DEVELOPMENTAL STUDIES AND LABORATORY 



INVESTIGATIONS CONDUCTED BY VETERINARY 



SERVICES DIAGNOSTIC LABORATORIES 



FISCAL YEAR 1973 



ANAPLASMOSIS 



r PRESERVATION OF SERUM SAMPLES FOR THE ANAPLASMOSIS MICROTITER COMPLEMENT-FIXATION 



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TEST. Blackburn, B. 0., Wright, H. S. and Sherman, K. C. (Project Report) 



Introduction 



The "Standardized Complement-Fixation Test for Anaplasmosis" published by the Animal and Plant Health 

 Inspection Service requires the addition of aqueous phenol to serum samples that are submitted for testing. In the 

 recent procedures described for conversion to the microtiter technique, this provision was retained. Since the 

 Veterinary Services Diagnostic Laboratory (VSDL), Ames, Iowa, now has responsibility for all export tests, it would 

 be convenient to drop the phenol requirement. One serum sample could then be used for the various serologic tests 

 required. This study was designed to determine whether serum not treated with phenol could be used effectively in 

 the microtiter complement fixation test for anaplasmosis. 



Materials and Methods 



Serum samples.— Ten serum samples were obtained from known positive animals at the Oklahoma State 

 University Veterinary Research Farm, Pawhuska, Okla. Ten negative serum samples were obtained from animals in 

 the National Animal Disease Laboratory herd. Each serum sample was divided into four parts and each part 

 randomly assigned one of four treatments as follows: (A) refrigeration, (B) refrigeration for 5 days followed by 

 adding aqueous phenol to a final concentration of 0.5 percent, (C) aqueous phenol added to a final concentration of 

 0.5 percent and (D) freezing. In effect, this gave 20 serum samples by each treatment. 



Test procedures. —Serum samples that had been kept refrigerated were tested first. In preparation for the test, 

 each of the 20 samples was further divided to form 25 replicate subsamples. Thus, the total number of subsamples 

 for the treatment group was 500. These subsamples were randomly ordered by using a random number table. The 

 subsamples were then tested following the procedures described by the Beltsville laboratory ("A Microtiter 

 Technique for the Complement Fixation Test for Anaplasmosis"). This same procedure was used for testing the 

 samples receiving the other three treatments. 



Results 



Results following the screening test (serum dilution 1:5) are summarized in table 1. None of the known 

 negative subsamples gave positive reactions and only one of the known positive subsamples gave a negative reaction. 

 The number of suspicious reactions among the known negatives was higher in the phenol treatment group than in 

 any of the other treatment groups. None of these differences are statistically significant. 



The titration results for the known positive subsamples are presented in table 2. Only one of the known 

 positive subsamples titrated had an antibody titer less than 1:20 and this subsample was in the phenol treatment 

 group. 



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