INTRODUCTION

There are many useful diagnostic and identification techniques employed for research and management purposes in the study of wildlife diseases; however, only a limited number of key procedures will be addressed.

Historically, much diagnostic work with wildlife has involved serology, the study of specific antibodies present in hosts. Immunoassays are the procedures used for identifying and measuring antibodies in specific hosts (Mahony and Chernesky 1999).

One minor but helpful distinction can be made between serum and plasma. When blood is collected from animals for serological studies, the erythrocytes must be removed. If the erythrocytes are removed by allowing them to clot first and then draining the remaining fluid, this remaining fluid, without the clotting elements, is called serum; the term “antiserum” often is used to denote “serum with antibodies.” But if an anticoagulant such as heparin is used and the clotting proteins still remain after the erythrocytes are removed by cen­trifugation, the remaining fluid is called plasma. Both serum and plasma are considered equally use­ful for serological studies. For simplicity, the term plasma generally will not be used in this appendix.

Serological surveys for specific antibodies can be used to determine the specific infective agents or toxins (antigens) to which hosts have been exposed. These surveys give insights on reservoirs, carriers, and past histories of diseases in particular species and habitats, and also give a clearer definition of the geographic distributions and habitats occupied by these particular antigenic agents. Serological stud­ies also are used to determine the effectiveness of immunization procedures for stimulating protective antibodies.

Serological tests are based on the assumption that a host animal will develop specific antibodies to anti­gens of parasites or foreign chemicals after it has been exposed to them. Serum from the animal being tested is exposed to a known specific antigen associated with a given parasite to test whether enough specific antibodies are present to produce an antigen-antibody reaction. Earlier tests for specific antibodies built on the observation that, in the laboratory, antigen-antibody reactions often form visible lattice frameworks that can be detected in test tubes or on slides.

Sensitivity and specificity are useful considerations when assessing serological results of immunoassays. Sensitivity is the proportion of samples truly positive for an antibody for which the positive results are detected with the test used; the closer to 1.0, the better the test at identifying individuals exposed to an antigen of an infective agent or toxin. Specificity is the proportion of truly negative animals that have negative results for an antibody with the test used; the closer to 1.0, the better the test at identifying individuals not exposed to an anti­gen. The false positive rate is the proportion of healthy animals that incorrectly are assessed as being positive for a test; this is equal to 1 minus specificity. Likewise, a false negative rate is the proportion of exposed (diseased) animals that incorrectly are assessed as being nega­tive for a test; this is equal to 1 minus sensitivity. The general goal is to minimize the probabilities of both false positives and false negatives; however, most test options maximize either specificity or sensitivity—at the expense of the other.

One goal is minimizing false positive or false negative tests. With some earlier tests, false positives commonly resulted from use of undiluted test serum and concentrated antigenic preparations, leading to low specificity. False positives can occur when low- level, cross-reacting antibodies in the test sera or impurities in the antigenic preparation are present in sufficient levels. Thus, antigen impurities may react with other, nontarget host antibodies; also, host anti­bodies against a similar antigen may cross-react with the antigen being tested. In other cases, an innate chemical bonding or electro-bonding of antigens or antibodies to other materials may occur. Likewise, false negatives occasionally occur when impurities in the antigen preparation inhibit formation of the normal antigen-antibody lattice framework.

Problems of false positives or false negatives can be reduced by purifying the antigens, or diluting the antigen preparation to reduce the impurities. Often one also makes serial dilutions of the antiserum. On diluting the antiserum, the inverse of the highest dilution giving a clear positive test is called the titer. Titers often were used as a relative indicator of the amount of antibody present in a serum sample—the higher the titer, the more antibodies were present in the original, undiluted serum.

However, in recent years, more specific and sen­sitive tests often have been reported in quantitative units rather than titers. Some, such as fluorescent antibody tests, typically are reported in positive or negative terms only.