SEROPREVALENCE OF PORCINE RESPIRATORY CORONAVIRUS AND TRANSMISSIBLE GASTROENTERITIS VIRUS INFECTIONS ON COMMERCIAL PIG FARMS IN CENTRAL SERBIA

Porcine respiratory coronavirus is an enzootic, viral, respiratory disease of pigs, which manifests with mild clinical signs, but it takes part in the etiopathogenesis of the porcine respiratory disease complex. Th e virus was fi rst discovered in Belgium in 1984 as a deletion mutant of the transmissible gastroenteritis virus. Th e two viruses are strongly antigenically related which is why they cross-react in serological tests. In this study, we tested 276 serum samples from diff erent categories of pigs using ELISA test, which allows diff erentiation between the porcine respiratory coronavirus infection and transmissible gastroenteritis. Th e seroconversion for coronavirus infection was determined in 80.4% of tested samples. Out of 222 positive samples, 219 samples (98.6%) were positive for porcine respiratory coronavirus antibodies, while 3 (1.01%) samples were positive for transmissible gastroenteritis virus antibodies. Depending on the production category, 97.7% of piglets, 83% of sows, and 35% of gilts tested positive for porcine respiratory coronavirus antibodies. In total, 2.3% of piglets tested positive for transmissible gastroenteritis virus antibodies. Taking into account the characteristics of the ELISA test, its sensitivity and specifi city, this result can be considered a false positive, because of a cross-reaction between the porcine respiratory coronavirus antibodies and the transmissible gastroenteritis virus. Specifi c antibodies in other swine production categories against the transmissible gastroenteritis virus were not determined.


INTRODUCTION
Porcine respiratory coronavirus (PRCV) causes an enzootic, mild, respiratory disease in pigs. Th e fi rst report of the virus was recorded aft er a prevalence study for transmissible gastroenteritis virus (TGEV) which revealed that over 60% of tested pigs had neutralising antibodies against TGEV without being vaccinated or developing the symptoms (Pensaert et al., 1986). It was reported that the virus that caused the infection was a mutant of TGEV, with a deletion (170-190 kDa) in the gene for spike (S) protein (Lin et al., 2015). Both PRCV and TGEV belong to the Coronaviridae family, and the Alphacoronavirus genus (International Committee on the Taxonomy of Viruses, ICTV.). Soon after the discovery of PRCV, it spread throughout the world suppressing TGEV in many regions (Whittaker, 2017). Th e TGEV causes gastrointestinal disease with a high affi nity for the intestinal tract, and a high mortality rate among piglets. Th e deletion in the S gene resulted in a change in the spike protein of PRCV, thus inhibiting binding to the sialic acid and entering enterocytes Th ere have been reports of diffi culties in diff erentiating TGEV antibodies from PRCV antibodies, caused by the similarity in the antibody response to the viruses (Valkó et al., 2019). To circumvent this, various ELISA tests were created that would allow a quick assessment of serological status of the herd.
Th is study aimed to determine the seroprevalence of PRCV and TGEV infection in domestic swine on commercial farms in Central Serbia by testing diff erent production categories of pigs for the presence of antibodies against PRCV and TGEV.

MATERIAL AND METHODS
Serum samples used in this study originated from Central Serbia, from commercial units, and were kept in the serum bank at the Serbian Institute of Veterinary Science. Th e total number of samples tested was 276. Th e samples were tested by blocking commercial ELISA test (INgezim Corona Diferencial 2.0, Ingenasa, Madrid, Spain), according to the manufacturer's instructions, and the optical density (OD) was measured at 450 nm with an ELISA reader (Multiscan, LabSystems). Based on the recommendations of the cut-off values by the manufacturer, the samples were considered positive or negative to PRCV or TGEV. Th e diagnostic sensitivity and specifi city, according to the manufacturer, are 94% and 98.2% respectively (INgezim Corona Diferencial 2.0, 2022).

RESULTS AND DISCUSSION
Out of 276 tested samples, 222 (80.4%) samples were positive for coronavirus antibodies. For PRCV antibodies, 219 samples tested positive, which makes up 79.3% of the total samples and 98.6% of all positive samples. For TGEV antibodies, 3 samples were positive, which makes up 1.09% of the overall sample or 1.4% of all positive samples. Depending on the production category, 97.7% of piglets, 83% of sows, and 35% of gilts tested positive for porcine respiratory coronavirus antibodies (Table 1.). Porcine respiratory coronavirus causes a subclinical disease in pigs, which in some cases results in fever, sneezing or mild coughing, and combined with other pathogens such as porcine respiratory and reproductive syndrome (PRRS) and swine infl uenza virus (SIV) can be a part of a post-weaning porcine respiratory disease complex (PRDC) that causes a signifi cant economic loss in swine industry (Brockmeier et al., 2002). However, strains 135 and 137 of PRCV are capable of producing similar pulmonary lesions to that of the swine infl uenza virus (Keep et al., 2022). Furthermore, a co-infection of PRCV and PRRS induces a disease with severe respiratory signs (Jung et al., 2009). Ever since the emergence of PRCV, the incidence of TGEV has decreased significantly through partial cross-protection by anti-PRCV antibodies. Nonetheless, there still might be a low TGEV circulation without a clinical manifestation usually explained by a high enough titre of antibodies against PRCV (Kim et al., 2000). Namely, only continuous reinfection allows for a high enough titre of antibodies for adequate protection against TGEV (Kim et al., 2000). Low seroprevalence of PRCV infection was recorded in wild boars in the region: 0.7% in Croatia (Roic et al., 2012), and 3% in Slovenia (Vengust et al., 2006). However, PRCV infection was not detected in wild boars in Serbia (Milicevic et al., 2016). On the contrary, a high seroprevalence of PRCV infection had been recorded in domestic swine in Slovenia at 65% (Vengust et al., 2006), while, in a study by Lőrincz et al. (2014) over 70% of tested gilts and 100% of tested sows seroconverted against PRCV in Hungary. Th ese fi ndings are in accordance with the results in this study where the overall seropositivity to PRCV infection was 79.3%, suggesting that there isn't a circulation of PRCV between the population of domestic swine and wild boars in Central Serbia. Th e reports regarding seroprevalence of TGE in neighbouring countries have been similar. Seroprevalence of TGE was 0.4% in Croatia (Roic et al., 2012;Brnić et al., 2020), and in wild boars in Slovenia TGE was not detected (Vengust et al., 2006). Th e results were similar in Serbia where TGEV was not detected either (Milicevic et al., 2010). In domestic swine, there was a single outbreak of TGE in the previous decade in Hungary (Lőrincz et al., 2014). In Serbia, there were reports of porcine epidemic diarrhoea virus (Prodanov-Radulović et al., 2017), but there were no reported outbreaks of TGE in domestic swine. In this study, 2.3% of piglets tested positive for TGE antibodies, which is similar to a study by Valkó et al., (2019) where a single serum positive for TGE antibodies was detected. Th e high-level serological cross-reactivity represents a hindrance in diagnosis since it allows for a false-positive result, especially at the individual pig level, which lowers the accuracy of blocking ELISA tests (Magtoto et al., 2019). In this study, coronavirus antibodies were detected in all tested production categories, with the highest seroprevalence in piglets, where all of the piglets tested positive for coronavirus antibodies, and 97.7% for antibodies against PRCV. Taking into account the characteristics of the ELISA test, its sensitivity and specifi city, it can be concluded that 3 piglets that tested positive for TGE antibodies were false-positive result, probably because of the crossreaction between the PRCV antibodies and the TGEV. Following this, none of the tested sows or gilts had antibodies against TGEV, which would be expected if there were positive piglets. On the contrary, a high percentage of PRCV seropositive piglets is in accordance with the high percentage of seropositive sows, probably caused by their continuous reinfection. Th e other 65% of gilts were negative for antibodies against TGEV and PRCV coronavirus-antibodies, which could be connected to the waning of antibodies under the detection level of the ELISA kit. Th is could represent a risk since there is a report from 2014 by Lőrincz et al. (2014), which describes a re-emergence event of TGEV in piglets from primiparous gilts which had low levels of PRCV antibodies.

CONCLUSION
Th e seroprevalence of PRCV infection in domestic swine on commercial farms in Central Serbia is high (79.3%). Antibodies against PRCV were detected in all tested production categories of pigs, with the highest seroprevalence being in piglets (97.7%). Th e 2.3% of piglets tested positive for TGEV antibodies. Antibodies against TGEV were not found in either sows or gilts. Considering the characteristics of the ELISA test, coupled with a high degree of cross-reactivity between PRCV antibodies and TGEV and the lack of antibodies against TGEV in other tested production categories, this result can be considered a false positive.