TRICHINELLOSIS IN SERBIA AND POSSIBILITIES TO IMPROVE CONTROL MEASURES

possibility Abstract Background. Trichinellosis is zoonotic disease caused by parasites of the genus Trichinella , which have a cosmopolitan distribution. In the Balkan region, including Serbia, trichinellosis is endemic. Although trichinellosis shows a decreasing incidence in Serbia, this disease remains a serious problem for human health and animal husbandry. Scope and Approach. To prevent and control Trichinella spp. infections in domestic and sylvatic animals, risk analysis for these zoonotic parasites should be conducted. In Serbia, a limited number of Trichinella have been identified to the species level so far. An institutional, multi-sectoral surveillance system, supported by an adequate legal framework for the detection, surveillance, prevention, control and reporting of this infection in animals and humans, harmonised with the EU legislation, is a priority. Key Findings and Conclusions. One of the main goals of the Serbian veterinary and public health services should be progressive improvement of animal rearing practices, food safety, and hunters’ and consumers’ education through the “One-health” approach.

Official diagnostic methods to detect Trichinella larvae in muscles of domestic pigs intended for human consumption are the trichinoscopy method and artificial digestion of muscle tissues. For detection in meat of wild boars and horses, artificial digestion is the mandatory diagnostic method (Official Gazette of the Republic of Serbia (RS) for the current year). At present, Serbian legislation includes the Law on Veterinary Matters (Official Gazette RS 91/2005, 30/2010and 93/2012 and regulations based on the Food Safety Law (Official Gazette RS 41/2009). As a part of the twinning project Enhancing the capacities of Serbian authorities in zoonoses and food borne disease control Serbia (Twinning reference SR 13 IB AG 01), the Veterinary Directorate, as the national competent authority, in partnership with the Ministry of Health, and with the support of Veterinary Institutes are making efforts in improving animal and human health, including further alignment of the Serbian legislation with the European Union legislation.
In Serbia, T. spiralis is the most frequent Trichinella species detected in domestic pigs and wild boars, whereas T. britovi was more frequently documented in wild carnivores (Zivojinovic 2013), and was detected in only one domestic pig and one wild boar in Zlatibor and Pirot districts (Cvetkovic et al., 2011). Mixed infections with T. spiralis and T. britovi were documented in three carnivores (Zivojinovic, 2013). In Serbia, Trichinella larvae have never been documented in slaughtered horses; however, T. spiralis larvae were detected in horses imported from Serbia to France and Italy (Pozio et al, 2001), where they caused human outbreaks of trichinellosis (Pozio, 2015). T. spiralis and T. britovi are the two main species circulating in Europe  and the Balkan countries bordering Serbia (Blaga et al., 2007). T. pseudospiralis has been documented in domestic pigs in Croatia and Bosnia and Herzegovina (Santrac et al., 2015;Beck et al., 2009), but there is still no available data for Serbia.
Trichinellosis is one of the most important zoonosis in Braničevo District. The reemergence of trichinellosis was officially recognised in 1999, and in 2003, the local government declared six parishes as endemic Trichinella areas and established control measures (Zivojinovic et al., 2009). In the course of an epidemiological survey on Trichinella spp. circulating in Braničevo District (Zivojinovic et al. 2010), muscle samples from domestic and wild animals were collected and tested by the artificial digestion method to collect Trichinella larvae according to the Commission Regulation (EC) No. 2075/2005 and the Manual of Standards for diagnostic tests and vaccines for terrestrial animals of the World Health Organization for Animal Health (European Commission, 2005;OIE, 2013). After digestion, larvae were washed 3-5 times in cold water, counted in triplicate and fixed in absolute ethyl alcohol for molecular identification at the species level by multiplex PCR at the International Trichinella Reference Center (ITRC), Rome, Italy, according to a validated protocol (http://www. iss.it/crlp). Geographical data of the Trichinella isolates from Braničevo District were collected using GARMIN Map Source (Garmin, Taiwan), and Trichinella isolates were mapped (Zivojinovic et al. 2010). These data allowed the identification of the endemic foci in the district.

Risk factors and control measures
The most important risk factors for the establishment of the Trichinella domestic cycle are: (a) the intentional feeding of food waste containing pork scraps or intentional or unintentional exposure to carcasses of dead swine or wildlife; these risks are usually encountered in free-range pasturing; (b) allowing pigs to scavenge on garbage dumps; (c) using wild game carcasses or scraps from hunting as animal feed; (d) feeding horses with pork scraps or with carcasses of fur animals; (e) using carcasses of slaughtered for animals as feed for other fur animals present on the farm (Gamble et al., 2000;Pozio & Murrell, 2006;Pozio, 2014).
In Serbia, the finding of T. spiralis in domestic pigs and in hunted wild boars near human settlements could be explained by the behaviour of raising pigs near rivers, small waterways and ponds where they can come into contact with wildlife . Additionally, free-ranging pigs and stray dogs are not prevented from scavenging on garbage dumps (Zivojinovic et al., 2010). The husbandry conditions of 90% of backyard farms with Trichinella infection are very poor (Zivojinovic et al., 2010). In Serbia, the practices of feeding pigs with food waste of home or restaurant origin that can contain raw pork, scavenging of pigs in garbage dumps and the improper disposing of pig carcasses in the field are on-going . These inappropriate rearing conditions of domestic pigs favour wild animals feeding on pork scraps and offal and on pig carcasses.
Pig meat samples can be examined by trichinoscopy and/or artificial digestion at licentiate veterinary services according to official regulations (Official Gazette RS for the current year). However, the low sensitivity of trichinoscopy as a diagnostic method and the possibility of missing non-encapsulated Trichinella (Forbes et al. 2003) contribute to less reliable data about prevalences of Trichinella spp. in Serbia. Through harmonisation of a strategic, institutional and legal framework for detection, surveillance, prevention, control and reporting of zoonoses, Serbia will be able to develop Trichinella control measures in compliance with the EU requirements. Further efforts should focus on harmonising with the EU requirements, improving hygienic conditions on farms, expanding systematic meat inspection of domestic pigs and wild boars and proceeding with risk communication on trichinellosis between veterinary services, farm owners and hunters.
In the case of a positive finding, information should be submitted to veterinary inspectors. According to the Serbian veterinary regulations, it is mandatory to report Trichinella findings, because of their zoonotic potential, to the national veterinary authority (Plavsic et al., 2009). All data are entered in the Animal Notification System (VetUP software application) and become available to all responsible services. Awareness amongst stakeholders and the general public of potentially zoonotic Trichinella infection (control activities, prevention, EU standards and requirements etc.) should be increased. The identification of Trichinella-infected animals allows identification of the foci of transmission, so veterinary services, the owners of pig farms and slaughterhouses and hunter's associations can be informed about the risk of transmission of these zoonotic agents. An awareness-raising campaign should be implemented in the whole country, particularly in risk areas. Improving biosecurity on animal farms, promoting prevention measures and implementing good farming practices will certainly improve animal health, human health and environmental protection in Serbia.
In the last 100 years in Europe, the increase in forests and fallow land, concomitant with a decrease in farms, has favoured the expansion of wild boar (Sus scrofa) populations and, consequently, increased Trichinella transmission among animals and humans. The common habits of hunters of leaving animal carcasses in the field after skinning or removing and discarding the entrails increases the probability of Trichinella transmission to new hosts (Pozio & Murrell, 2006). According to OIE data and the Institute of Public Health of Serbia (IPHS, 2018), 15 human Trichinella infections in Serbia were documented in 2017, showing a decrease of this zoonosis in the last ten years when the yearly incidence was between 46 and 190 infections. The decrease of human trichinellosis in Serbia suggests the efforts of veterinary and public health services are producing positive results.

CONCLUSION
As a part of the twinning project Enhancing the capacities of the Serbian authorities in zoonoses and food borne disease control Serbia (Twinning reference SR 13 IB AG 01) Serbia is making an effort to improve animal and human health. Only through harmonisation of a strategic, institutional and legal framework for detection, surveillance, prevention, control and reporting of zoonoses, Serbia will be able to be in compliance with EU requirements. An improvement of the professional capacity of the competent authorities to implement and enforce standards and regulations for the control of Trichinella infections in animals and humans is expected.

Authors contributions
MZ designed the paper, selected references for the presentation and wrote manuscript. ID assisted in data collection and analysis. ZK made substantial contribution to the conception and interpretation of data. SR made critical revise of the concept, gave substantial contribution to analysis and interpretation and was involved in drafting the manuscript and revising critically. TB assisted in data collection and provided legislative framework. BP made substantial contribution to the conception, acquisition and interpretation of data.