Pig OF has been regarded for some years as one of the most appropriate samples for pathogen detection [3, 20, 21]. Major research in this field started towards 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 provided some insight into OF collection, pre-diagnostic procedures and results regarding different pathogens; results were mostly promising; nevertheless, unanswered questions regarding methodology remain, including incomplete standardised procedures in terms of pre-diagnosis and the variability of its results when compared to those of other individual or group samples, e.g. serum, nose swabs, post-mortem isolation from organs, and group faeces samples [23, 24].
In our study, group OF samples and group faecal samples were compared with individual serum samples from different pig categories from six Slovenian farms, each with different numbers of disease history data. PRRSV, PCV2 and HEV were chosen since these viruses represent quite common pathogens in the pig industry and are either economically important for pig health, or constitute a possible food safety threat, such as HEV. All diseases have their own pathophysiological characteristics and, therefore, are 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 regarding the diseases considered. Group sampling in the case of OF is seen to be more effective than the use of group faecal samples, probably due to the presence of greater 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 the results favoured OF over other samples in terms of PRRS and HEV detection. Compared with individual serum samples, OF showed complementary results. Variation was noted 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  (a maximum up to 20 pigs in a group pen). OF was sampled pursuant to all pigs being observed chewing the ropes for collecting OF. Faeces from the rectum of the animals in the pen were added directly to containers to ensure that samples were representative. Even though sensitivity fell by an estimated 27‒100 % when five sera samples were pooled, as when compared to individual samples, our results are of great value, especially in terms of studies of prevalence .
When considering PRRSV, PCV2 and HEV, several studies have investigated simultaneous co-infection with two viruses. This took place on Farm 6, where PCV2 and HEV were found in weaning pigs of all ages. Salines et al. described the same virus combination in 2019, finding a statistically significant concentration of HEV in faeces during infection. Seroconversion for anti-HEV antibodies took longer and the transmission rate for HEV was approximately three times higher in co-infected pigs . Another study by the same author states that the presence of immunosuppressive virus increases the viral concentration of HEV in the liver at slaughter . Accordingly, the fact that the degree of infection by HEV on Farm 6 was high, could be due to PCV2 coinfection which led to modification of immune system caused by the immunosuppressive effect of PCV2. This was harder to assess because our primary focus was co-infection and our environment was uncontrolled. But looking from a viewpoint opposite to that of Salines, PCV2 was detected in weaner’s sera and, generally, in more categories than on the other two farms. The farm owner stated that he experienced trouble with a higher mortality rate, poor average daily gain and wasting during this stage. Yang et al. in 2015 provided evidence for fatalities in weaned pigs co-infected with PCV2 and HEV . HEV is usually depicted as benign and not as a swine pathogen, but it may be part of a multifactor clinical outbreak catalyst, thus negatively affecting pork quality.
PRRSV was only found on Farm 5, a farm that had previously experienced disease outbreak. Even though this farm acclimates its gilts before transferring them to the breeding herd, transmission to offspring still occurs. Youngest weaners are disease-free due to immunity provided by colostral; the oldest 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 also evidenced in study by Christianson et al. (1993) where PRRSV only appears in faeces intermittently . Fatteners appear to eliminate virus from sera, presumably due to the appearance of antibodies in the sera; even so, the virus appears to persist in OF for longer periods of time . This means that OF can be a sample of choice for diagnostic use, especially for longer periods following infection, that is, during the weaning-fattening period and for replacement of gilts before entering the breeding herd, when the virus is eliminated from the bloodstream, but present in other tissue.
Although PCV2 is considered to be ubiquitous, its presence was only detected on half 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, none of the younger weaners were viremic. The virus was found in the OF and faeces of 9 w/o weaners. However, the situation in the case of 11 w/o weaners was different, since PCV2 was not found in OF and faeces. This virus was present in 80% (11 w/o weaners) and 30% (fatteners) of pig sera. It appears that the immunity acquired by means of preventive programs varies 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 appears as if their prevention programme does not completely protect pigs from the viremic phase; viral DNA could be detected later in OF and faeces of 9 w/o and in sera of 11 w/o weaners and of fattening pigs. Feng et al. (2016) suggest that 3 w/o piglets should also be vaccinated also, since it extends protection against the pathogen until pigs are up to 25 weeks old, regardless of the presence of maternally-derived antibody , and that protocol of vaccination against PCV2 has since been applied on many Slovenian farms as a preventive measure.
PCV2 was also detected on Farm 4 despite the fact that a local veterinarian stated that this virus had never before been detected in any samples taken from pigs in this farm. These pigs were, otherwise, not vaccinated. PCV2 was only detected in the sera of 60 % of 9 w/o weaners, and not in OF. This situation was reversed for 11 w/o weaners, which could mean that pigs become infected by PCV2 at around 9 w/o and eliminate the virus from their bloodstreams in a short period of time. This was in accordance with a study by Grau-Roma et al. (2007). Pigs are protected by maternal immunity until week 8 and then by blood viral load peaks when 10 w/o; from then on, the virus is eliminated from the bloodstream and then from the organism . Nonetheless, OF appears to be a good diagnostic tool for determining farm prevalence. Research by Nielsen et al. stated that it is even better than serum .
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 in sera. Our statistical analysis indicates that OF is more effective than faeces are for detecting viruses (p=0.001). The 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 was, in addition to being PCV2-positive, the only HEV-positive farm, despite the latter virus being described as ubiquitous . HEV DNA was shown, using RT-PCR, to be present in OF and faeces of the three youngest categories of weaners. The results show that disease is absent in older categories, in accordance with previous reports: pigs become 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 at week 9, which indicates that HEV only spreads sporadically into the bloodstream. Although the virus is supposed to replicate in the lower gastrointestinal tract , OF concentration is apparently 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 the 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 mentioned earlier, statistical analysis supports the usefulness of pig OF for detecting PCV2 DNA, but neither proved nor disproved correlation in cases of HEV RNA detection in OF, faeces or serum due to the low number of positive samples in our study.