Review of the the KHV virus neutralization assay on previous pages illustrates a portion of the process. The serum or plasma specimen from each individual fish is diluted in the laboratory in serial dilutions starting at a dilution of 1:10 and put in tissue culture. Antibody titers are rarely high except in that period associated with acute infection. Six weeks after an acute infection, titers in many survivors may be as low as 1:10 or 1:20 and remain at these levels for an extended period. WIth herpesvirus, any antibody level is significant (as is a "touch" of pregnancy). Ultimately circulating antibody titers may fade below detectable threshold levels and immunity is conferred through other poorly understood cell mediated mechanisms.
Consider what would happen if a dealer or individual, in an attempt to reduce testing costs, combined the serum from koi obtained from the same breeder and maintained in the same quarantine system. Let us assume that equal amounts of blood from each of three fish were combined in a vacutainer and sent for testing.
For discussion purposes, assume one of these three fish was a KHV survivor and had anti KHV antibody detectable at 1:20 dilution. Assume the other two fish had never been exposed to KHV and had no detectable anti KHV antibody. The blood of the KHV carrier fish is diluted by the blood from two unexposed fish such that effective titer is below the assay detection threshold and the result would be reported as negative. In short , the KHV survivor would be missed. Indeed, the only time one could be certain combined specimens give a reliable result is that unlikely case where all fish are positive (KHV survivors with detectable antibody). In that case one might get an approximation of the average antibody titer. For practical purposes, each specimen from each fish should be individually tested. The predictable result of combining specimens is false negatives.
As a matter of perspective, in one small study the incidence rate of KHV serology positives from specimens imported from some 15 well known Niigata area breeders (all facilities certified KHV free by PCR testing by their government) from fall 2006 was approximately 3% (n=97). In a slightly larger group, a similar incidence rate was seen in fish imported in 2006 from Taiwan. Again all facilities were certified KHV free by their respective governments. Virtually all imported koi tested in these studies were two or more years of age. The incidence appears to low but very real.