stages of production, processing and distribution (18), help to minimize the production and distribution of unsafe or poor quality products. Epidemiology of foodborne viruses Although shellfish consumption can contribute to a healthy diet they are often associated with outbreaks of foodborne disease. Viral foodborne outbreaks associated with shellfish consumption have occurred in many countries (11) despite existing strategies to prevent contamination. They are often attributed to water contamination by sewage and/or during processing and serving. According to epidemiological evidence, NoVs as the predominant agents of nonbacterial gastroenteritis in humans along with Hepatitis A virus (HAV), both as agents of greatest concern from the consumption of shellfish, are important agents of foodborne human viral illness worldwide (17, 19, 20, 21, 22, 23). Of the approximately 600 million cases of illness caused by foodborne hazards in 2010 worldwide, infectious agents that cause diarrheal diseases accounted for the vast majority (550 million), in particular noroviruses (120 million cases) and Hepatitis A virus 14 million cases (24). A total of 5251 foodborne outbreaks were reported in 2014 in the EU (20) within the framework of member states' national monitoring. In 2014, food-borne viruses were, for the first time, identified as the most commonly detected causative agent in the reported food-borne outbreaks. 1070 food-borne outbreaks caused by viruses were reported in 2014, implicated 11740 cases, 2486 hospitalizations and 2 deaths. In strong-evidence outbreaks caused by viruses, 'crustaceans, shellfish, mollusks and products thereof was the most commonly implicated food vehicle (44.7% of outbreaks), followed by 'buffet meals' (15.8% of outbreaks), 'mixed food' (13.2%) and 'fruit' and 'berries and juices' (both 5.3%). The place of exposure most frequently reported was 'restaurant, café, pub, bar, hotel', followed by the household. Norovirus was the most commonly reported virus implicated in the strong-evidence outbreaks and accounted for 97.6% of cases. National statistics on foodborne viral disease are not easily available and, where present, likely to reflect significant under-reporting (17), because there is a lack of systematic surveillance for foodborne viral disease (25). Considering the scientific opinion from the EFSA, RASFF notifications and results of official controls, since 2013 the Slovenian National Zoonoses Monitoring Programme has included food sampling for the presence of NoVs and HAV in live shellfish at the retail level and distribution of local and foreign origin, which are recognized as potentially zoonotic viruses (26). In Slovenia, the Zoonoses Monitoring Programme (27) has been conducted at the national level since 1985. It is designed for the systematic collection, monitoring, analysis and communication of data on the emergence of zoonosis, zoonotic agents, and related antimicrobial resistance and comprises the recently emerging zoonotic agents, including foodborne viruses. The ultimate purpose is to capture high-quality information about infections in humans as well as in animals and the contamination of foods, providing important information that is integrated across sectors. It should provide a fundamental basis for making public health decisions with actions for reducing the risks to public health, document the impact of an intervention, track progress towards specified goals, and elucidate the epidemiology of health problems. The results obtained within Slovenian official national monitoring have shown that live shellfish, purchased at retail stores in Slovenia during independent sampling times throughout the year were contaminated with NoVs, but HAV was not found in any of the samples analyzed (Table 1), which correlates with the low number of human infections in Slovenia (Table 2). In studies, contamination of mussels' samples varies from 16.9% (9), to 34.4% NoVs in Italy (28), to 35.0% of contaminated mussels in France (29). Henigman et al. (8) reported 198 M. Ambrožič, U. Henigman, M. Jevšnik, A. Kirbiš, P. Raspor Table 1: Presence of NoVs and HAV in live shellfish samples within Slovenian official surveillance Year Virus NoVs HAV 2013 5/17 (29%) 0/15 2014* 3/12 (25%) 0/12 2015* 3/10 (40%) 0/10 ^Preliminary results Table 2: Reported infections caused by NoVs and HAV in humans in Slovenia from 2007-2014 (27) N° of cases Year 2007 2008 2009 2010 2011 2012 2013 2014* NoVs 1094 1043 1393 2012 2231 1611 2146 1316 HAV 15 17 12 9 12 11 23 10 ^Preliminary results practices are segregated along the food supply chain and are not connected to a comprehensive system, resulting in the existence of exposure to potential of food hazards, especially emerging hazards, such as viruses (12, 30). The development in different areas within the food production chain and in particularly in technological and technical means is moving very quickly. We encounter innovations in materials, and supporting measures almost daily. Consequently, the gap between knowledge and skills is widening. We are willing to accept the paradigm that drifting is the most dangerous challenge in analytical instruments. However, it is also extremely influential in technological practices. It occurs side by side with "industrial blindness", which develops as a personal characteristic of employees who do not see particular items although they are commonly present in routine operations. Consumers play an important role in the transmission of hazards, including viruses. Implemented viral food safety guidelines (12) are not purposely designed for informing consumers, although studies in recent years have highlighted gaps in food safety knowledge and some critical safety violations regarding food handling at home (31, 32, 33, 34, 35, 36). Consumer behaviour and attitudes toward food safety have shown that the levels of understanding, motivation and trust need to be further cultivated, and their training and informing due to changes in lifestyle and food that mussels collected in Slovenian coastal waters were contaminated with NoVs, the highest at Debeli Rtič (25.9%), 21.2% in Strunjan, and only 8.1% in the Seča harvesting area. The difference in positive results is interlinked to the location of harvesting areas due to sea current, dense shipping, and the influx of streams and rivers. Control and prevention of foodborne viruses Virus contamination as a consequence of human handling can occur at any stage of food production, processing, and even preparation. At present, we are faced with insufficient knowledge and awareness of food safety issues among food handlers and accompanied by consumers being insufficiently informed about food safety principles in the home. Today, we manage food safety through good practices at different levels within a food supply chain that can be described as a network of food-related businesses involved in the creation and consumption of food products that move from farm to table and are linked by information, material, and capital flows. Good practices are described in several different codes of practice designed by producers' organizations, importers and retailer's consortia and government bodies at different levels of production, processing and consumption within the food supply chain. All current active Viral contamination in mussel production chain on the Slovenian coastline 199 consumption patterns encouraged (30, 37, 38, 39). EFSA reported (20) that viral foodborne outbreaks most frequently occurred in 'restaurant, café, pub, bar, hotel", followed by the household. However, outbreaks of foodborne illness occurring in private homes are less likely to be reported than those in commercial and public premises, and it is believed that infections attributed to private homes are three times more frequent than those attributed to canteens (40). Food handlers also play an important role in the transmission of enteric viruses in the shellfish supply chain (16, 20, 41, 42), especially because shellfish specialities are generally prepared and served in restaurants. During production, harvest and packaging preparation, food can become contaminated with viruses by food handlers or after contact with virus-contaminated water and surfaces. A major contributor to the spread of disease in food production is poor hygiene practices or being in contact with faecal material or vomit (15). Food handlers are unaware of controls specific to enteric viruses (16). That is why training on all important aspects of NoVs and HAV according to the recently developed Codex Alimentarius guidelines to control viruses in food is strongly recommended. The primary purpose of the codex guidelines for the control of viruses in food is to give guidance on how to prevent or minimize the presence of human enteric viruses in food, especially NoVs and HAV, and to emphasize that management strategies regarding foodborne viruses and associated illnesses should be different from those for bacterial pathogens. In 2011 Poklar Vatovec with co-workers (42) carried out the research to evaluate the offer of shellfish specialities in Slovene Istria restaurants and to assess food safety knowledge and behaviour of food handlers in preparing shellfish dishes. Results indicated poor food safety knowledge regardless the education of food handlers. The origin of shellfish is important in ensuring food safety; and restaurants should be convinced of good raw meat to exclude foodborne poisoning. Therefore, shellfish should be bought only at registered plants, since these are under official supervision. However they observed that shellfish were not always bought at registered plants, but supplied from the so called illegal »black market«. Next to that it was also observed that employed personnel were hardly acquainted with HACCP principles which represent major food safety hazard. Cooking (at least 90 °C for at least 90 seconds) is a critical point for ensuring food safety. The survey showed that the mid temperature was measured by only 26.8% of the interviewees with formal education and 7.3% with informal education. The remaining did not perform this procedure or it was not known whether it was performed. The results of the survey demonstrated that only 4.9% of the interviewees, regardless their education, are familiar with the correct temperature for heat treatment of shellfish. Research also pointed out that food handlers employed in Slovene Istria restaurants have insufficient knowledge on storing temperatures, storing time and the adequate methods of storing shellfish. Pilot study: Semi-structured interview with Slovenian mussel farmers Pilot study illustration In order to determine eventual connection between comprehension of viral food safety and the shellfish growing practices, the four semi-structured interviews were carried out with Slovenian mussel farmers. The interview guide covered the following topics: - Factors responsible for food safety within the shellfish food supply chain, - Conditions related to food safety hazards with an emphasis on foodborne viruses due to virus-commodity combination, which has been identified as one of the greatest public health concerns. In this pilot study, an empirical grounding was important because an exploration of local farmers' viral food safety perceptions and their good hygiene practices, together with results from the National Zoonoses Monitoring Programme, outlined and gave insight into the current situation in Slovenia. The semi-structured interviews were chosen due to the sensitivity and complexity of the subject discussed. The semi-structured interview started with questions concerning food safety in general. The first open thematic question was: "Tell me as much as you can about the importance of the mussels production process, and about the factors that could affect its food safety." Discussions continued with the questions: "Could you please 200 M. Ambrozic, U. Henigman, M. Jevsnik, A. Kirbis, P. Raspor explain what food safety means from your point of view and when mussels are considered safe for a consumer?" and: "Have you ever heard about viruses that are transmitted by shellfish?" Follow-up questions were posed to complement and facilitate the dialogue. The discussions were concluded with the question: "Do you use working documents that have resulted from food safety legislation, like the HACCP plan, good practices that includes viruses?" The semi-structured interview ended with a question on whether the interviewee had something to add. Efforts were made to create trust, since issues of guilt and failure may easily arise. The results of national monitoring are supported by the responses recorded by the interviewers, which were clearly marked due to the assurance of anonymity. The letter "I" (I1) signifies "interview", while the number represents a running number of interviews. The semi-structured interviews with the local mussel farmers were qualitatively analyzed, using the grounded theory method. This theory produces theoretical models of individuals' perspectives of a given phenomenon and the strategies they use to resolve or cope with the problem in a distinct and bounded context (43, 44, 45). Interviews were recorded using a Dictaphone and later transcribed. Transcripts of the interviews were analyzed by coding the statements of the respondents using identified notions. These notions were gathered into topic categories (44, 45, 46). The validity is justified by triangulation. Three researchers with different disciplinary basic knowledge and different experiences in research were included in the analysis and interpretation process. Findings from semi-structured interviews Eight topics (Scheme 1) were identified following text analysis of the respondents' answers during semi-structured interviews: compliance with the legislation, transport, traceability, employer's awareness, food handlers' awareness and competencies, microbiological contamination, communal infrastructure, and disregarding an environmental code of conduct in the sea. These topics were obtained after identifying the topics in the statements. The interviewee's answers were very guarded; consequently, more comprehensive data processing cannot be achieved. With the intention to show comprehension within interviewee reflections, these identified topics (Scheme 1) were integrated with a specified section in Guidelines for the control of viruses in food ( 15) as follows: Primary production / Harvesting area; Establishment: Design and facilities; Control of operation; Establishment: Maintenance and sanitation; Establishment: Personal hygiene; Product information and consumer awareness; and Training. Specific topics (microbiological contamination, communal infrastructure, and disregarding an environmental code of conduct in the sea) represent an unrecognized threat to V-/ Primary production (microbiological ¡_ contamination of the waters; in which they grow) | Establishment: Design and ! facilities Control of operation | (cooking temperature versusr eating habits) V V Establishment: Maintenance!, and sanitation Personal hygiene ' Product information and . j consumer awarenenss (lot ! ; identification, labelling and [ | education) ; I Training (preventing faecal ! ! contamination, contagious > food handlers) Current recommendations CL O MNs; p<0.001) and anatomical conformation of the lumbosacral area, which was correlated with the incidence of DLSS. GSDs had significantly higher bodies of L7 (p<0.001) and S1 (p<0.01), higher L7/S1 step (p<0.01) and shorter CrS1/SL distance (p<0.05) than MNs. There was a significant association between DLSS and spondylosis deformans (p<0.05) and sclerosis of S1 cranial endplate (p<0.05). In MNs, no dog was radiographically confirmed for DLSS, although the age of dogs of both breeds was comparable. Fewer eventual radiological changes of the lumbosacral spine were also found in MNs. Regarding our findings, MNs seem to be more suitable for working dogs. The limitations of our study are the small number of MNs and the lack of MNs with DLSS. Our study confirmed radiographic differences of the lumbosacral junction between GSDs and MNs. Nevertheless, we could not confirm any radiographic parameter as a predisposing sign. Key words: lumbosacral junction; radiography; myelography; working dogs; German Shepherd dogs; Belgian Shepherd dogs (Malinois); degenerative lumbosacral stenosis Introduction Cauda equina syndrome (CES) refers to a complex of clinical signs resulting from compression of cauda equina nerve roots. Degenerative lumbosacral stenosis (DLSS) is the most common cause of cauda equine syndrome reported in working dogs. German Shepherd dogs (GSDs) are the most commonly affected by clinical signs related to DLSS (1, 2, 3, 4, 5, 6, 7, 8, 9). To Received: 14 March 2016 Accepted for publication: 29 July 2016 our knowledge there is no data published about incidence of DLSS in MNs. DLSS is the most common abnormality of the LS junction in dogs, particularly working GSDs (4, 6, 8, 9, 10). It is a multifactorial degenerative disorder resulting in stenosis of the spinal canal and compression of the cauda equina or its blood supply (4, 6, 8). Spinal cord diseases and CES in particular are common causes for early exclusion of working dogs (11, 12, 13). The most common and typically earliest finding in dogs with DLSS is pain during palpation and hyperextension of the LS junction (10). The clinical 220 E. Pogorevc, B. Lukanc, A. Seliskar, R. Pelc, B. Zorko signs of dogs that are affected by DLSS may show considerable variation related to the severity of compression of the cauda equina. The most common clinical signs are pelvic limbs lameness, abnormal gait, and caudal lumbar pain (3, 4, 5). Each dog can have one or more clinical signs. Diagnosis is based on history, clinical and neurological assessment and the correlation of clinical findings and ancillary diagnostic imaging findings. Radiography, stress radiography and contrast studies (myelography, epidurography) may aid in ruling out pathological osseous changes of the LS region as cause of CES (14, 15, 16). Myelography represents a useful technique for assessing stenotic lesions in the lumbar spinal canal as well as detection of DLSS when a dural sac extends into the sacrum. The diagnostic sensitivity of myelography may be enhanced by using flexion-extension myelography of the lumbosacral junction (17)»=B. Slovenian police GSDs and MNs are examined radiographically for hip and elbow dysplasia before accepted to working unit (18). To our knowledge, there are no published data elsewhere in the world regarding working dogs being regularly evaluated for spinal changes in lateral projection of the spine (ie. lumbal transitional vertebra (LTV), evaluation of LS conformation) before acceptance to the working unit. The aim of this study was to radiographically assess LS area in working police GSDs and MNs and compare the radiographic (myelography) and clinical findings of DLSS. This study also attempts to identify whether radiography of whole lumbar and sacral area in lateral projection before acceptance to the working unit is of clinical relevance. Materials and methods Animals Thirty-six working police dogs, 35 intact males and 1 intact female, were included in the study. Twenty-four of them were GSDs and 12 MNs. Mean age of the examined dogs was 68.3±33.5 months (76.6±31.9 months in GSDs and 51.8±31.3 months in MNs). All dogs were regularly used as patrol and attack dogs at a Slovenian police unit during the time of examination. The consent of the owner was obtained, and protocols were approved by the Veterinary Administration of the Republic of Slovenia (No. 34401-25/2010/3). Evaluation of the dogs consisted of the observation of a handler, clinical and neurological examination and native and contrast (mielography) radiography. Final diagnosis of DLSS was based on clinical signs of pain and mielographically confirmed compression of cauda equina. Compression was pronounced with extension and relieved with flexion. In this study, dogs were assigned into three groups based on a dog breed and confirmed lumbosacral stenosis, namely 1) GSDs without DLSS(GSD/ NOrDLSS; n=14), 2) GSDs with DLSS(GSD/rDLSS; n=10) and 3) MNs without DLSS (MN/NOrDLSS; n=12). Study protocol Handlers were asked about the general health status of their dog and any concurrent disorders diagnosed by their veterinarian. They were also asked about their dog's performance of expected duties and if they noticed any signs of pain, lameness or weakness (especially of the pelvic limb and tail). Each dog underwent a neurological examination that included assessments of attitude, posture and gait outside the clinic. Later on, the quality of conscious proprioception, spinal reflexes, the anal and tail tone were assessed. Deep palpation, lumbosacral hyperextension and hyperextension of the tail were performed to evaluate lumbosacral hyperesthesia. A physical examination, including auscultation of the heart and lungs, palpation of the peripheral pulse and blood sampling for evaluation of general health status before anaesthesia was performed. A pre-sedation complete blood count, white cell differential count, and serum biochemistry profile including blood urea nitrogen, creatinine, total protein, albumin, glucose, sodium, potassium, chloride, alkaline phosphatase and alanine aminotransferase (data not shown) were determined to exclude underlying diseases. Radiography Following the neurological examination all the dogs underwent survey and contrast radiography (myelography) under general anaesthesia. Dogs were premedicated with methadone (Heptanon; Pliva, Zagreb, Croatia) 0.28-0.3 mg/kg Radiological comparison of lumbosacral anatomy between German and Belgian Shepherd (Malinois) working dogs 221 subcutaneously. General anaesthesia was induced with midazolam (Dormicum; F. Hoffmann-La Roche, Basel, Switzerland) 0.07-0.2 mg/kg and thiopental (Nesdonal, Merial, Lyon, France) 7.817.4 mg/kg given intravenously. After intubation with a cuffed endotracheal tube, anaesthesia was maintained with isoflurane (Forane; Abbott Laboratories, Baar, Switzerland) in 100 % oxygen, using a circle circuit. Radiographic images were taken by AXIOM Iconos R100, Siemens AG, Munich, Germany and films developed by CLASSIC E.O.S., Agfa, Munich, Germany. First, a right lateral (RL) radiograph of the lumbosacral area with hind legs in a neutral position was obtained. After collecting cerebrospinal fluid (data not shown), a non-ionic contrast medium iohexol (Omnipaque 240 mgI/ ml, Nycomed Inc, Princeton, NJ) 0.3-0.5ml/kg was injected into the subarachnoid space at the cisterna magna. Dogs were tilted at 15-20° with their heads up and the fluoroscopy/radiograph was taken immediately. When contrast reached the lumbosacral region, three more radiographs of the lumbosacral junction were obtained: 1) a RL with hind legs in neutral position, 2) a RL with hind legs in flexion, 3) a RL with hind legs in extension. In total, the radiographic examination included four lateral radiographs in three different positions. All radiographs were assessed by the same radiologist (BZ) for evidence of spondylosis, L7 and S1 endplate sclerosis, LTV and sacral osteochondrosis (SOC). The number of lumbar vertebra was counted in each dog in order to identify LTV (8 lumbar vertebra). Next, the ventral displacement of the sacrum in respect to L7, also named misalignment of L7 or L7/S1 step formation, was evaluated. It was defined as a distance between two lines: a first line was drawn along the dorsal aspect of the body of L7, and a second line, parallel to the first, was drawn at the height of the craniodorsal edge of the sacrum. The height of the caudal endplate of L7 and the cranial endplate of S1 were measured axially. At the same place, the height of the spinal canal at the level of L7 and S1 was measured, defined as L7/S1 spinal canal ratio. The extension of the dural sac over the lumbosacral junction was evaluated on contrast radiographs with hind legs in neutral position. All measured parameters are shown in Figure 1. Cranially prolonged sacral lamina with or without sacral overhang could contribute to the compression of cauda equina, so the measurement of CrS1/SL distance was also made between the cranial endplate of S1 and cranial extent of the sacral lamina(19), as shown in Figure 2. Statistical analysis Data were analysed with the commercial software SPSS 22.0. (Chicago, Illinois, USA). Descriptive statistics was used to describe the basic features of the data. The association of the all three dog groups with anatomical conformation of the LS region was examined using a x2-square test. The difference between MN/NOrDLSS, GSD/NOrDLSS and GSD/rDLSS in body weight, vertebral body height of L7, vertebral body height of S1 and CrS1/SLdistance was examined using a one-way ANOVA. Spinal canal height at L7, spinal canal height at S1 and L7/S1 misalignment were examined using a non-parametric Mann-Whitney U test. A Shapiro-Wilks test was used to test the normality. Statistical differences were considered significant with p< 0.05. Results History Handlers of 12 of 36 (33%) dogs (11 GSDs and 1 MN) reported problems potentially associated with a lumbosacral disorder. These included mild lameness or occasional weakness of the pelvic limbs (10 dogs) difficulty jumping (6 dogs), and signs of lower back pain (4 dogs). Handlers of 33 of 36 (92%) dogs considered their dogs to be able to perform their duties without restrictions and 3 (all GSDs) dogs were reported to be on restricted duties (dogs were used as patrol dogs, but excluded from heavy attack training). Reason for restricted duty was DLSS in 2 dogs and thoracolumbar disc disease in 1 dog, all diagnosed previously. Gait assessment Of 36 dogs, posture and gait were normal in 20 (55%). Changes were seen intermittently in 8 (22%) dogs, persistent but mild changes (intermittent foot misplacement and toe knuckling, mild ataxia) were seen in 6 (17%) dogs, persistent and obvious (ataxia, uneven distribution of weight on the limbs, improper limb positioning, toe knuckling, occasional circling) changes in 2 (6%). 222 E. Pogorevc, B. Lukanc, A. Seliskar, R. Pelc, B. Zorko Figure 1: Schematic presentation of parameters of the lumbosacral junction measured on RL radiographs. L7- seventh lumbar vertebra; Si-first segment of sacrum; a- L7/S1 misalignment; b- L7 height; c- S1 height; d- spinal canal height L7; espinal canal height S1 Figure 2: Schematic presentation of CrSi/SL distance on a RL radiograph. CrS1/SL distance (a) was defined as distance between the cranial endplate of sacrum (CrS1) and cranial point of sacral lamina (SL); L7- seventh lumbar vertebra; S1- first segment of sacrum Clinical signs Sixteen GSDs showed clinical signs of DLSS. On clinical exam, the most consistent finding was lower back pain (15 of 16), elicited by hyperextension of the lumbosacral junction (11 of 15; 2 GSD/NOrDLSS and 9 GSD/rDLSS), by digital palpation of the paraspinal muscles at the level of the lumbosacral joint (2 of 15) or by hyperextension of the tail (2 of 15). Digital palpation of paraspinal muscles was painful in 2 GSD/NOrDLSS and hyperextension of the tail was painful in two other GSD/NOrDLSS. Neurological examination The neurological exam was abnormal in 17 of 36 (47%) dogs, including 11 dogs with a history of pain or pelvic gait abnormalities. Abnormal findings included proprioception deficits (5 of 17; 2 GSD/NOrDLSS and 3 GSD/rDLSS), reduced withdrawal reflex (1 of 17; GSD/rDLSS) and gait abnormalities seen in 5 GSD/NOrDLSS and 5 GSD/rDLSS. Each dog could have one or more clinical signs. None of the MN/NOrDLSS showed any clinical abnormalities. Radiological comparison of lumbosacral anatomy between German and Belgian Shepherd (Malinois) working dogs 223 Table 1: Body weight and vertebral parameters at the level of L7-S1 in GSD/NOrDLSS, GSD/rDLSS and MN/ NOrDLSS dogs. Data are presented as mean ± S.E.M. GSD/NOrDLSS (n= 14) GSD/rDLSS (n= 10) MN/NOrDLSS (n= 12) BW [kg] 37.5 ± 1.2 36.0 ± 1.2 31.2 ± 0.8a L7/S1 misalignment [mm] (rate) 0.5 ± 0.2 0.9 ± 0.3 0.1 ± 0.1c incidence of L7/S1 misalignment [%] 43 70 8 CrS1/SL distance [mm] 8.1 ± 0.8 8.6 ± 1.1 10.5 ±0.4c vertebral body height [mm] L7 21.1 ± 0.4 21.3 ± 0.6 18.5 ± 0.3a S1 19.0 ± 0.5 19.6 ± 0.7 16.6 ± 0.3b spinal canal height [mm] L7 9.6 ± 0.2 10.2 ± 0.4 10.1 ± 0.3 S1 7.1 ± 0.2 7.1 ± 0.4 7.3 ± 0.2 ap< 0.001; different than GSD/NOrDLSS and GSD/rDLSS bp< 0.01; different than GSD/NOrDLSS and GSD/rDLSS cp< 0.05; different than GSD/NOrDLSS and GSD/rDLSS Survey and contrast radiography The group of GSD/NOrDLSS consisted of 14 dogs without myelographically detected DLSS. Two dogs showed clinical signs of CES (both had painful hyperextension of the tail and one had mild gait abnormalities), but were radiographically normal. In this group, there was also one dog with LTV. Radiographic changes were seen in 5 dogs, none of them showing clinical signs. Spondylosis of lumbar area was found in 4 dogs. Mild sclerosis of the adjacent endplates was found in 4 dogs. The dural sac extended over lumbosacral junction in all GSD/NOrDLSS. The group of GSD/rDLSS consisted of 10 dogs with radiographic/myelographic signs of lumbosacral stenosis. All of them showed clinical signs of CES; painful lumbar extension was observed in 8 dogs and painful extension of the tail in 5 dogs. Spondylosis of the lumbar area was found in 7 dogs. Sclerosis of the adjacent endplates was found in 8 dogs. The dural sac extended over the lumbosacral junction in 9 dogs. None of GSD/ rDLSS dogs had a LTV. Cauda equina compression was greater at lumbosacral spine in extension in comparison to lumbosacral spine in flexion. The group of MN/NOrDLSS consisted of 12 dogs without myelographically detected lumbosacral stenosis. None of them showed any clinical signs of CES. On the radiographs, none of them had spondylosis, and one of them had a LTV. There was mild sclerosis of S1 endplate, but not seen on the caudal endplate of L7. The dural sac extended over the lumbosacral junction in 10 dogs. Statistical analysis showed significant association between radiographically confirmed DLSS and the presence of 1) spondylosis (p< 0.05) and 2) sclerosis of S1 endplate (p< 0.05) in GSDs only. The presence of a dural sac extending over the lumbosacral junction, osteophytes of the articular processes, sclerosis of the L7 endplate or LTV were not associated with DLSS in GSDs. No dog in our study had radiographic evidence for SOC. The incidence of L7/S1 misalignment was the highest in GSD/rDLSS dogs (70%), which also exhibited the largest maximal S1 displacement (GSD/rDLSS = 3 mm, GSD/NOrDLSS = 2 mm, MN/NOrDLSS = 1 mm). In line with this, GSDs had significantly higher L7/S1 misalignment (p< 0.05) and shorter CrS1/SL distance (p< 0.05) than MNs (Table 1). GSDs had significantly higher vertebral bodies (L7; p< 0.001, S1; p< 0.01) but not corresponding spinal canals at L7 and S1 than MNs (Table 1). GSDs were also heavier (p< 0.001) than MNs (Table 1). Discussion German Shepherd dogs and Belgian Shepherd dogs (Malinois) are the most common breeds used as working police dogs worldwide. A common cause for the exclusion of police dogs from working 224 E. Pogorevc, B. Lukanc, A. Seliskar, R. Pelc, B. Zorko units is CES (11, 12, 13). In our study, some dogs showed difficulty and reluctance to get up and to jump. The dogs seemed to have more problems with extension of the caudal lumbar spine than with flexion. This is probably due to the increase of cauda equina compression that occurs when the caudal lumbar spine is extended. Dogs that are affected with DLSS may not simultaneously show all clinical signs, but caudal lumbar pain is usually predominant (3, 4, 5). Most often, pain arises as a result of compression of the nerve roots of the cauda equina, although other potential sources of pain include the lumbosacral disc and the articular facets (10). Pain is usually evoked during palpation and hyperextension of the lumbosacral junction, which is highly sensitive, with responses to painful stimuli in 91% to 100% dogs with DLSS (6). In our study 13 GSDs showed pain and 9 (69%) of them were later mielographically confirmed for DLSS. During clinical examination, exerting pressure over the lumbosacral region, and hyperextension of the caudal lumbar spine and hip joint extension also evoked signs suggesting pain and discomfort in dogs with DLSS. The dogs were radiographed for absence of hip displasia before they were recruited as working dogs, so there is strong evidence that hip dysplasia was not the cause of pain. DLSS commonly affects medium sized to large breed dogs, at a mean age of six to seven years (20) and occurs more often in male dogs (3, 4, 5). All dogs in our study were large breed dogs. The mean age of affected GSDs was 95.2 months (61 to 117months). Male-to-female predisposition from 1.7:1 up to 5:1(3, 5, 7), with a higher mean body weight for male dogs than for female dogs with DLSS, suggests that biomechanical loading plays a role in the pathogenesis (7). In our study, male dogs were overrespresented, so we cannot define male-to-female comparison. Nevertheless, our GSDs had significantly (p<0.001) higher body weight than MNs, which would support the suggestion about biomechanical loading as another predisposing factor for greater incidence of DLSS in GSDs than in MNs. Mainly large breed dogs with a high level of physical activity are predominately affected. The influence of the increased load is supported by the fact that DLSS is extremely rare in small dogs and cats and also in large dogs with less physical activity. The incidence of the radiologically confirmed DLSS in GSDs in our study was 42 % which is higher than in previous reports (5, 7, 21). This supports the suggestion of genetic predisposition of GSDs to DLSS (5, 7). The overall incidence of DLSS in our study was 28% which is probably due to presence of only normal MNs. Diagnostic investigation of CES begins with survey radiographs of the lumbosacral joint, to rule out bone-associated neoplasia, discospondylitis, trauma, and vertebral abnormalities (14). The next indication to take survey radiographs is to identify conditions which may predispose a dog for development of DLSS such as LTV segments and osteochondritis dissecans of the endplate of S1 or L7 (17, 22). Survey radiography (+/- mielography) as a widely available modality still has potential as a screening technique (6). In our study, survey radiography revealed L7/S1 misalignment in 14 of 36 dogs (13 GSDs and 1 MN), 7 GSDs also had clinical signs. This is considered to be a sign of lumbosacral instability, although the size of the lumbosacral step formation does not always correlate with the clinical signs (3, 6, 7). In our study, lumbosacral step formation was no higher than 3 mm. It was previously reported that lumbosacral step formation is of clinical relevance only when higher than 4 mm (21). In contrast, Suwangkong et al. (2006) suggested that lumbosacral misalignment as low as 2 mm may be clinically relevant. The height of the body of L7 (p<0.001) and the height of the body of S1 (p<0.01) were significantly greater in GSDs in comparison to MNs. In contrast, there was no significant difference between breeds when comparing the height of the spinal canal at the level of caudal L7 and cranial S1 endplate. These findings support the hypothesis of primary stenosis of the spinal canal in GSDs (1). The L7/S1 spinal canal ratio was less than 2 in all dogs in our study, the highest being 1.7. There were no significant differences between groups nor between breeds. Primary canal stenosis is assumed to be a hereditary disease in large breed dogs and may be a cause of CES (14, 20). The only anatomical conformation that is correlated with DLSS is a LTV (22, 23). In our study, we diagnosed 2 of 36 dogs (5%) with LTV (1 GSD/NOrDLSS and 1 MN/NOrDLSS). A LTV is an abnormally formed vertebra between the last normal lumbar vertebra and the first normal sacral vertebra (22). While both symmetrical and asymmetrical LTV may be associated with DLSS, asymmetrical LTV results in a specific pattern of Radiological comparison of lumbosacral anatomy between German and Belgian Shepherd (Malinois) working dogs 225 DLSS consisting of unilateral protrusion of a disk and degeneration of the adjacent bone marrow (24). Lacking ventrodorsal or dorsoventral projection, we could not define whether it was symmetrical or asymmetrical LTV. None of the dogs in our study had SOC, which is in accordance with study of Scharf (25), although SOC was reported in over 30% of predominately male GSDs with CES and 6.4% in clinically normal GSDs (26). Myelography is a useful diagnostic procedure in the assesment of the stenotic lesions of the spinal canal, but it depends on the extension of the dural sac over the lumbosacral junction. In large dogs the spinal cord ends at L6 and the dural sac extends further caudally (8). Myelography is not diagnostic when the dural sac ends cranial to the lumbosacral junction, when the sac is elevated from the vertebral floor or the compressive lesion is located in the lateral recess or the intervertebral foramen (6). DLSS can be detected when the dural sac extends into the sacrum, which is observed in 80% of dogs with DLSS (17). In our study dural sac extended over LS junction in 33 of 36 (91%) dogs. The diagnostic sensitivity of myelography may be enhanced by using flexion-extension position of the lumbosacral junction (17). The CrS 1 / SL distance could play a role in development of DLSS. The greater the distance, there is less possibility of compression during extension. In our study GSDs had significantly (p<0.05) smaller distance and additionally higher lumbosacral step. This could lead to excessive stenosis during the extension of the spine and cause pain even in younger dogs or dogs without radiographically visible degenerative changes. Advanced imaging procedures, as CT and MRI, are used for more exact evaluation of soft tissues of lumbosacral and foraminal area as well as for the evaluation of the facet joints geometry (6, 16, 27, 28). Due to their accuracy, they are very imporant in diagnostics as in surgical treatment planning, but so far they remain expensive and frequently not available everywhere. MRI provides excellent soft tissue contrast, so it appears to be superior in detection of spinal stenosis caused by soft tissue proliferation or recognition of early disc degeneration (16, 29, 30, 31). However, there is also lack of correlation between the severity of clinical signs and the severity of compression as determined even by MRI (30). Regarding to our findings, MNs seem to be more suitable for working dogs. There were only 12 MNs included in this study, but they were approximately of the same age as GSDs (range from 15 to 119 months). Nevertheless, we did not confirm any clinical or radiographical signs of DLSS in this breed. Slovenian police GSDs and MNs are examined radiographically for hyp displasia and elbow dysplasia before accepted into working unit. In addition, radiographic examination of whole lumbosacral spine would be recommended in order to determine LTV, although there was no correlation found in our study. Acknowledgement Author would like to thank Rok Korljan for his assistance and technical support during this study. This research was financially supported by the Slovenian Research Agency, program group P4-0053. References 1. Ondreka N, Amort KH, Stock KF, et al. Skeletal morphology and morphometry of the lumbosacral junction in German shepherd dogs and an evaluation of the possible genetic basis for radiographic findings. Vet J 2013; 196(1): 64-70. 2. Indrieri RJ. Lumbosacral stenosis and injury of the cauda equina. Vet Clin North Am Small Anim Pract 1988; 18(3): 697-710. 3. Watt PR. 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Sisson AF, LeCouteur RA, Ingram JT, et al. Diagnosis of cauda equina abnormalities by using electromyography, discography, and epid-urography in dogs. J Vet Intern Med 1992; 6(5): 253-63. 28. Seiler GS, Hani H, Busato AR, et al. Facet joint geometry and intervertebral disk degeneration in the L5-S1 region of the vertebral column in German Shepherd dogs. Am J Vet Res 2002; 63(1): 86-90. 29. Adams W H, Daniel G B, Pardo A D, et al. Magnetic resonance imaging of the caudal lumbar and lumbosacral spine in 13 dogs (1990-1993). Vet Radiol Ultrasound 1995; 36(1): 3-13. 30. Mayhew PD, Kapatkin AS, Wortman JA, et al. Association of cauda equina compression on magnetic resonance images and clinical signs in dogs with degenerative lumbosacral stenosis. J Am Anim Hosp Assoc 2002; 38(6): 555-62. 31. Suwankong N, Voorhout G, Hazewinkel HA, et al. Agreement between computed tomography, magnetic resonance imaging, and surgical findings in dogs with degenerative lumbosacral stenosis. J Am Vet Med Assoc 2006; 229(12): 1924-9. Radiological comparison of lumbosacral anatomy between German and Belgian Shepherd (Malinois) working dogs 227 RADIOLOŠKA PRIMERJAVA ANATOMSKIH ZNAČILNOSTI LUMBOSAKRALNEGA OBMOČJA MED NEMŠKIMI IN BELGIJSKIMI (MALINOIS) OVČARJI - DELOVNIMI PSI E. Pogorevc, B. Lukanc, A. Seliškar, R. Pelc, B. Zorko Povzetek: Namen raziskave je bili oceniti medpasemske rentgenske razlike in pojavnost kliničnih in rentgenskih znakov degenerativne lumbosakralne stenoze (DLSS) pri 36 aktivnih policijskih delovnih psih; 24 pasme nemški (GSD) in 12 pasme belgijski ovčar (Malinois; MN). Po razgovoru z vodniki psov smo opravili natančen klinični in nevrološki pregled ter rentgensko slikanje lumbalnega in sakralnega dela hrbtenice brez in s kontrastnim sredstvom (mielografija). Svoje delo je brez omejitev opravljalo 33 psov, trije pa omejeno in sicer dva zaradi DLSS in eden zaradi bolezni diska v prsno-ledvenem delu hrbtenice. Šestnajst GSD je imelo znake DLSS, najpogostejši klinični znak je bila bolečina v zadnjem delu hrbta (15 od 16; 94%). Rentgensko smo potrdili DLSS pri 10 GSD (GSD/rDLSS). Potrdili smo razlike v anatomski konformaciji lumbosakralnega dela med pasmama, ki so korelirale s pojavnostjo DLSS pri GSD. Nemški ovčarji so imeli statistično značilno višja telesa sedmega ledvenega vretenca (L7; p<0.001) in prvega segmenta križnice (S1; p<0.01) kot pa MN. Pri GSD je bila stopnica L7/S1 (p<0,01) višja in razdalja CrS1/SL nižja (p<0,05) kot pri MNs. Nemški ovčarji so imeli tudi večjo telesno maso kot MN (p<0.001). Ugotovili smo tudi statistično značilno povezavo med DLSS in spondilozo deformans (p<0.05) ter brstenjem kranialne površine S1 (p<0.05). Pri MN bolezni nismo potrdili, čeprav je bila starost psov obeh pasem med seboj primerljiva. Tudi sicer smo pri MN rentgensko našli manj sprememb na hrbtenici kot pri nemških ovčarjih. Glede na naše ugotovitve, so MN bolj primerni za delovne pse. Omejitev naše študije je majhno število MN, in to, da v študijo ni bil zajet noben MN z DLSS. Med pasmama smo potrdili rentgenske razlike v konformaciji lumbosakralnega predela. Rentgenskega parametra, ki bi lahko bil predispozicijski faktor za razvoj bolezni, nismo odkrili. Za oceno pojavnosti bolezni pri MN bodo potrebne nadaljnje študije. Ključne besede: lumbosakralni predel; rentgenologija; mielografija; delovni psi; nemški ovčar; belgijski ovčar (Malinois); degenerativna lumbosakralna stenoza Slov Vet Res 2016; 53 (4): 229-35 UDC 636.7.09:616.993.1-078:57.088.7 Case Report Pentatrichomonas hominis COINFECTION IN A PUPPY FROM A SLOVENIAN ANIMAL SHELTER Maja Brloznik1, Sinisa Faraguna2, Brigita Slavec3, Rok Kostanjsek5, Aleksandra Vergles Rataj4, Igor Gruntar4 1PRVA-K, Small Animal Clinic, Gorkiceva 6, Ljubljana, 2Veterinary faculty, 3Institue for Health Care of Poultry, institute of Microbiology and Parasitology, Veterinary Faculty, Gerbiceva 60, Ljubljana, 5Department of Biology, Biotechnical Faculty, Jamnikarjeva 101, Ljubljana, Slovenia Corresponding author, E-mail: igor.gruntar@vf.uni-lj.si Summary: A 3-month-old dog originating from a Slovenian animal shelter presented with acute bloody, soft, foamy and malodorous diarrhoea. The clinical examination, haematology and serum biochemistry were unremarkable. Ultrasonography of the abdomen showed prominent mesenteric lymph nodes and the presence of echogenic content within the small intestine. Light microscopy of a native smear and a wet mount darkfield microscopy examination of the faecal material showed motile trichomonad-like organisms with a particular circular motion. The flotation and SAF (Sodium acetate - acetic acid - formalin solution) method using light microscopy revealed eggs of nematode Toxocara canis and protozoan oocysts of Isospora spp. Trichomonad-like organisms were successfully isolated and cultivated in axenic culture. Light microscopy of Giemsa-stained trichomonads showed the presence of five flagella, and Pentatrichomonas hominis (P. hominis) was presumptively diagnosed. The diagnosis was confirmed by the Polymerase Chain Reaction (PCR) followed by DNA sequencing and the Scanning Electron Microscopy (SEM) of cultured trichomonad isolates. The PCR and sequencing results confirmed a 99% homology of the P. hominis isolates with isolates from other studies, originating both from humans and animals, which suggests thatP hominis could have zoonotic potential and have been transmitted from animals to people via the per-oral route. This is also the first report on P. hominis involvement in clinical diseases in dogs in Slovenia. Key words: Pentatrichomonas hominis; dog; isolation; cultivation; PCR; DNA sequencing; scanning electron microscopy Introduction Young dogs are commonly infested with different zoonotic intestinal parasites. Conventional canine intestinal parasites of zoonotic importance, such as Toxocara, Taenia, Ancylostoma, Giardia, Cryptosporidium, etc., have been studied thoroughly, but less is known about the zoonotic trichomonad species Pentatrichomonas hominis (P. hominis), formerly Trichomonas intestinalis Received: 19 February 2015 Accepted for publication: 435 April 2016 or Trichomonas hominis (1). This flagellated protozoan has recently been identified in faeces of dogs with diarrhoea (2, 3, 4, 5). The pathogenicity of the parasite remains unclear. Due to the lack of evidence of cases where P. hominis was the only infecting agent, this trichomonad is presumed to be a commensal organism that may overgrow in patients with other causes of diarrhoea. As enteropathogens have always been found in dogs infected by P. hominis (6), the pathogenic potential of this trichomonad species has to be further evaluated by experimental infection studies (4). For such trials an axenic P. hominis culture of 230 M. Brloznik, S. Faraguna, B. Slavec, R. Kostanjsek, A. Vergles Rataj, I. Gruntar dog origin is needed, which, to the best of our knowledge, has not yet been available. In humans, P. hominis has been reported as the causative agent of gastrointestinal disturbances in children (7, 8, 9). Therefore the assessment of the zoonotic potential of canine P. hominis is important. It is also necessary to establish whether host-specific genotypes exist as demonstrated among Tritrichomonas foetus isolates from cats and cattle (10, 11). To prove this, the characterisation of as many isolates as possible from diverse hosts should be performed. A study carried out last year (12), however, demonstrates that even when using the high-resolution gene locus of the ITS (internal transcribed spacer) regions, all P. hominis strains from diverse hosts are genetically identical. This suggests that zoonotic transmission between humans and animals may occur in the area investigated. Consequently, further research is required to clarify the role of P. hominis in human and animal diseases. This article provides the first description of P. hominis involved in a clinical disease in dogs in Slovenia. The trichomonad was successfully isolated and cultivated in axenic culture. The presumptive diagnosis based on light microscopy was confirmed by SEM and PCR. Material and methods Case Description and Sampling A 3-month old, 9 kg mixed breed and regularly vaccinated dog that had recently been adopted from a Slovenian shelter, presented at a small animal clinic with acute bloody, soft, foamy and malodorous diarrhoea. Clinical examination, haematology, blood serum biochemistry and abdominal ultrasonography were performed. According to the results viral etiology was ruled out. Consequently, coprological and bacteriological analyses of a faecal sample were carried out. The faecal sample was analysed using light microscopy, bacteriological examination, flotation, sedimentation and the SAF (Sodium acetate-acetic acid-formalin solution) method. Bacteriological analyses of faecal samples were conducted on blood agar and Drigalski agar plates, incubated aerobically and anaerobically at 37 oC overnight. The isolation of trichomonads followed. SEM and PCR were used for definitive diagnosis. Isolation of Trichomonads Trichomonads were isolated using the following procedure: a set of 3 tubes containing Modified Diamond's growth medium (MDM) was inoculated with a loopful (approx. 0.1 g) of faecal sample and incubated at 37°C for seven days (13). In parallel, another set of tubes was inoculated in the same way, this time with MDM supplemented with meropenem (6 ^g/ml; MeMDM), to provide additional prevention against bacterial contamination (14). The inoculated tubes were checked for trichomonad growth at intervals during this period. An aliquot was taken from the bottom of the tube and wet mount-examined by darkfield microscopy. When motile flagellates were observed, an aliquot (0.1 ml) of fresh culture was transferred to fresh MDMs/MeMDMs. Staining of Isolated Trichomonads Thin smears of cultivated trichomonad suspension were air-dried, fixed and stained with Giemsa, trichrome and methylene blue stain. Scanning Electron Microscopy For scanning electron microscopy, the trichomonads in the cultivation suspension were washed with phosphate buffered saline (PBS) by centrifugation at 100 xg for 5 min before overnight fixation in a combination of 1 % glutaraldehyde and 0.5 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4 °C. Fixed cells were transferred to pre-cleaned cover slides, washed by PBS and postfixed in 2 % OsO4 for one hour at 4 °C. After being washed in deionized water, the cells on the slides were dehydrated in a graded series of ethanols, then dried by hexamethyldisilazane (HMDS), mounted on aluminum stubs and coated with platinum, as described above (15). The samples were examined with the JEOL JSM-7500F field emission scanning electron microscope. Molecular Diagnosis For the molecular detection of trichomonads, two hundred microlitres of protozoal culture suspension were used. Total DNA was extracted using the QIAamp® Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions for Pentatrichomonas hominis coinfection in a puppy from a Slovenian animal shelter 231 blood and body fluid spin protocol. Finally, the DNA was eluted with 100 pl of AE buffer and stored at -20 °C until examination. Specific pairs of primers TFR1 and TFR2 amplifying the 350 bp long ITS1-5.8S-ITS2 region were used (16). PCR was performed in a total volume of 20 pl containing 10 jl of 2X Thermo Scientific DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, CA, USA), 0.8 jl of each primer (0.4 pM), 6.4 jl of nuclease-free water and 2 jl of extracted DNA. The reaction was performed on an ABI 2720 Thermo Cycler (Applied Biosystems, Foster City, CA, USA). The cycling profile included initial denaturation at 95 °C for 5 min, which was followed by 40 cycles of heat denaturation at 94 °C for 30 sec, oligonucleotide annealing at 55 oC for 1 min, oligonucleotide extension at 72 °C for 1 min, and final oligonucleotide extension step at 72 °C for 10 min. The extracted DNA of Trichomonas gallinae was used as positive control in the PCR assay. The PCR products were analysed by electrophoresis on a 1.8 % ethidium bromide-stained agarose gel. DNA fragments were excised from the gel and, after being purified with the Wizard PCR Preps DNA Purification System (Promega, Madison, WI, USA), sent for sequencing to the Macrogen laboratory (Macrogen Inc, Amsterdam, the Netherlands). The nucleotide sequences were downloaded using Chromas software (Technelysium Pty Ltd., Queensland, Australia), and the nucleotide sequence data were analysed by BLAST (17) to find similar sequences in the Genbank NCBI sequence database. Results The clinical examination of the diarrhoeic 3-month old mixed breed dog was unremarkable. Haematology and serum biochemistry did not reveal any abnormalities. Ultrasonography of the abdomen showed prominent mesenteric lymph nodes and the presence of echogenic content within the small intestine. Light microscopy of the native faecal smear and darkfield microscopy (wet mount examination) of the diarrhoeic material showed numerous motile trichomonad-like organisms with a particular circular motion. Flotation and the SAF method using light microscopy revealed Toxocara canis nematode eggs (Figure 1), Isospora sp. oocysts (Figure 2) and the above-mentioned trichomonad species. Bacteriological analyses of faecal samples were negative for aerobic and anaerobic pathogens. Trichomonad isolation and cultivation attempts were successful. Numerous motile trichomonads were observed after seven days incubation in MDM (xenic culture; with other organisms -bacteria - present). In MeMDM, the trichomonads were less abundant but other organisms were absent (axenic culture). The cultures were stored using 10 % dimethylsulphoxide (18). The isolated Figure 1: Toxocara canis egg, size 90 x 80 |m, flotation Figure 2: Isospora sp. oocyst, size 23 x 18 |m, flotation method, x400 method, x400 232 M. Brloznik, S. Faraguna, B. Slavec, R. Kostanjsek, A. Vergles Rataj, I. Gruntar » „ > V ~ $ Figure 3: Pentatrichomonas hominis trophozoite, Giemsa, x1000 trichomonads were stained with Giemsa, trichrome and methylene blue stain. When examined by light microscopy, the Giemsa stain improved the visibility and enabled the enumeration of flagella (Figure 3). The SEM and PCR assay confirmed the diagnosis of P. hominis. SEM observations revealed the presence of five flagella in the anterior part of the trichomonad (four flagella in a group and a single independent flagellum) and one in the posterior part (Figure 4). The latter run from the anterior part, alongside the cell in a posterior direction, forming a distinct undulating membrane displaying three undulations. It ended freely at its distal end. The axostyle was observed as a discrete tip in the posterior region of the cell. With PCR and sequencing, a high similarity of obtained sequence (Accession No. KU670675) with P. hominis was confirmed. The comparison of the 300 bp long sequence of the complete ITS1-5.8S-ITS2 gene had 100 % homology with P. hominis isolate from empyema thoracis (Accession No. AF156964), and 99 % homologies with the P. hominis isolates from a human (Acc. No. JN007007) and a dog (Acc. No. KJ404270). The dog was treated with metronidazole 200 mg/12 h p/o for 5 days and Dehinel plus® (febantel 150 mg, pyrantel embonate 144 mg, praziquantel 50 mg) 1 tbl/day for 5 days. The dog's condition improved immediately, and the faecal exam was negative two weeks after the treatment. Since then, the dog has been asymptomatic for one year. Discussion P. hominis, a flagellated protozoan of the order Trichomonadida, inhabits the large intestine of many mammalian hosts, including humans (6, 7, 8, 9, 19). Its prevalence in humans is low in developed countries (20), but is much higher in subtropical and tropical zones (21). In dogs, the prevalence of the trichomonad infection in a study analysing 215 puppies from French kennels was 15.8% (4). This study also reports that P. hominis was the only trichomonad infecting the studied canine population, whereas some older papers suggest that P. hominis was far more frequent than Tritrichomonas foetus in diarrhoeic dogs suffering from trichomonosis (2, 3, 6). In the majority of studies (2, 3, 4), the reported age of dogs with trichomonosis ranged from 7 weeks to 6 months which is consistent with the age of the 3-month-old dog in this case. A more recent study reported that trichomonosis was also diagnosed in 10-year-old dogs (6). Differential diagnoses for bloody, soft, foamy and malodorous diarrhoea are dietary intolerance, infection, partial obstruction, motility disorders, inflammatory/immune-mediated disease, drugs/ toxins, idiopathic disease, neoplasia and extra-gastrointestinal disease (22). Given the age of the dog in this study, the clinical signs and results of the clinical investigation, infection was considered the most likely cause. Considering that the dog has been routinely vaccinated against viral diseases, the absence of leukopenia in haematology results, as well as lack of other usual clinical signs, parvoviral enteritis was not suspected and additional diagnostics regarding shedding the parvoviruses was not performed. The excretion of other viruses is generally not evaluated in practice, since viral diarrhoea is usually self-limiting and does not require a positive diagnosis. The negative results of the bacteriological analyses led to the conclusion that the mixed trichomonad-parasitic-coccidial infection was responsible for the symptoms. However, it is difficult to presume about the origin of the diarrhea from amongst the three organisms. It is most likely that the concurrency of the pathogens was significant. Microscopical observations of unusual and copious trichomonad-like organisms in the native sample was a surprising finding, which triggered additional efforts to characterise these organisms. Microscopy cannot be used to identify the Pentatrichomonas hominis coinfection in a puppy from a Slovenian animal shelter 233 Figure 4: Pentatrichnmonas hominis trophozoite, A - anterior flagella, B - posterior flagellum, C - undulating membrane, D - axostyle, E - independent flagellum, SEM x 550 trichomonad species in native samples (23, 24, 25). Flagella are not well discernible in such preparations, but their visibility improves with the use of stains, thus supporting the species differentiation. Trichomonad species can accurately be determined by SEM: for the purposes of this study the isolate was characterised as a pear-shaped organism with distinctive undulating membrane, an axostyle, a single posterior (recurrent) flagellum and five anterior flagella, comprising a group of four flagella of unequal length and a single independent flagellum. The undulating membrane displayed three undulations. An axostyle was an elongated rod-like structure projecting out of the posterior end to form a pointed spine. The axostyle is an important criterion for distinguishing tritrichomonads from pentatrichomonads (5, 26). In this study, the axostyle of the trichomonad had a relatively heavy terminal segment and was observed as a discrete tip in the posterior region of the cell. By contrast, the axostyle of the T. foetus has a short conical projection with a small spherical structure at the end (27). Furthermore, it has only 3 anterior flagella. The long undulating membrane is also suggestive of P. hominis because the other trichomonads have a shorter undulating membrane (27). For the detection of P. hominis in biological specimens, a highly specific and sensitive PCR assay can be used (28, 29, 30). In this case, the PCR assay for amplification of the 5.8S rRNA gene and two flanking internal transcribed spacer regions ITS1 and ITS2 of trichomonads was used. This region is one of the most used for taxonomic classification of trichomonads (16). The results confirmed high homology of this isolate with the previously described genetic loci of P. hominis from various mammalian hosts (humans, dogs, cattle, pigs, cats, goats, water buffaloes), suggesting a low genetic diversity in P. hominis isolates and a very broad host range for this species (5, 12, 28, 29, 30). Therefore, it is possible that P. hominis strains may circulate between different hosts, including humans, where they, under certain circumstances, may cause clinical disease (7, 8, 9) or exacerbate symptoms of an existing illness (31). To elucidate the actual role and pathogenicity of P. hominis in human and animal disease, further research is needed. Conclusion Routine coprological examinations, at least microscopic evaluation of the stool for protozoans, parasites and the ova (eggs, cysts) of parasites, are essential for a correct diagnosis in a dog with diarrhoea. In the case of trichomonads, SEM or PCR assays are required for the definite diagnosis of trichomonad species. Molecular diagnostic data of trichomonads suggest that P. hominis is a zoonotic species with the potential for transmission via the per-oral route from animals to people and vice-versa. 234 M. Brloznik, S. Faraguna, B. Slavec, R. Kostanjsek, A. Vergles Rataj, I. Gruntar References 1. Pentatrichomonas. 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Structure of trichomonads as revealed by scanning electron microscopy. J Protozool 1979; 26: 56-62. 28. Crucitti T, Abdellati S, Ross DA, Changal-ucha J, van Dyck E, Buve A. Detection of Pentatri- chomonas hominis DNA in biological specimens by PCR. Lett Appl Microbiol 2004; 38: 510-6. 29. Gookin JL, Birkenheuer AJ, St John V, Spector M, Levy MG. Molecular characterization of trichomonads from feces of dogs with diarrhea. J Parasitol 2005; 91: 939-43. 30. Mostegl MM, Wetscher A, Richter B, Ne-dorost N, Dinhopl N, Weissenböck H. Detection of Tritrichomonas foetus and Pentatrichomonas homi-nis in intestinal tissue specimens of cats by chro-mogenic in situ hybridization. Vet Parasitol 2012; 183: 209-14. 31. Jongwutiwes S, Silachamroon U, Putaporn-tip C. Pentatrichomonas hominis in empyema tho-racis. Trans R Soc Trop Med Hyg 2000; 94: 185-6. SOOKUŽBA S PROTOZOJEM Pentatrichomonas hominis PRI PSU IZ SLOVENSKEGA ZAVETIŠČA M. Brložnik, S. Faraguna, B. Slavec, R. Kostanjšek, A. Vergles Rataj, I. Gruntar Povzetek: Na kliniko smo zaradi akutne krvave, penaste in smrdljive driske sprejeli tri mesece starega psa, ki so ga lastniki nedavno posvojili iz zavetišča. Klinični pregled ter hematološke in biokemične preiskave krvi so bili brez posebnosti. Pri pregledu trebuha z ultrazvokom smo ugotovili velike mezenterialne bezgavke, tanko črevo pa je bilo polno ehogene vsebine. Z mikroskopskim pregledom nativnih preparatov smo v vzorcu blata opazili številne gibljive organizme, podobne trihomonasom, s specifičnim krožnim gibanjem. Z mikroskopiranjem ter s flotacijo in metodo SAF smo diagnosticirali še jajčeca nematodov Toxocara canis in oociste praživali Isospora sp. Trihomonasom podobne organizme smo uspešno izolirali in jih vzgojili v čisti kulturi. Mikroskopski pregled organizmov, obarvanih po Giemsi, je omogočil določitev števila bičkov in vzpostavitev suma na okužbo s Pentatrichomonas hominis (P. hominis). Diagnozo smo dokončno potrdili s polimerazno verižno reakcijo (PCR) in določitvijo baznega zaporedja, ter z vrstičnim elektronskim mikroskopom. PCR in določitev baznega zaporedja sta pokazala 99 % skladnost našega izolata z izolati P. hominis iz drugih gostiteljev/študij. Ta podatek kaže na zoonotski potencial P. hominis in na možnost peroralnega prenosa med živalmi in ljudmi. Članek predstavlja tudi prvo poročilo o trihomonadni okužbi s P hominis pri psih v Sloveniji. Ključne besede: Pentatrichomonas hominis; pes; izolacija; gojenje; PCR; določanje zaporedja DNK; vrstična elektronska mikroskopija Slov Vet Res 2016; 53 (4): 237- 40 AUTHOR INDEX VOLUME 53, 2016 Acinger - Rogic Z, see Novosel D, Petrovic T, Acinger - Rogic Z, Stukelj M.......... 185 Adnan M, see Tariq A, Adnan M, Mussarat S....................... 119 Aksu F, see Kahraman T, Issa G, Altunatmaz SS, Kahraman BB, Aksu F, Aydin A, Aksu H . . . 5 Aksu H, see Kahraman T, Issa G, Altunatmaz SS, Kahraman BB, Aksu F, Aydin A, Aksu H . . . 5 Altunatmaz SS, see Kahraman T, Issa G, Altunatmaz SS, Kahraman BB, Aksu F, Aydin A, Aksu H..........................5 Amato E, see Rocchigiani G, Nardoni S, Amato E, Tempori C, Mancianti F...............63 Ambrozic M, Henigman U, Jevsnik M, Kirbis A, Raspor P......................... 195 Antonov A, Georgiev P, Dineva J, Conze T, Dimitrova R, Wehrend A. Dynamics of some vaginal parameters in non-pregnant bitches after mid-luteal aglepristone treatment........................ 161 Aybazov M, see Trukhachev V, Skripkin V, Kvochko A, Kulichenko A, Kovalev D, Pisarenko S, Volynkina A, Selionova M, Aybazov M, Shumaenko S, Omarov A, Mamontova T, Yatsyk O, Krivoruchko A. . . 77 Aydin A, see Kahraman T, Issa G, Altunatmaz SS, Kahraman BB, Aksu F, Aydin A, Aksu H 5 Bajc Z, Jencic V, Sinigoj Gacnik K. The heavy metal contents (Cd, Pb, Cu, Zn, Fe and Mn) and its relationships with the size of the rudd (Scardinius erythrophthalmus) from lake Cerknica, Slovenia...................69 Bajc Z, see Dobeic M, Vadnjal S, Bajc Z, Umek P, Pintaric S, Uranjek I, Sinigoj Gacnik K . . . 29 Barazorda Romero S, Kvapil P, Cizek A, Knotek Z. The prevalence and antimicrobial resistance of Salmonella species isolated from captive reptiles at Ljubljana Zoo..............43 Batkowska J, see Listos P, Gryzinska M, Batkowska J.......................85 Benic M, see Cvetnic L, Samardzija M, Habrun B, Kompes G, Benic M................. 131 Bogut I, see Župan B, Ljubojevič D, Pelič M, Čirkovič M, Dordevič V, Bogut 1.........19 Brenesselova M, see Korenekova B, Mačanga J, Brenesselova M, Sopoliga I........... 111 Brložnik M, Faraguna S, Slavec B, Kostanjšek R, Vergles Rataj A, Gruntar I. Pentatrichomonas hominis coinfection in a puppy from a Slovenian animal shelter.............229 Brudnicki W, see Skoczylas B, Brudnicki W, Kirkillo-Stacewicz K, Nowicki W, Wach J. .13 Caracappa G, see Pugliese M, Spadola F, Morici M, Piazza A, Caracappa G, Persichetti MF, Lommi A.........................205 Ceh K, see Mohoric L, Zorko B, Ceh K, Majdic G. Blinded placebo study of bilateral osteoarthritis treatment using adipose derived mesenchymal stem cells............. 167 Conze T, see Antonov A, Georgiev P, Dineva J, Conze T, Dimitrova R, Wehrend A. Dynamics of some vaginal parameters in non-pregnant bitches after mid-luteal aglepristone treatment........................ 161 Cvetnič L, Samardžija M, Habrun B, Kompes G, Benič M. Microbiological monitoring of mastitis pathogens in the control of udder health in dairy cows................ 131 Čižek A, see Barazorda Romero S, Kvapil P, Čižek A, Knotek Z........................43 Čirkovič M, see Župan B, Ljubojevič D, Pelič M, Čirkovič M, Dordevič V, Bogut I. Common carp response to the different concentration of linseed oil in diet.................... 19 Dimitrova R, see Antonov A, Georgiev P, Dineva J, Conze T, Dimitrova R, Wehrend A .... 161 Dineva J, see Antonov A, Georgiev P, Dineva J, Conze T, Dimitrova R, Wehrend A......161 Dobeic M, Vadnjal S, Bajc Z, Umek P, Pintarič Š, Uranjek I, Šinigoj Gačnik K. Antibacterial properties of a Non-thermal, Atmospheric, Openair®, plasma jet in surface decontamination of eggs in shell........29 Dordevič V, see Župan B, Ljubojevič D, Pelič M, 238 Slov Vet Res, Author Index Volume 53, 2016 Čirkovič M, Dordevič V, Bogut 1.........19 Faraguna S, see Brložnik M, Faraguna S, Slavec B, Kostanjšek R, Vergles Rataj A, Gruntar 1.........................229 Gambi R, see Rossi G, Laus F, Piccinini A, Piccinini R, Pasquinelli F, Gambi R, Paggi E, Tesei B...........................99 Georgiev P, see Antonov A, Georgiev P, Dineva J, Conze T, Dimitrova R, Wehrend A...... 161 Gombač M, Gruntar I, Kvapil P, Švara T. Brevundimonas vesicularis septicaemia in a kid with congenital goitre............ 175 Gruntar I, see Brložnik M, Faraguna S, Slavec B, Kostanjšek R, Vergles Rataj A, Gruntar I . 229 Gruntar I, see Gombač M, Gruntar I, Kvapil P, Švara T.......................... 175 Gryzinska M, see Listos P, Gryzinska M, Batkowska J.......................85 Habrun B, see Cvetnič L, Samardžija M, Habrun B, Kompes G, Benič M 131 Henigman U, see Ambrožič M, Henigman U, Jevšnik M, Kirbiš A, Raspor P......... 195 Issa G, see Kahraman T, Issa G, Altunatmaz SS, Kahraman BB, Aksu F, Aydin A, Aksu H ... 5 Jakovac Strajn B, see Pavšič Vrtač K, Jakovac Strajn B, Salobir J, Šrimpf K, Tavčar Kalcher G.........................49 Jenčič V, see Bajc Z, Jenčič V, Šinigoj Gačnik K..........................69 Jevšnik M, see Ambrožič M, Henigman U, Jevšnik M, Kirbiš A, Raspor P......... 195 Kahraman BB, see Kahraman T, Issa G, Altunatmaz SS, Kahraman BB, Aksu F, Aydin A, Aksu H..........................5 Kahraman T, Issa G, Altunatmaz SS, Kahraman BB, Aksu F, Aydin A, Aksu H. Effects of oregano essential oil, grapefruit seed extract and their combination on the growth and survival of Salmonella Typhimurium and Listeria monocytogenes in poultry fillets under modified atmosphere packaging..........5 Kirbiš A, see Ambrožič M, Henigman U, Jevšnik M, Kirbiš A, Raspor P. Viral contamination in mussel production chain on the Slovenian coastline ......................... 195 Kirkillo-Stacewicz K, see Skoczylas B, Brudnicki W, Kirkillo-Stacewicz K, Nowicki W, Wach J........................... 13 Knotek Z, see Barazorda Romero S, Kvapil P, Čižek A, Knotek Z...................43 Kompes G, see Cvetnič L, Samardžija M, Habrun B, Kompes G, Benič M...............131 Korenekova B, Mačanga J, Brenesselova M, Sopoliga I. The effects of two ways of storage on physicochemical changes in pheasant meat............................ 111 Kostanjšek R, see Brložnik M, Faraguna S, Slavec B, Kostanjšek R, Vergles Rataj A, Gruntar 1.........................229 Kovalev D, see Trukhachev V, Skripkin V, Kvochko A, Kulichenko A, Kovalev D, Pisarenko S, Volynkina A, Selionova M, Aybazov M, Shumaenko S, Omarov A, Mamontova T, Yatsyk O, Krivoruchko A. . . 77 Krivoruchko A, see Trukhachev V, Skripkin V, Kvochko A, Kulichenko A, Kovalev D, Pisarenko S, Volynkina A, Selionova M, Aybazov M, Shumaenko S, Omarov A, Mamontova T, Yatsyk O, Krivoruchko A. . . 77 Kulichenko A, seeTrukhachev V, Skripkin V, Kvochko A, Kulichenko A, Kovalev D, Pisarenko S, Volynkina A, Selionova M, Aybazov M, Shumaenko S, Omarov A, Mamontova T, Yatsyk O, Krivoruchko A. . . 77 Kvapil P, see Barazorda Romero S, Kvapil P, Čižek A, Knotek Z...................43 Kvapil P, see Gombač M, Gruntar I, Kvapil P, Švara T.......................... 175 Kvochko A, see Trukhachev V, Skripkin V, Kvochko A, Kulichenko A, Kovalev D, Pisarenko S, Volynkina A, Selionova M, Aybazov M, Shumaenko S, Omarov A, Mamontova T, Yatsyk O, Krivoruchko A. . . 77 Laus F, see Rossi G, Laus F, Piccinini A, Piccinini R, Pasquinelli F, Gambi R, Paggi E, Tesei B99 Li Q, see Tong J, Zhang H, Wu Y, Wang Y, Li Q, Liu Y............................ 141 Listos P, Gryzinska M, Batkowska J. Postmortem decrease in temperature in the orbit of dogs for use in determining time of death............................85 Liu Y, see Tong J, Zhang H, Wu Y, Wang Y, Li Q, Liu Y............................ 141 Ljubojevic D, see Župan B, Ljubojevic D, Pelič M, Čirkovič M, Dordevič V, Bogut I.........19 Lommi A, see Pugliese M, Spadola F, Morici M, Piazza A, Caracappa G, Persichetti MF, Lommi A.........................205 Lukanc B, see Pogorevc E, Lukanc B, Seliškar A, Pelc R, Zorko B...........219 Mačanga J, see Korenekova B, Mačanga J, Brenesselova M, Sopoliga I........... 111 Majdic G, see Mohoric L, Zorko B, Ceh K, Majdic G......................... 167 239 Slov Vet Res, Author Index Volume 53, 2016 Mamontova T, see Trukhachev V, Skripkin V, Kvochko A, Kulichenko A, Kovalev D, Pisarenko S, Volynkina A, Selionova M, Aybazov M, Shumaenko S, Omarov A, Mamontova T, Yatsyk O, Krivoruchko A. . . 77 Mancianti F, see Rocchigiani G, Nardoni S, Amato E, Tempori C, Mancianti F.......63 Mohoric L, Zorko B, Ceh K, Majdic G. Blinded placebo study of bilateral osteoarthritis treatment using adipose derived mesenchymal stem cells........................ 167 Morici M, see Pugliese M, Spadola F, Morici M, Piazza A, Caracappa G, Persichetti MF, Lommi A.........................205 Mussarat S, seeTariq A, Adnan M, Mussarat S........................ 119 Mutluay D. Distribution of primitive endoderm and epiblast lineage specific factors in late stage blastocysts...................211 Nardoni S, see Rocchigiani G, Nardoni S, Amato E, Tempori C, Mancianti F.............63 Novosel D, Petrovič T, Acinger - Rogič Ž, Štukelj M. Epidemiology and status of porcine reproductive and respiratory syndrome in the Western Balkan region: Challenges and prospects........................ 185 Nowicki W, see Skoczylas B, Brudnicki W, Kirkillo-Stacewicz K, Nowicki W, Wach J . . 13 Omarov A, see Trukhachev V, Skripkin V, Kvochko A, Kulichenko A, Kovalev D, Pisarenko S, Volynkina A, Selionova M, Aybazov M, Shumaenko S, Omarov A, Mamontova T, Yatsyk O, Krivoruchko A. . . 77 Paggi E, see Rossi G, Laus F, Piccinini A, Piccinini R, Pasquinelli F, Gambi R, Paggi E, Tesei B...........................99 Pasquinelli F, see Rossi G, Laus F, Piccinini A, Piccinini R, Pasquinelli F, Gambi R, Paggi E, Tesei B...........................99 Pavšič Vrtač K, Jakovac Strajn B, Salobir J, Šrimpf K, Tavčar Kalcher G. Study of trace and ultratrace elements in silage intended for cattle nutrition.....................49 Pelc R, see Pogorevc E, Lukanc B, Seliškar A, Pelc R, Zorko B....................219 Pelič M, see Župan B, Ljubojevič D, Pelič M, Čirkovič M, Dordevič V, Bogut 1.........19 Persichetti MF, see Pugliese M, Spadola F, Morici M, Piazza A, Caracappa G, Persichetti MF, Lommi A ......................... 205 Petrovič T, see Novosel D, Petrovič T, Acinger -Rogič Ž, Štukelj M.................. 185 Piazza A, see Pugliese M, Spadola F, Morici M, Piazza A, Caracappa G, Persichetti MF, Lommi A ......................... 205 Piccinini A, see Rossi G, Laus F, Piccinini A, Piccinini R, Pasquinelli F, Gambi R, Paggi E, Tesei B...........................99 Piccinini R, see Rossi G, Laus F, Piccinini A, Piccinini R, Pasquinelli F, Gambi R, Paggi E, Tesei B...........................99 Pintarič Š, see Dobeic M, Vadnjal S, Bajc Z, Umek P, Pintarič Š, Uranjek I, Šinigoj Gačnik K..........................29 Pisarenko S, see Trukhachev V, Skripkin V, Kvochko A, Kulichenko A, Kovalev D, Pisarenko S, Volynkina A, Selionova M, Aybazov M, Shumaenko S, Omarov A, Mamontova T, Yatsyk O, Krivoruchko A. . . 77 Pogorevc E, Lukanc B, Seliškar A, Pelc R, Zorko B. Radiological comparison of lumbosacral anatomy between German and Belgian Shepherd (Malinois) working dogs............................219 Pugliese M, Spadola F, Morici M, Piazza A, Caracappa G, Persichetti MF, Lommi A. Evaluation of the conjunctival bacterial flora in 140 rabbits (Oryctolagus cuniculus) farmed in Sicily Island....................205 Raspor P, see Ambrožič M, Henigman U, Jevšnik M, Kirbiš A, Raspor P............... 195 Rocchigiani G, Nardoni S, Amato E, Tempori C, Mancianti F. Occurrence of anti Toxoplasma antibodies in owned dogs from Italy: A retrospective study..................63 Rossi G, Laus F, Piccinini A, Piccinini R, Pasquinelli F, Gambi R, Paggi E, Tesei B. Metastasizing ovarian carcinoma in an Eurasian brown bear (Ursus arctos arctos): A case report........................99 Salobir J, see Pavšič Vrtač K, Jakovac Strajn B, Salobir J, Šrimpf K, Tavčar Kalcher G . . . . 49 Samardžija M, see Cvetnič L, Samardžija M, Habrun B, Kompes G, Benič M........131 Sandikci M, see Yildiz M, Sandikci M......151 Selionova M, see Trukhachev V, Skripkin V, Kvochko A, Kulichenko A, Kovalev D, Pisarenko S, Volynkina A, Selionova M, Aybazov M, Shumaenko S, Omarov A, Mamontova T, Yatsyk O, Krivoruchko A. . . 77 Seliškar A, see Pogorevc E, Lukanc B, Seliškar A, Pelc R, Zorko B...........219 Shokri H. Frequency of yeasts and filamentous fungi in the external ear canals of cattle in 240 Slov Vet Res, Author Index Volume 53, 2016 Iran..............................91 Shumaenko S, see Trukhachev V, Skripkin V, Kvochko A, Kulichenko A, Kovalev D, Pisarenko S, Volynkina A, Selionova M, Aybazov M, Shumaenko S, Omarov A, Mamontova T, Yatsyk O, Krivoruchko A. . . 77 Skoczylas B, Brudnicki W, Kirkillo-Stacewicz K, Nowicki W, Wach J. Arterial supply of the cerebral cortex in cattle (Bos primigenius f. dom.)..............13 Skripkin V, see Trukhachev V, Skripkin V, Kvochko A, Kulichenko A, Kovalev D, Pisarenko S, Volynkina A, Selionova M, Aybazov M, Shumaenko S, Omarov A, Mamontova T, Yatsyk O, Krivoruchko A. . . 77 Slavec B, see Brložnik M, Faraguna S, Slavec B, Kostanjšek R, Vergles Rataj A, Gruntar I . 229 Sopoliga I, see Korenekova B, Mačanga J, Brenesselova M, Sopoliga I........... 111 Spadola F, see Pugliese M, Spadola F, Morici M, Piazza A, Caracappa G, Persichetti MF, Lommi A.........................205 Šinigoj Gačnik K, see Bajc Z, Jenčič V, Šinigoj Gačnik K..........................69 Šinigoj Gačnik K, see Dobeic M, Vadnjal S, Bajc Z, Umek P, Pintarič Š, Uranjek I, Šinigoj Gačnik K..........................29 Šrimpf K, see Pavšič Vrtač K, Jakovac Strajn B, Salobir J, Šrimpf K, Tavčar Kalcher G.......49 Štukelj M, see Novosel D, Petrovič T, Acinger - Rogič Ž, Štukelj M.................. 185 Švara T, see Gombač M, Gruntar I, Kvapil P, Švara T.......................... 175 Tariq A, Adnan M, Mussarat S. Use of ethnoveterinary medicines by the people living near Pak- Afghan border region ....... 119 Tavčar Kalcher G, see Pavšič Vrtač K, Jakovac Strajn B, Salobir J, Šrimpf K, Tavčar Kalcher G.........................49 Tempori C,see Rocchigiani G, Nardoni S, Amato E, Tempori C, Mancianti F........................63 Tesei B, see Rossi G, Laus F, Piccinini A, Piccinini R, Pasquinelli F, Gambi R, Paggi E, Tesei B...........................99 Tong J, Zhang H, Wu Y, Wang Y, Li Q, Liu Y. Oestrogens and prolactin regulate mammary gland epithelial cell growth by modulation of the Wnt signal pathway.............. 141 Trukhachev V, Skripkin V, Kvochko A, Kulichenko A, Kovalev D, Pisarenko S, Volynkina A, Selionova M, Aybazov M, Shumaenko S, Omarov A, Mamontova T, Yatsyk O, Krivoruchko A. Polymorphisms of the IGF1 gene in Russian sheep breeds and their influence on some meat production parameters........................77 Umek P, see Dobeic M, Vadnjal S, Bajc Z, Umek P, Pintarič Š, Uranjek I, Šinigoj Gačnik K . 29 Uranjek I, see Dobeic M, Vadnjal S, Bajc Z, Umek P, Pintarič Š, Uranjek I, Šinigoj Gačnik K . 29 Vadnjal S, see Dobeic M, Vadnjal S, Bajc Z, Umek P, Pintarič Š, Uranjek I, Šinigoj Gačnik K..........................29 Vergles Rataj A, see Brložnik M, Faraguna S, Slavec B, Kostanjšek R, Vergles Rataj A, Gruntar 1.........................229 Volynkina A, see Trukhachev V, Skripkin V, Kvochko A, Kulichenko A, Kovalev D, Pisarenko S, Volynkina A, Selionova M, Aybazov M, Shumaenko S, Omarov A, Mamontova T, Yatsyk O, Krivoruchko A. . . 77 Wach J, see Skoczylas B, Brudnicki W, Kirkillo- Stacewicz K, Nowicki W, Wach J........13 Wang Y, see Tong J, Zhang H, Wu Y, Wang Y, Li Q, Liu Y.......................... 141 Wehrend A, see Antonov A, Georgiev P, Dineva J, Conze T, Dimitrova R, Wehrend A......161 Wu Y, see Tong J, Zhang H, Wu Y, Wang Y, Li Q, Liu Y............................ 141 Yatsyk O, see Trukhachev V, Skripkin V, Kvochko A, Kulichenko A, Kovalev D, Pisarenko S, Volynkina A, Selionova M, Aybazov M, Shumaenko S, Omarov A, Mamontova T, Yatsyk O, Krivoruchko A. . . 77 Yildiz M, Sandikci M. Changes in the uterus and vagina of rats with experimentally induced diabetes and the effect of lycopene on the changes......................... 151 Zhang H, see Tong J, Zhang H, Wu Y, Wang Y, Li Q, Liu Y.......................... 141 Zorko B, see Mohoric L, Zorko B, Ceh K, Majdic G......................... 167 Zorko B, see Pogorevc E, Lukanc B, Seliškar A, Pelc R, Zorko B...........219 Župan B, Ljubojevič D, Pelič M, Čirkovič M, Dordevič V, Bogut I. Common carp response to the different concentration of linseed oil in diet.............................. 19 SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Slov Vet Res 2016; 53 (4) Review Article Novosel D, Petrovic T, Acinger - Rogic Z, Stukelj M. Epidemiology and status of porcine reproductive and respiratory syndrome in the Western Balkan region: Challenges and prospects....................................................................... 185 Original Research Articles Ambrozic M, Henigman U, Jevsnik M, Kirbis A, Raspor P. Viral contamination in mussel production chain on the Slovenian coastline.............................................................................................................................. 195 Pugliese M, Spadola F, Morici M, Piazza A, Caracappa G, Persichetti MF, Lommi A. Evaluation of the conjunctival bacterial flora in 140 rabbits (Oryctolagus cuniculus) farmed in Sicily Island............................................................. 205 Mutluay D. Distribution of primitive endoderm and epiblast lineage specific factors in late stage blastocysts..................... 211 Pogorevc E, Lukanc B, Seliskar A, Pelc R, Zorko B. Radiological comparison of lumbosacral anatomy between German and Belgian Shepherd (Malinois) working dogs................................................................................. 219 Case Report Brloznik M, Faraguna S, Slavec B, Kostanjsek R, Vergles Rataj A, Gruntar I. Pentatrichomonas hominis coinfection in a puppy from a Slovenian animal shelter.................................................................................................... 229 Author Index Volume 53, 2016............................................................................................................... 237