Pig OF has been regarded as one of the most appropriate samples for pathogen detection for some years now [3, 20, 21]. Major research in this field started toward the end of the previous decade with the goal of finding a way to make sample collection less stressful for pigs, and easier, faster and cheaper for those performing sampling [4, 22]. Previous studies provide some insight into OF collection, pre-diagnostic procedures and results regarding different pathogens; results are mostly promising; nevertheless, unanswered questions regarding methodology remain, including incomplete standardised procedures in terms of pre-diagnosis and its variable results when compared to other individual or group samples, e.g. serum, nose swabs, post-mortem isolation from organs, and group faeces samples [23, 24].
Our study compared group OF samples with group faecal samples and individual sera samples from different pig categories from six Slovenian farms, each with varying amounts of disease history data. PRRSV, PCV2 and HEV were chosen because these viruses represent quite common pathogens in terms of the pig industry and are either economically important for pig health, or represent a possible food safety threat, such as HEV. All diseases have their own pathophysiological characteristics and, therefore, detectable in a variety of infection stage samples. Even though OF samples were not taken from individual pigs, results show they are of great use for determining viral presence on farms in relation to our considered diseases and group sampling is seen to be more effective than group faecal samples collected from pig farms, probably due to the presence of higher amounts of inhibitory substances in faeces [23‒25]. Statistically significant correlation between OF and faeces samples was shown with more than 95% probability (p=0.001); in other cases, correlation was disputed by a Fisher’s test value of p < 0.05, although results favoured OF over other samples in terms of PRRS and HEV detection. In comparison to individual sera samples, OF showed complementary results, variation noticed in PCV2 DNA detection. OF samples were collected from a relatively small number of pigs compared to the study by Nielsen et. al in 2018 [20] (a maximum up to 20 pigs in a group pen); samples were collected pursuant to all pigs being observed chewing the ropes and faeces were added directly from the rectum of the animals in the pen to assure samples were representative. Even though sensitivity falls by an estimated 27‒100 % when five sera samples were pooled when compared to individual samples, our results are of great utility, especially in terms of prevalence studies [26].
When talking about PRRSV, PCV2 and HEV, several studies have investigated simultaneous co-infection with two viruses. This happened on Farm Six, where PCV2 and HEV was found in weaning pigs of all age. Salines et al. described the same virus combination in 2019, finding a statistically significant concentration of HEV in faeces during infection, and that seroconversion for anti-HEV antibodies takes longer and the transmission rate for HEV was approximately three times higher in co-infected pigs [27]. As HEV is an observed differential in this case, the findings could be a result of immune system modification caused by the immunosuppressive effect of PCV2. Another study by the same author states that immunosuppressive virus presence increases the viral concentration of HEV in the liver at slaughter [28]. The situation was harder to assess because our primary focus was co-infection and our environment was uncontrolled. But looking from a viewpoint opposite to Salines, PCV2 was detected in weaner sera and, generally, in more categories than on the other two farms. The farm owner stated that he did experience trouble with a higher mortality rate and poor average daily gain and wasting during this stage. Yang et al. evidenced fatalities in weaned pigs co-infected with PCV2 and HEV [29] in 2015. HEV, is usually depicted as benign and not as a swine pathogen, but it may be part of a multifactor clinical outbreak catalyst and negatively affect pork quality.
PRRSV was only found on Farm 5, a farm that had previously experienced disease outbreak. Even though Farm 5 acclimates its gilts before transferring them to the breeding herd, transmission to offspring still occurs: youngest weaners are disease-free pursuant to colostral immunity, the eldest get infected. Viral RNA was present in OF and sera tested positive for weaners aged 7, 9 and 11 weeks; PRRSV RNA was only found in the faeces of 11 w/o weaners, as evidenced in a previous study, whereby PRRSV only appears in faeces intermittently [30]. Fatteners seem to eliminate the virus from sera, presumably after the appearance of antibodies in sera; even so, the virus seems to persist in OF for longer periods of time [19], meaning OF can be a sample of choice for diagnostic use, especially for longer periods after infection, that is, during the weaning-fattening period and for replacement gilts before entering the breeding herd, when the virus is eliminated from the bloodstream, but present in other tissue.
Although PCV2 is considered ubiquitous, its presence was only detected on half of the farms concerned, of which only one used anti-PCV2 vaccination as part of their preventive program. On Farm 2, where pregnant sows were vaccinated against PCV2 with a commercially-sourced vaccine, the virus was found in the OF and faeces of 9 w/o weaners, but none of the younger weaners were viremic. However, the situation was different in relation to 9 and 11 w/o weaners, where PCV2 was not found in OF and faeces but was present in 80% and 30% of pig sera respectively. It seems that the immunity acquired by means of preventive programs wanes between 9 and 11 w/o and, as the virus is still present in the environment, weaners and fatteners become viremic. In the case of Farm 2, it looks as if their prevention programme does not protect pigs from the viremic phase entirely, even though 3 w/o suckling piglets are vaccinated; viral DNA could be later detected in the OF and faeces of 9 w/o, 11 w/o weaners and fattening pigs, as well as in the sera of the last two categories. A 2016 study by Feng et al. suggests that vaccination should also be facilitated for 3 w/o piglets, as it extends protection against the pathogen until pigs are up to 25-weeks old, regardless of maternally-derived antibody presence [31], as vaccination against PCV2 is now used as a preventive measure on Slovenian farms. PCV2 was detected on Farm 4; a local veterinarian stated that PCV2 had never been detected in any samples taken from it and that it did not vaccinate. The virus was only detected in the sera of 60 % of 9 w/o weaners on Farm 4, but not in OF; the situation reversed for 11 w/o weaners, which could mean that pigs get infected by the virus somewhere around 9 w/o and eliminate it from their bloodstreams in a short period of time, also confirmed in a study by Grau-Roma et al. Pigs are protected by maternal immunity until week 8 then by blood viral load peaks when 10 w/o; thereon, the virus is eliminated from the bloodstream and then from the organism [32]. Nonetheless, OF seems to be a good diagnostic tool for farm prevalence determination; research by Nielsen et al. stated that it is even more efficient than sera utilisation [20] in 2018. On Farm 6, PCV2 was detected in the youngest categories of weaners (5, 7 and 9 w/o): pigs on this farm had not been vaccinated. PCV2 was detected in both faeces and OF, and sera; our statistical analysis indicates that OF is more effective for detecting viruses than faeces (p=0.001); infection timeline and viremia onset are in accordance with previous studies: most pigs were infected at 4-11 w/o and repetitive viremia is present from day 7 to day 70 [32, 33].
Farm 6, also PCV2-positive, was the only HEV-positive farm, despite the virus being described as ubiquitous [34]. The three youngest categories of weaners were determined HEV-positive in terms of OF and faeces by means of RT-PCR. The results show that disease vines out in older categories, in accordance with previous reports: pigs get infected at around 2‒3 months of age and this persists in some excrement for 3‒7 weeks. If viremia appears, it is usually present for short periods of time, between one and two weeks [18, 34]; none of the weaners were viremic by week 5; one in ten were viremic at 7 weeks, and two in ten were viremic at week 9, which indicates that HEV only sporadically spreads into the bloodstream. Although the virus is supposed to replicate in the lower gastrointestinal tract [35], OF concentration is seemingly high enough for molecular detection. Compared to group faeces samples, OF is collected more easily and all pigs from the group chew on the ropes. Faeces for collection are not evenly distributed over pens’ floors: some excrement will be old, some will fall through floors’ slats. If faeces are obtained directly from the rectum of animals, the procedure can be time-consuming and stressful for pigs. As aforementioned, statistical analysis supports the usefulness of pig OF for detecting PCV2 DNA, but did not prove or disprove correlation in cases of HEV RNA detection in OF, faeces or serum due to the low number of positive samples in our study.