Longitudinal Study of Hepatitis E Virus Infection in Spanish Farrow‐to‐Finish
4.3.2 RT‐PCR detection
4.3.4 Histopathological results
4.3.4 Histopathological results
At slaughter age, none of the animals had significant macroscopic lesions in the liver. Slight periportal hepatic fibrosis (stage I of Rosell et al., 2000) was histollogically seen in nine pigs (15%), while only one animal (1.7%) presented mild periportal hepatitis (stage II of Rosell et al., 2000). Since only one pig that presented a slight periportal hepatic fibrosis was also RT‐
PCR positive in liver, no correlation between liver lesions and presence of HEV RNA in liver could be established.
4.4 Discussion
This study represents the first longitudinal survey on swine HEV infection dynamics conducted in different farms. Animals were followed from nursery to slaughter in order to in order to detect virus and antibodies during their productive life and its presence in the carcass. Other studies also have described serologic and excretion patterns of HEV infection between farms, but by means of a cross‐sectional studies (Takahashi et al., 2003; Nakai et al., 2006;
Fernandez‐Barredo et al., 2007; Leblanc et al., 2007; Di Bartolo et al., 2008).
Our data of HEV seroprevalence in Spanish swine herds agree with a previous study conducted by the same research group, where an average of 47.8% seropositive sows was found in 208 farms (Study I). Although most sow antibody titres were correlated with their piglets, some positive sows had negative piglets as well as some negative sows had positive piglets. These discrepancies might have been caused by an insufficient or inadequate colostrum intake or to a cross‐fostering practice during suckling stage.
Slight differences in antibody dynamics of HEV infection were found in the 6 studied farms.
Five of these farms showed evidence of HEV infection at 7 weeks of age, whereas in farm 2 pigs started to develop IgM antibodies some weeks later, at 13 weeks of age. Assuming that IgM presence is related with viraemia (de Deus et al., 2008a), pigs from farm 2 may have been in contact with the virus later than in the other 5 farms. In consequence, animals can be
seronegative at the slaughter age but being infected during the latest fattening period. In two studies conducted in Japan, similar differences in virus detection in faeces or IgG appearance were found (Takahashi et al., 2003; Nakai et al., 2006). A lower infection pressure in farm 2 and also in 4 may have reduced the probability of infection and consequently prolonged the period between exposure and infection (Bouwknegt et al., 2008a). Satou and Nishiura (2007) also described that a decline in the force of HEV infection in a farm would elevate the age at infection. These hypotheses could probably explain the observed difference in HEV serological curves. Even though Satou and Nishiura described that a lower force of infection could increase the number of infected pigs at the slaughter age, we have not observed any apparent differences in this percentage among farms. Further research is necessary to elucidate if different infection dynamics may affect the proportion of infected pigs at slaughter age.
How the virus persists within a herd while being able to infect each production batch is still not clarified. Some studies suggest that breeding sows could play a role as HEV reservoirs being able to transmit the virus to suckling piglets (Fernandez‐Barredo et al., 2007; Di Bartolo et al., 2008). In the present study only 2 pre‐farrowing sows (17%) and 5 breeding sows were shedding virus in faeces (16%), similar percentages as showed in other studies (Fernandez‐
Barredo et al., 2006 and 2007). How these breeding sows could be infected despite having IgG HEV‐antibodies is still uncertain. Physiological changes during pregnancy (high level of steroid hormones) or farrowing (stress) might induce a reactivation of HEV replication, as suggested by Fernandez‐Barredo et al. (2006) and Navaneethan et al. (2008). Stress during farrowing may explain why all sows that were shedding virus in faeces during suckling period were not excreting virus during pre‐farrowing stage. Even though transmission from sows to piglets has not been excluded, none of the studied piglets born from positive sows became infected at 3 weeks of age. Despite of this, an efficient disinfection of pens between batches should be recommended to avoid virus persistence.
Viraemia and virus excretion in experimentally infected pigs are expected to last for 1‐2 weeks and 2‐3 weeks, respectively (Halbur et al., 2001); virus in liver can be detected until 4 weeks post infection (Bouwknegt et al., 2009). In the current study, most HEV positive animals at slaughterhouse had not had virus detection before. Although some pigs had already developed an antibody response at the abattoir, none of them had virus in serum or in faeces,
suggesting that they had probably finished the acute phase of HEV infection. Interestingly, three animals that had virus in serum or faeces at 13 weeks of age also showed virus in liver or in bile 12 weeks after their first detection. What is more, sequences from the same animal at different sampling ages were not identical. We can speculate that these sequences could have had enough mutations to change into other HEV strain, but the calculated mutation rate in these cases (10‐1) would be higher than the calculated for positive‐sense single‐stranded RNA virus like HEV (10‐3) (Duffy et al., 2008). Therefore, it is easier to assume that these pigs could be infected with two different strains of HEV over their life. It could happen that a higher amplification rate of each isolate in liver at different time points results in a different strain detection. The presence of virus in liver until 3 months after its detection in faeces may be due to a possible persistence of virus in target organs. A short protective immunity could also facilitate new HEV infections during its productive life, as described by Chandra et al. (2008).
In both cases, animals infected during early life could still contain HEV at slaughter age, representing a risk for public health.
Phylogenetic analysis of 19 positive samples, representing the different farms tested, indicated that multiple HEV strains can be present in a same farm (Huang et al., 2002; Nakai et al., 2006; Di Bartolo et al., 2008). Huang et al. (2002) demonstrated that in 37 swine farms from the United States, HEV isolates from the same geographic region showed higher percentages of identities than those from different geographic region. Although the distance between the 6 farms in our study is quite large (25‐30 Km), they are located in an area with high density of pig production and consequently with substantial concentration of slurry in the environment. Water sources may have been contaminated with swine faeces that have facilitated HEV transmission between farms. It is worthy to note that pigs that have isolates of group 3 could be infected for two different HEV strains at the same time, since they displayed two different nucleotides at the same variable regions than group 2. However, this must be confirmed cloning this sequences and isolating the two supposed different strains separately.
In the current study, a total of 12% of pigs had presence of RNA in liver or in bile at the slaughter time. Whether this HEV RNA had an extrinsically (cross‐contamination) or intrinsically (natural infection) source or whether it continuous to be infectious or not, remains to be examined. Nonetheless, the prevalence is slightly higher than found in other
European countries (Banks et al., 2007; Bouwknegt et al., 2007), but similar to the proportion showed in United States (Feagins et al., 2007). A nutritional survey performed in Catalonia during 2002‐2003 revealed that viscera consumption was very low (0‐2.9 grams/person/day (g/p/d)) compared with meat and fish consumption (57.6‐86.6 g/p/d and 56.1‐84.3 g/p/d, respectively) (Anonymous, 2005). These data suggested that the risk for infection by consumption of pig livers in Catalonia (Spain) might be extremely small. However, manipulation of HEV‐infected livers or other organs from pigs could be considered a possible route of transmission, as has been reported recently in a butcher and in a slaughterhouse worker (Jary, 2005; Pérez‐Gracia et al., 2007).