ISSN 15804003 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Volume 54 Slov Vet Res • Ljubljana • 2017 • Volume 54 • Number 3 • 99-138 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Volume 54 Slov Vet Res • Ljubljana • 2017 • Volume 54 • Number 3 • 99-138 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, Joško Račnik, Ivan Toplak, 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 2017; 54 (3) Original Research Articles Paller T, Hostnik P, Pogačnik M, Toplak I. The prevalence of ten pathogens detected by a real-time PCR method in nasal swab samples collected from live cattle with respiratory disease.....................................................................101 Yeh JY, Hwang JM, Kim JG. Detection of Lawsonia intracellularisDNA in ileal tissues of dead wild birds in the Republic of Korea.............................................................................................................109 Lukanc B, Butinar J, Nemec Svete A, Prosek M, Seliskar A. The influence of isoflurane anaesthesia on intestinal permeability in healthy dogs........................................................................................................117 Ho CNQ, Hoang SN, Nguyen TTP, Doan CC, Nguyen MTP, Le TH, Nguyen HTT, Le LT. The ORF5 variation of Vietnamese porcine reproductive and respiratory syndrome virus strains...................................................................125 Petrovic D, Kopitovic A, Pericin-Starcevic I, Vujcic M, Dragic N, Gouni O, Topalidou A, Sekulic S. Guinea pig fetus does not change its presentation during second half of gestation........................................................................133 Slov Vet Res 2017: 54 (3): 101 -7 UDC 636.2.09:616.2-002:616.214-076:577.21 Original Research Article THE PREVALENCE OF TEN PATHOGENS DETECTED BY A REAL-TIME PCR METHOD IN NASAL SWAB SAMPLES COLLECTED FROM LIVE CATTLE WITH RESPIRATORY DISEASE Tomislav Paller, Peter Hostnik, Milan Pogačnik, Ivan Toplak* National Veterinary Institute, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia Corresponding author, E-mail: ivan.toplak@vf.uni-lj.si Summary: Respiratory diseases often correspond to primary infections with different pathogens of cattle, causing heavy economic losses in young stock and breeding herds. Between 2012 and 2014, nasal swab samples were collected from twenty-eight herds from 133 affected live cattle that were clinically suffering from symptoms of respiratory disease, pyrexia, cough, serous nasal and lacrimal discharge, increased respiratory rate, and breath sounds. Individual swab samples were tested in the laboratory using three commercial and one in-house real-time PCR methods, to detect nucleic acids of a total of ten different respiratory pathogens. Pasteurella multocida (P. multocida) was detected in 58.65% of samples, Mannheimia haemolytica (M. haemolytica) in 15.04%, while Mycoplasma bovis (M. bovis) and Histophilus somni(H. somni) were positive in 9.77% of nasal swab samples. Among viral pathogens, the highest prevalence (40.60%) was observed for bovine respiratory syncytial virus (BRSV), followed by bovine coronavirus (BCV) 12.03%, bovine para-influenza 3 (PI-3) 3.01%, and bovine viral diarrhea virus (BVDV) with 1.50% of positive samples. The less frequently detected viral pathogens were bovine herpes virus type 1 (BHV-1) and bovine adenovirus (BAdV) with 0.75% positive samples each. The new implemented molecular methods can be an important diagnostic tool for laboratories and farmers to improve the therapy, control, and prevention of respiratory disease in cattle herds. Key words: bovine respiratory disease; nasal swab samples; diagnostics; real-time PCR detection; cattle Introduction Bovine respiratory disease (BRD) is the major cause of serious respiratory tract infections worldwide, often leading to high morbidity and mortality rates in cattle. The disease is considered to be a multifactorial disorder, produced with either stress or reduced immunity, allowing several pathogens to emerge. It inflicts considerable mortality and financial losses mainly in calves in dairy and beef herds. Viral and bacterial pathogens Received: 5 April 2016 Accepted for publication: 7 October 2016 together with mycoplasma and environmental risk factors are the most common cause of diseases, ranging from common colds to life-threatening pneumonia (1). A large number of both RNA and DNA viruses uses the respiratory tract to initiate host infection. Infection may be restricted to certain sections of the airway system such as the trachea, bronchi, or alveoli. For some viruses, the respiratory tract may merely serve as a primary entry site from where infection spreads to other organs or tissues. An important defense strategy is the mucociliary clearance system. While some epithelial cells are specialized to produce and release mucins, other cells are equipped with cilia 102 T. Palier, P. Hostnik, M. Pogacnik, I. Toplak that enable them to contribute to the transport of the mucus with pathogens out of the respiratory tract. The most important viral pathogens associated with BRD are bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus 3 (PI-3), bovine herpesvirus 1 (BHV-1), bovine adenovirus (BAdV), bovine coronavirus (BCV) and bovine viral diarrhea virus (BVDV) (2, 3). While BHV-1 and BVDV have already been eradicated in some European countries, infections with BRSV and BCV are endemic in the cattle population globally (4, 5). The infection of cattle with BAdV usually results in disease of the gastrointestinal or respiratory tract. Different serotypes of BAdV are divided into two subgroups. BAdV type 1, 2, 3, 9 and 10 comprise Group A, BAdV type 4, 5, 6, 7 and 8 comprise Group B (6). Only a few publications on the simultaneous detection of more than three different pathogens in case of respiratory or enteric diseases are available (7, 8, 9). Several studies present antibodies against etiologic agents of BRD, but these only indirectly confirm the previous infections with specific viral and bacterial agents in animals or herds (45, 10-12). Newly developed molecular methods significantly improved the diagnosis of respiratory tract infections, providing a fast and cost-effective tool for different pathogens, to determine the prevalence of respiratory viruses, bacteria, and mycoplasma in clinically affected cattle (13). Few reports about the prevalence on the respiratory disease of cattle in Slovenia exist, mainly obtained several years ago with conventional methods of bacteria or virus isolation (14-18). However, the traditional farming system in Slovenia with small isolated cattle herds is disappearing; herds are enlarging gradually and, in several cases, animals of different herds and ages are kept in a pen. In the new rearing system, young calves at the age of 1-3 weeks originating from several herds, are transported to beef units and grouping together, frequently with a combination ofimported beef cattle. Sometimes, vaccines against respiratory disease are used, but almost no data about effectiveness is available. Antimicrobials are generally not used for disease prevention; sick animals are mostly treated individually with antibiotics. The aim of this study was to determine the prevalence of ten respiratory pathogens detected by new implemented real-time PCR methods using nasal swab samples collected from affected cattle with respiratory disease. Materials and methods Sampling was conducted mostly in winter and spring periods from 2012 to 2014. All swab samples were collected into sterile swabs (Sigma Virocult®, MW 951S, UK) and were immediately sent to a laboratory. Nasal swab samples were collected from 133 live affected animals, originating from twenty-eight different Slovenian cattle herds identified with bovine respiratory disease. Five of them are feedlot cattle herds, four are dairy herds, and nineteen are traditional, combined herds, with milk and meat production. All of the sampled animals had abnormal sound on auscultation of the respiratory tract and most had either one or several of the following symptoms: fever >39 °C, elevated respiratory rate (> 40/min), cough or nasal and/or lacrimal discharge. In the case of an acute outbreak on a farm, 1 to 18 samples were collected from the same herd, only from clinically affected animals. As a control group of the study, ten animals from three farms without clinical signs of respiratory disease in the previous two months were selected, and nasal swab samples were collected from healthy animals and screened for ten pathogens. After arrival at the laboratory, samples were homogenized and stored in a freezer at < -15 °C until testing. Total nucleic acids were extracted from 140 ^l of homogenate using a commercial kit for RNA extraction (QIAamp® Viral RNA Mini Kit (Qiagen, Germany)) according to the manufacturer's instructions. Individual swab samples were tested by one in-house and three commercial real-time PCR methods, detecting specific nucleic acids of a total of ten different respiratory pathogens, including detection of endogenous internal positive control (IPC) for controlling the efficiency of extraction and the absence of inhibitors in individual samples. Samples were tested on a 96-tube microplate. On each microplate, the positive controls for all tested pathogens were included. A commercial TaqMan® real-time PCR kit for the detection of seven major ruminant pathogens (LSI VetMAX™ Screening Pack - Ruminants Respiratory Pathogens, LSI, France) allows the simultaneous detection of the M. bovis, H. somni, P. multocida, M. haemolytica, BCV, BRSV, and PI-3. For the detection of BVDV and BHV-1, another two commercial real-time kits (Kit TaqVet® BVDV "Screening" and LSI VetMAX™ IBR gB, both produced by LSI, France) were used The prevalence of ten pathogens detected by a real-time pcR method in nasal swab samples collected from live cattle 103 according to the producer's instructions. For the detection of BAdV, an in-house protocol was implemented with previously designed primers detecting BAdV, serotypes 4-8 (19). Real-time PCR was performed using the forward primer BAV4-8F 5'-CRA GGG AAT AYY TGT CTG AAA ATC-3', the reverse primer BAV4-8R 5'-AAG GAT CTC TAA ATT TYT CTC CAA GA-3' and the probe FAM-TTC ATC WCT GCC ACW CAA AGC TTT TTT-BHQ-1 targeting the hexon gene of BAdV (9). The reaction was performed in a total volume of 15 ^l, using QuanTitec® Virus Kit (Qiagen, Germany) as follows: 8 ^l of nuclease free water, 3 ^l of 5x PCR Master Mix, 0,5 ^l of the stock solution with 20 ^M of BAV4-8F primer, 0,5 M of the 20 mM of BAV4-8R primer, 0,5 ^l of the stock solution with 10 MM of probe and 2,5 M of the RNA/DNA template. The real-time PCR running program for BAdV was 95 °C for 15 min; followed by 45 cycles of 95 °C for 10 s, 54 °C for 30 s and 60 °C for 30 s. All real-time cyclings were performed on an Mx3005P thermocycler (Stratagene, USA) using protocol according to the manufacturer's instructions for commercial kits and the above-described protocol for BAdV detection. The fluorescent signal was detected after each annealing, and the results were presented as a cycle threshold value for individual samples. Analysis of real-time amplification curves was performed using commercial thermal cycler Table 1: The results of the detection of ten different pathogens in 133 nasal swabs samples, collected from live cattle suffering from respiratory disease together with a control group are presented. The ranges of cycle threshold values obtained by specific real-time PCR methods are presented for individual pathogens Name of pathogen Cattle with respiratory disease Control group (healthy animals) Number of tested samples Number of positive samples % of positive samples Cycle threshold (Ct) range (mean) Number of tested samples Number of positive samples Cycle threshold (Ct) M. bovis 133 13 9.77% 20.88-37.81 (29.60) 10 0 - H. somni 133 13 9.77% 26.63-41.97 (35.35) 10 1 35.35 P. multocida 133 78 58.64% 19.76-43.04 (30.41) 10 1 32.99 M. haemolytica 133 20 15.04% 27.12-43.54 (34.05) 10 0 - BCV 133 16 12.03% 25.16-38.82 (31.61) 10 0 - BRSV 133 54 40.60% 20.48-39.89 (29.53) 10 0 - BPI-3 133 4 3.01% 20.81-42.05 (34.05) 10 0 - BVDV 133 2 1.50% 28.73-35.34 (32,03) 10 0 - BHV-1 133 1 0.75% 29.91 (29.91) 10 0 - BAdV 133 1 0.75% 29.43 (29.43) 10 0 - system software, and an "auto baseline" was used to determine fluorescence baselines. Results A total of 133 swab samples from live cattle with symptoms of respiratory disease and 10 swab samples from healthy cattle (control group) were successfully screened for 10 pathogens with the real-time PCRs method. In cattle with respiratory disease, P. multocida was detected in 78/133 (58.65%) of samples, M. haemolytica in 20/133 (15.04%), while M. bovis and H. somni were positive in 13/133 (9.77%) of nasal swab samples. The highest prevalence of viral pathogens was observed for BRSV 54/133 (40.60%), following BCV 16/133 (12.03%), PI-3 with 4/133 (3.01%) and BVDV with 2/133 (1.50%) of positive samples. The less frequently detected viral pathogens were BHV-1 and BAdV with 1/133 (0.75%) positive samples (Table 1). At least one pathogen was detected in 110/133 (82.70%) of clinically affected cattle (Figure 1). In 43 samples (32.33%), only one pathogen was detected; P. multocida in 17 samples and BRSV in 16 samples, and M. haemolytica in five samples. The simultaneous detection of two different pathogens was observed in 46 samples (34.59%); 104 T. Palier, P. Hostnik, M. Pogacnik, I. Toplak 40 % of samples 35 1/ ■ 14 c c c c c c 00 ™ ™ / # / / / / / / / / xr jf gf? gOv «p* J* ¡¿F ¿>v / y / / 0.05). Fetuses were brought into a head-down and head-up 154 times. In none of these attempts did a fetus change presentation during the five minutes of observation. Conclusion: Guinea pig fetuses do not spontaneously or at provocation change presentation after 30th GD. Key words: fetal presentation; gestation; guinea pig; ultrasound Introduction Etiology of fetal presentation in mammals has not been fully elucidated yet (1,2). Adequate presentation at birth is significant since anterior presentation in herd animals enables physiological delivery. Posterior presentation is accompanied by an inadequate dilatation of the birth channel, and consequently, a difficult delivery (3). In a previous paper, it was postulated that fetal presentation is a Received: 6 October 2016 Accepted for publication: 24 February 2017 consequence of postural development (1). In herd animals, locomotor-postural development occurs during the second half of gestation. In the anterior presentation, the cranial part of the body is above the caudal part. The hind legs are the source of the main propulsive force. When the fetus has its hind legs below its cranial part, it has an optimal mobility. In up-side down position the fetus has more difficulties to move (1). Guinea pig are precocial rodents that have full locomotion at birth. In previous studies with guinea pigs it was shown that a reflex of turning from supination to pronation can be induced in the 134 D. Petrovic, A. Kopitovic, I. Pericin-Starcevic, M. Vujcic, N. Dragic, O. Gouni, A. Topalidou, S. Sekulic guinea pig fetus and that its intrauterine behavior may be gravity-dependent (4). For guinea pigs, there is no data about spontaneous or provoked changes in fetal presentation during gestation. The aim of this paper is to investigate spontaneous changes in fetal presentation and situs during the second half of gestation. In addition, a secondary aim is to determine the existence of active turning of the fetus through 180 degrees, from its passive positioning into an upside-down position. It is assumed that in the second half of gestation a fetus that is passively positioned in an upside-down position will turn around though 180 degrees. Material and method Experimental animals The study subjects were albino guinea pigs (Caviaporcellus) obtained from the Department of Biochemistry, Faculty of Medicine, Clinical Center of Vojvodina, Novi Sad. The experiments with animals were approved by the Ethics Committee of the University of Novi Sad No 04-29/62). The guinea pigs were kept in 400Wx1000Lx300H mm plastic containers in a harem system: two females and one male. Pregnant females were moved to 300Wx300Lx300Hmm plastic containers after the 60th day of gestation, where they were kept separate until delivery, and afterwards for the first 15 days with the offspring. The animals had a standard commercial pellet diet and ad libitum water enriched with vitamin C (30 mg/100 ml water). Artificial cycles with 12 hours of light (08:00-20:00) and 12 hours of dark were provided. The room temperature was maintained at 22 ± 2°C. The air was recirculated 10 times per hour. Inspection of vaginal introitus was performed daily, and the day of vaginal membrane perforation was taken as the first day of gestation. Individual guinea pigs were identified by yellow patterns on their backs. Eight pregnant guinea pigs with one fetus were included. The number of fetuses was determined by ultrasound examination. Pregnant females were shaved before examination; the shaving of the abdominal region was made during a short-term inhalatory ether narcosis on the 25th day of gestation. Ultrasound examination Ultrasound examinations were started on the 26th day of gestation, until when the first movements of the guinea pig fetus usually occurs (5). During gestation that usually lasts for 66 days each fetus was examined 2-3 times in each 5-day interval (gestation days 26-30, 31-35, 36-40, 4145, 46-50, 51-55, 56-60, >61). Immediately before the examination the pregnant guinea pigs were supported in a supine position on a 15x30cm board using plastic strips with clasps, fastened over the thoracic area and both hind legs. The strips were pulled through holes in the board near the body of the animal and fastened on the other side of the board. Ultrasound examinations were performed with a Toshiba Nemio SSA-550A apparatus with a 6-11 Hz linear probe. Pregnant females were brought into supination with the board on which they were fastened to. The orientation of fetus was determined by tracking along the longitudinal and transversal axes of the fetus with the ultrasound probe. The position of the fetus was determined on the basis of the positions of its head, spine, heart, forelimbs and hind legs (Image 1). Then the board was rotated until the fetus was brought into a head-down position relative to gravity and any changes in fetal presentation were observed for 5 minutes. The same subjects were used as controls. After a 2-minute pause, the examination was repeated with the fetus in the head-up position. Statistical analysis Statistical tests were completed using the SPSS software (version 21, 2012, IBM, Armonk, NY, USA). Results with a p - value of p<0.05 was accepted to be statistically significant. Results Spontaneous changes of presentation Table 1 - summarizes data related to fetal presentation and situs at the beginning of ultrasound examination, with pregnant females in supination. A transversal lie with the head on the right side was the most frequent situs. At the last examination before delivery there was one anterior and one posterior presentation of the Guinea pig fetus does not change its presentation during second half of gestation 135 Table 1: Fetal presentation and situs at the beginning of ultrasound examination, with pregnant females in supination Days of gestation Longitudinal lie No (%) Transverse lie No (%) Total No (%) AP PP HL HR SP USDP 26-30 5 (23.80%) 7 (33.33%) 3 (14.28%) 6 (28.56%) 0 0 21 31-35 4 (17.39%) 6 (26.08%) 3 (13.04%) 8 (34.74%) 2 (8.69%) 0 23 36-40 6 (28.57%) 5 (23.80%) 4 (19.04%) 6 (28.57%) 0 0 21 41-45 5 (31.25%) 4 (25%) 1 (6.25%) 6 (37.50%) 0 0 16 46-50 4 (23.52%) 4 (23.52%) 3 (17.64%) 6 (35.29%) 0 0 17 51-55 3 (16.66%) 2 (11.11%) 5 (27.77%) 8 (44.44%) 0 0 18 56-60 5 (26.31%) 3 (15.78%) 2 (10.52%) 9 (47.36%) 0 0 19 > 61 2 (10.52%) 3(15.78%) 4 (21.04%) 10 (52.63%) 0 0 19 fetus, as well as six transverse lies; in 4 cases with the head on the right side and in two cases with the head on the left side. In the period from the 26th to the 30th day of gestation a total of seven changes in presentation and situs (in five fetuses) were observed, that indicate turning around the longitudinal axis of the body for 180°. From the 31st day of gestation none of the fetuses in our sample changed their body orientation by 180°. They oscillate around one position for 45° during whole second period of gestation. This means that no fetus made a spontaneous turn around its longitudinal axis. The chi-squared test showed that difference in changes of presentation and situs before vs after 30th day of gestation had a statistical significance of x2 = 25.16 p <0.05. The strength of these association is very strong (phi = - 0.77, Cramer's V = 0.77, p < 0.001). Frequency of ultrasound examinations before and after 30th day of gestation was not statistically significant (Fisher exact chi square p = 0.46, p > 0.05). Oscillations around one position by 45° were present in all experimental animals during the whole examined period. There is no 136 D. Petrovic, A. Kopitovic, I. Pericin-Starcevic, M. Vujcic, N. Dragic, O. Gouni, A. Topalidou, S. Sekulic statistically significant difference in frequency of oscillations around one position for 45° between each examined period of pregnancy (Fisher exact chi square p = 0.46, p > 0.05). Provoked changes in presentation Fetuses were brought into a head-down position for a total of 154 times and in none of these attempts did a fetus through 180° degrees during the five minutes of observation. The fetuses did not change their presentation. The same results were obtained when fetuses were positioned in a head-up position after a 2-minute pause. Discussion The results of this study indicate that there are two periods of gestation in the guinea pig with regard to its situs and presentation. After the occurrence of the first movements on gestation days 25 and 26, the fetus is capable of passive or active turning around its longitudinal axis for 180° degrees only for a few days. After that, throughout the second half of gestation, its intrauterine position is fixed and it is no longer capable of turning around its longer axis. This finding is contrary to other mammalian species in which postural development occurs prenatally. The incidence of different presentations and lies during gestations with one fetus in human species (6,7) and herd precocial mammals (810) indicates the existence of three stages. The beginning of the first stage is characterized by equal proportions of longitudinal and transverse lies with equal proportions of breech and cephalic presentations within the longitudinal lie. During this stage, there is an increasing incidence of longitudinal lie with a proportional decrease in transverse lie. In the second stage, which occurs in the second half of gestation, a transverse lie is almost completely absent, whereas in the longitudinal lie there is an increasing incidence of anterior (cephalic) presentation and a proportional decrease in posterior (breech) presentation. By the end of this stage, around 95% of fetuses are in a longitudinal lie with cephalic presentation. In the third stage, during the last weeks of gestation, there is a further mild increase in the incidence of longitudinal lie with cephalic presentation (2,11). Published data show that in humans and herd mammals there is spontaneous turning around the longer axis in the second half of gestation (2,9). Studies with exteriorization of guinea pig fetuses have shown that turning around the shorter axis occurs when gestation is terminated around the 60th day. Taking an upright position and maintaining balance occurs around the 63rd day of gestation (12). In a study dealing with intrauterine induction of righting reflex from supination to pronation, turning to one side was present already around the 40th day of gestation, and it was the most frequent after the 60th day of gestation (4). Despite the presence of postural reactions in the guinea pig fetus in the second half of gestation and its ability to turn around the shorter body axis, turning around the longer body axis could not be induced by positioning the fetus in an upside-down position. In a sheep fetus, in the period of an exclusive increase in the incidence of anterior presentation, it is possible to induce the fetus to turn around its longer axis by positioning it in a head-down position (2). Therefore, it is likely that the turning around the longitudinal body axis in the guinea pig fetus is not possible due to physical characteristics i.e. lack of space. The guinea pig fetus is therefore not a suitable and adequate experimental model for confirmation of the concept that presentation of the human and herd mammals fetuses is influenced by their intrauterine postural development. A limitation of the present study is that it was not able to register changes in fetal position greater than 180° degrees. A 270° degree turn would have been registered as a 90° degree turn. The absence of erratic changes in fetal position indicates that this problem does not affect the results of the study. Oscillations of 45° from the basic position are probably caused by changes in the position of the internal organs. In order to confirm or discard the hypothesis on the influence of postural development on presentation of the fetus, it is necessary to advance the experimental model of the sheep fetus. A problem with the sheep fetus as an experimental model is that the fetus assumes a transverse lie when the pregnant female is positioned into a sitting position. This manipulation with the pregnant female is performed in order to provoke a fetus in anterior presentation to assume a head-down position and turn for 180° degrees. In order to overcome this problem, investigators used external pressure with their palms on the Guinea pig fetus does not change its presentation during second half of gestation 137 abdominal walls of pregnant females (2). However, this is an inappropriate method because of external, physical stimulation of fetal movements. It is necessary to surgically fix the horn of the uterus (hornopexia) to accomplish stable longitudinal situs of the fetus prior to studying its reaction to changes of gravity vector in the intrauterine environment. Acknowledgments This article is based upon work from COST Action IS1405 BIRTH: Building Intrapartum Research Through Health - an interdisciplinary whole system approach to understanding and contextualising physiological labour and birth. References 1. Sekulic SR. Possible explanation of the cephalic and noncephalic presentation during pregnancy: a theoretical approach. Med Hypotheses 2000; 55: 429-4. 2. Sekulic SR, Mikov A, Petrovic D. Probability for breech presentation and its significance. J Mater Fetal Neonatal Med 2010; 23: 1160-4. 3. Kalbe P, Schultz J. Neonatological aspects of the anterior presentation of the calf. Tierarztl Prax K 2000; 28: 9-11. 4. Sekulic S, Naumovic N, Lukac D, et al. Ultrasound assessment of the effect of fetal position on the supine to prone righting reflex in guinea pig fetus. Period Biol 2010; 112: 97-104. 5. Sekulic S, Kekovic G, Filipovic D, et al. The progressive increase of the longest episode of spontaneous movements in the guinea pig fetus within the first eleven days after the appearance of its first movements. Arc Biol Sci 2013; 65: 1459-62. 6. Boos R, Hendrik HJ. Schmidt W. Behavior of fetal position in the 2d half of pregnancy in labor with breech and vertex presentations Geburtshilfe Frauenheilkd 1987; 47: 341-45. 7. Miller EC, Kouam L. Frequency of breech presentation during pregnancy and on full term. Zentral Gynekol 1981; 103: 105-9. 8. Reimers TJ, Dziuk PJ, Bahr J, et al. Tran-suterine embryonal migration in sheep, antero-posterior orientation of pig and sheep fetuses and presentation of piglets at birth. J Anim Sci 1973; 37: 1212-7. 9. Scanlon PF. Orientation of cattle fetuses in utero in relation to stage of pregnancy. J Dairy Sci 1975; 58: 571-3. 10. Scanlon PF. Orientation of sheep fetuses in-utero in relation to stage of pregnancy. J Anim Sci 1976; 42: 1217-9. 11. Sekulic S, Bozic A, Zarkov M, et al. Changes in the anterior presentation in sheep fetuses due to their ventro-sacral position in the second half of gestation. Philipp J Vet Med 2012; 49: 51-6. 12. Avery GT. Responses of foetal guinea pigs prematurely delivered. Genet Psychol Monogr 1928; 3: 245-331. 138 D. Petrovic, A. Kopitovic, I. Pericin-Starcevic, M. Vujcic, N. Dragic, O. Gouni, A. Topalidou, S. Sekulic PLODOVI MORSKIH PRAŠIČKOV NE SPREMENIJO LEGE V DRUGI POLOVICI BREJOSTI D. Petrovic, A. Kopitovic, I. Pericin-Starcevic, M. Vujcic, N. Dragic, O. Gouni, A. Topalidou, S. Sekulic Povzetek: Namen prispevka je: 1. raziskati spontane spremembe v legi plodu v drugi polovici brejosti pri plodovih morskih prašičkov; 2. opazovati vzpodbujene spremembe lege plodov v položaj od zgoraj-navzdol in v »položaju sedenja«.V raziskavo je bilo vključenih osem plodov iz ene brejosti. Z ultrazvočnimi pregledi (aparat Toshiba Nemio SSA-550A) smo pričeli 26. dan brejosti. Vsak plod je bil pregledan 2-3-krat v 5-dnevnih razmikih do konca brejosti. Najprej je bila ugotovljena spontana orientacija plodu z določanjem njegove vzdolžne in prečne osi z ultrazvočno sondo. Potem je bil plod za 5 minut obrnjen v položaj z glavo navzdol, sorazmerno s težo. Pregled plodu je bil ponovljen v položaju z glavo navzgor. V obdobju od 26. do 30. dneva brejosti smo v 7 od 21 opazovanj opazili spontane spremembe lege plodu. Od 31. dneva brejosti dalje pri vseh 133 opazovanjih ni bilo opaziti spontanih sprememb lege plodu. Analiza Hi-kvadrat je pokazala, da je razlika v spontanih spremembah lege plodu pred in po 30. dnevu brejosti statistično značilna (x2 = 25,16 p <0,05). Pogostnost ultrazvočnih preiskav pred in po 30. dnevu brejosti ni bila statistično značilna (Fisherjev natančni Hi-kvadrat test p=0,46). Plodovi so bili postavljeni v položaj z glavo navzdol in navzgor 154-krat. V nobenem od teh poskusov ni prišlo do spontane spremembe lege plodu med petminutnim opazovanjem. Ti rezultati torej kažejo, da plodovi morskih prašičkov ne spremenijo lege spontano ali po provokaciji po 30. dnevu brejosti. Ključne besede: sprememba lege plodu; brejost; morski prašiček; ultrazvok SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Slov Vet Res 2017; 54 (3) Original Research Articles Paller T, Hostnik P, Pogačnik M, Toplak I. The prevalence of ten pathogens detected by a real-time PCR method in nasal swab samples collected from live cattle with respiratory disease.....................................................................101 Yeh JY, Hwang JM, Kim JG. Detection of Lawsonia intracellularisDNA in ileal tissues of dead wild birds in the Republic of Korea.............................................................................................................109 Lukanc B, Butinar J, Nemec Svete A, Prošek M, Seliškar A. The influence of isoflurane anaesthesia on intestinal permeability in healthy dogs........................................................................................................117 Ho CNQ, Hoang SN, Nguyen TTP, Doan CC, Nguyen MTP, Le TH, Nguyen HTT, Le LT. The ORF5 variation of Vietnamese porcine reproductive and respiratory syndrome virus strains...................................................................125 Petrovic D, Kopitovic A, Pericin-Starcevic I, Vujcic M, Dragic N, Gouni O, Topalidou A, Sekulic S. Guinea pig fetus does not change its presentation during second half of gestation........................................................................133