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INTRODUCTION
Wild boars can be a reservoir of agents that are infectious to domestic pigs and other livestock (1). The disease status of wild boars has been surveyed using blood samples collected from animals shot during the hunting season (23). However, seroprevalence analysis in hunted wild boars is difficult owing to the limited hunting area, the restricted hunting season, and a biased age distribution (24). Testing feces obtained from the habitat of wild boars is an alternative to testing serum samples, since fecal samples are easy to collect in large quantities and can also be used for the direct detection of bacteria, viruses, and parasites (4).
Before testing fecal samples for a specific disease, gross examination is commonly used as a method to screen the species of origin. Additionally, the sequence of cytochrome b or chromosomes carrying endogenous retroviruses has been used for scientific proof of the animals' identification (56). However, gross examination is not accurate, and the gene sequencing method is complicated and costly, whereas ELISA can be used to screen a large number of samples simultaneously. In the present study, secretory fecal IgA was tested by ELISA as an alternative method for distinguishing the species origin of fecal samples from wild boars (Sus scrofa coreanus) and domestic pigs (Sus scrofa domestica). IgA was selected as a target protein owing to its presence in large amounts in feces (7) and its conserved domains that allow for comparison across a broad range of species (89).
MATERIALS AND METHODS
Sample collection
Fecal samples from 22 different animals in Korea, including wild boars and domestic pigs, were tested (Table I) for their cross-reactivity with the porcine fecal IgA antibody. Forty fresh fecal samples of farmed wild boars from one farm were collected to evaluate IgA differences in wild boars of different ages. In addition, 50 fecal samples were collected from domestic pigs of different age groups on a commercial pig farm. The samples were transported and stored at 4℃ until analyzed.
Quantitative ELISA
Each fecal sample was diluted to a concentration of 0.5 g/mL with phosphate-buffered saline containing 0.05% Tween 20 and 1% BSA, vortexed, and centrifuged at 1,500 g for 20 min at 4℃. The supernatants were centrifuged at 16,000 g for 15 min to remove large solid particles and then transferred into Eppendorf tubes. Fecal samples from the wild boars and domestic pigs were diluted 200-fold (final dilution) and tested in triplicate using the Pig IgA ELISA Quantitation Set (Bethyl Laboratories, Montgomery, TX, USA). The IgA concentrations were quantified according to the following age groups: 1 day~1 month, 1~3 months, 3~6 months, 6 months~1 year, and >1 year (Table II). The average optical density at 450 nm (OD450) of 1% BSA as a negative control was subtracted from the average OD450 values of all other fecal samples. Twelve fecal samples were collected <1 h after excretion from four different individuals of 20 different animals (3 orders, 8 families, 15 genera, 16 species, 19 subspecies) in Seoul Grand Park and analyzed in the same way as described above. Data are presented as the mean and standard deviation (SD).
Statistical analysis
The two-tailed Student's t-test was used to compare differences in the OD450 values between Sus scrofa and the other animals. The genetic distance based on the cytochrome b peptide sequence from Sus scrofa was calculated for each subspecies using the MEGA 6 software with the Jones–Taylor–Thornton amino acid substitution model with gamma distribution (Table III).
RESULTS AND DISCUSSION
There were no differences in OD450 values between wild boars and domestic pigs (p=0.000) in each age group. Except for the Manchurian leopard cat (Prionailurus bengalensis Manchuria), all fecal samples from the other animal species had low reactivity with porcine IgA antibodies, showing statistically significant difference with that of wild boar in all age groups (p<0.001). Despite its similar genetic distance from Sus scrofa, the OD450 of the Manchurian leopard cat was 10 times higher than that of the other animal species (Table III). This result contradicts the finding of the previous report (10) showing the significant correlation between cytochrome b sequence and cross-reactivity with dolphin Ig G antibodies. However, there has been no known report that the structure of Ig A is evolutionarily related with cytochrome b sequence. Therefore, to elucidate the high affinity of pig Ig A antibodies with immunoglobulin-like molecules of Manchurian leopard cat, further studies about the genetic relationship between Ig A and cytochrome b sequence will be needed.
The fecal IgA concentrations in the suckling period were high, whereas they were lower in weanling pigs (1~3 months old) and higher again in pigs older than 6 months (Fig. 1, Table II), which agrees with previous reports of lower porcine secretory fecal IgA during the weanling period (7). Additionally, it was identified that the OD450 of the weanling pigs did not show a statistical difference with that of Manchurian leopard cat (Table I, p=0.0652). For this reason, in case of a fecal sample not showing statistically significant difference in OD450 with that of weanling pigs, we cannot convince it as droppings from wild boar. However, the IgA concentration in fecal samples of wild boars of all ages, except those 1~3 months old, was distinguishable from that of all wild animal species used for comparison in this paper, which means that the porcine IgA ELISA could be a useful method for differentiating wild boar feces from the feces of other wild animal species.
Fecal IgM concentrations are higher than IgA concentrations in weanling pigs (7) and could therefore be more useful than IgA for species identification in pigs and wild boars at 1~6 months of age. Conversely, the low level of fecal IgA in animals at 1~3 months of age could be useful to differentiate feces of the weaning period from those of the adult period. The prevalence of many infectious diseases in wild boar populations depends on the density and abundance of juveniles (11). In this situation, the population structure of weaners, as estimated from the IgA concentration, may contribute to understanding the disease status of wild boars.
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