A comparison of the direct agglutination test and the indirect hemagglutination test for the detection of blood serum antibodies to Escherichia coli organisms indicated that these serological tests were comparable. In some instances the indirect hemagglutination test provided higher endpoint readings. Preparation of the antigens for the indirect hemagglutination test was more time consuming than for the direct agglutination test. Crude extract and purified polysaccharides were comparable as red blood cell sensitizing agents

Ebeltoft, H.

Graves, D.

Kelling, Clayton L.

Schipper, I. A.

English

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Virology Papers Virology, Nebraska Center for March 1973 Comparison of the Direct Agglutination and Indirect Hemagglutination Tests in the Determination of Blood Serum Titers to Escherichia coli Organisms I. A. Schipper North Dakota State University, Fargo Clayton L. Kelling University of Nebraska-Lincoln, ckelling1@Unl.edu H. Ebeltoft North Dakota State University, Fargo D. Graves North Dakota State University, Fargo Follow this and additional works at: https://digitalcommons.unl.edu/virologypub  Part of the Virology Commons Schipper, I. A.; Kelling, Clayton L.; Ebeltoft, H.; and Graves, D., "Comparison of the Direct Agglutination and Indirect Hemagglutination Tests in the Determination of Blood Serum Titers to Escherichia coli Organisms" (1973). Virology Papers. 107. https://digitalcommons.unl.edu/virologypub/107 This Article is brought to you for free and open access by the Virology, Nebraska Center for at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Virology Papers by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. APPLIED MICROBIOLOGY, Mar. 1973, p. 458-460Copyright 0 1973 American Society for MicrobiologyVol. 25, No. 3Printed in U.S.A.Comparison of the Direct Agglutination andIndirect Hemagglutination Tests in theDetermination of Blood Serum Titers toEscherichia coli Organisms'I. A. SCHIPPER, C. KELLING, H. EBELTOFT, AND D. GRAVESDepartment of Veterinary Science, North Dakota State University, Fargo, North Dakota 58102Received for publication 5 September 1972A comparison of the direct agglutination test and the indirect hemagglutina-tion test for the detection of blood serum antibodies to Escherichia coliorganisms indicated that these serological tests were comparable. In someinstances the indirect hemagglutination test provided higher endpoint readings.Preparation of the antigens for the indirect hemagglutination test was moretime consuming than for the direct agglutination test. Crude extract andpurified polysaccharides were comparable as red blood cell sensitizing agents.Colibacillosis is one of the major digestivedisturbances of the neonate of several species(1, 3, 6, 9, 13, 16). Attempts to utilize naturalpassive immunity as a means of protectionhave been reported (5, 7, 8). Evaluation of theeffectiveness of vaccination must be deter-mined in part through blood serum titers (11,12). The indirect hemagglutination test forsomatic (0) Escherichia coli antibody has beenadvocated as a more sensitive method of serumtiter determination than the direct conven-tional agglutination test (9, 10).These investigations were initiated to com-pare the efficacy of the indirect hemagglutina-tion test versus the conventional tube aggluti-nation test, by utilizing blood serum of adultswine and their piglets. The adult swine hadbeen vaccinated with heat-inactivated anti-gens of specific serotypes of E. coli.MATERIALS AND METHODSExperimental animals. Eight pregnant second-litter sows, of Duroc or Chester White breeding, andtheir piglets were used as investigational animals.The sows were obtained from two farm herds andhoused in a conventional swine housing facility for atleast 30 days prior to farrowing. The sows weighedbetween 140 and 160 kg each. Piglets of the sowsremained with the sows for at least 4 weeks postfar-rowing.' Published with the approval of the director of the NorthDakota Agricultural Experiment Station as Science Series379.Inoculation. Bacterins were prepared from E. coli055: B5 (Difco) grown on veal infusion agar. Afterincubation (18 h), the bacterial growth was removed,placed in sterile phosphate-buffered saline (PBS),and heated for 2.5 h at 121 C. This was then dilutedto a nephelometer density of tube three (3). Each sowreceived 10 cm' of the heat-killed bacterin intramus-cularly at least 3 weeks prefarrowing. The 055: B5serotype was selected because of its infrequent exist-ence in the average swine environment, avoidingexistence of blood titers previous to vaccination, andbecause purified lipopolysaccharides of this typewere readily available. This serotype is also noted forits minimum of reciprocal antigen relationship toother E. coli organisms.Blood was collected from the anterior vena cavaprior to vaccination, immediately following farrow-ing, and 1- and 4-week postfarrowing. Serum wasobtained by the conventional method of coagulation,cooling, and centrifugation. Collected serum wasfrozen and stored at -20 C until use.Antigens. Crude antigens of E. coli 055: B5 wereprepared according to the procedures of Vosti (14)with modifications. After incubation (18 h) in brainheart infusion broth, the organisms were recoveredby centrifugation and were suspended in sterile PBSat 1 ml of sediment per 10 ml of PBS. They were thenboiled for 2.5 h. After centrifugation, the supernatantfluid (crude extract) was stored at -20 C.Purified lipopolysaccharides of E. coli 055: B5(Difco) were prepared according to the technique ofWestphal (15).Serological tests. All serum samples were heatinactivated at 56 C for 30 min. The indirect hemag-glutination test was one of the tests used to detect the0 antibody (9, 10, 14). A 3% suspension of sheep redblood cells (RBC) was washed 3 times in sterile PBS458AGGLUTINATION VS. HEMAGGLUTINATION TESTSby centrifugation, and then was added to a 10%suspension of the respective crude or purified poly-saccharide. The suspension was incubated at 37 C ina water bath for 30 min. This was then washed 3times in sterile PBS, and suspended in 2.5% PBS;Serums to be evaluated were diluted by a twofoldserological dilution in sterile PBS (pH 7.3), and 0.5ml of each dilution was added to a test tube (10 by75 mm). Sensitized sheep RBC (0.05 ml) was alsoadded to each tube. The tubes were well shaken andincubated at 37 C for 2 h. Controls consisted of PBS,negative and inactivated serum with the sensitizedsheep RBC. All tubes were read by gross observationby using a Quebec bacterial colony counter andreread after 12 h of refrigeration at 4 C. A flocculentcovering of uniform thickness which exhibited ag-glutination when suspended and which nearly cov-ered the entire tube bottom was considered positive.A test was designated as negative when the RBC wasin a compact disk-like formation and formed auniform suspension when suspended.The direct tube agglutination test was performedwith antigen made from smooth colonies grown ontryptose agar. The colonies were removed from theagar with sterile saline, centrifuged, and diluted to 1ml of cells to 9 ml of sterile PBS (pH 7.3). The dilutedcell suspension was heated to 121 C for 2 h (2).Serums to be investigated were diluted twofold. A0.5-ml sample of each dilution was added to the testtubes (10 by 75 mm) in addition to 0.05 ml of theantigen suspension. The tubes were well shaken andthen incubated in a water bath at 48 C for 18 h (4).Titers for all tests were recorded as the reciprocal ofthe highest serum dilution exhibiting a positiveagglutination.RESULTSComparisons of the direct agglutination testto indirect hemagglutination test with 44serum samples indicated that 36.3% of theserum samples had identical titers as wasdetermined by both methods of titration. Ti-ters of 70.5% of the samples were either identi-cal or within one dilution of being identical. Agreater number of the indirect hemagglutina-tion tests had higher dilution end points thanwere detected for the direct agglutination test(Table 1). The indirect hemagglutination testprovided a more readily detectable end point,but considerable time and effort were requiredto prepare the antigens and RBC indicatorfraction.Comparative tests were made for RBC sensi-tivity by using RBC of individual speciesincluding swine, human (O group), and sheep.The sheep RBC provided the greater detectablereactions of those investigated.Comparisons were made of purified lipopoly-saccharides and crude extracts of E. coli testorganisms for sensitization of the RBC indica-tor fraction for the indirect hemagglutinationtest. Of the 32 antiserums investigated, 37.5%had equal endpoint readings, and 34.4% had a1-dilution difference. Tests made with crudeextracts provided 15.7% more endpoint read-ings of two dilutions greater than the purifiedlipopolysaccharides preparations (Table 2).There were no observable differences in theutilization of either testing procedure with thepurified or crude extracts of the test organism.DISCUSSIONThe direct agglutination and indirect hemag-glutination tests provided very similar (twodilutions) end-point readings for 90% or more ofthe serums evaluated. This is in contradictionto previous investigations (6, 9, 10, 11). Theseinvestigations indicate a greater number ofindirect hemagglutination tests had higherendpoints than the direct agglutination test.This is not necessarily indicative of greatersensitivity; however, it is possibly indicative ofmore readily detectable endpoint reactions.The indirect hemagglutination test provided amore readily determined endpoint, but a muchgreater effort was necessary in preparing theantigens for hemagglutination than that re-quired for the direct agglutination test. Theseinvestigations concur with the work of KuninTABLE 1. Comparison of direct agglutination toindirect hemagglutination tests in the detection of E.coli antibodiesDirect Indirect hemag-agglutination glutinationEnd-point readings Percent- Percent-No. of age of No. of age oftests total tests totaltests testsEqual 16 36.4 16 36.41-Dilution greater 7 15.9 8 18.22-Dilutions greater 2 4.5 8 18.23-Dilutions greater 0 0 3 6.8TABLE 2. Comparison of crude extracts to purifiedpolysaccharides of E. coli organisms for thesensitization of RBC indicator cellsDirect Indirect hemag-agglutination glutinationEnd-point readings Percent- Percent-No. of age of No. of age oftests total tests totaltests testsEqual 12 37.5 12 37.51-Dilution greater 5 15.6 6 18.82-Dilutions greater 1 3.1 6 18.83-Dilutions greater 0 0 2 6.3VOL. 25, 1973 459460 SCHIPPER ET AL.(7), who suggested that the problem of hetero-genic reactions deterred from the effectivenessof this test procedure.Other investigators utilized RBC of variousspecies including human (9), sheep (11), andswine (6). Comparative tests utilizing thesespecies of RBC indicated that the sheep RBCprovided the most readily detectable hemag-glutination under the condition of this investi-gation.Comparisons of the crude E. coli extracts tothe purified lipopolysaccharides extracts in-dicated that they were comparable RBC sensi-tizers, the crude extract being preferable inmost laboratories. These results concur withthose of Vosti (14).ACKNOWLEDGMENTSFunds for these investigations were provided in part byRegional Research Funds (NC-62-Enteric Diseases of BabyPigs).LITERATURE CITED1. Annon. 1960. A survey of the incidence and causes ofmortality of pigs. Vet. Rec. 72:(2)1240-1247.2. Ewing, W. H. 1956. Production of Escherichia Coliantiserums. Pub. Health Lab. Bull. Conf. State Prov.Public Health Lab. Dir. 14:128-141.3. Ewing, W. H., and P. R. Edwards. 1957. Isolation andpreliminary identification of Escherichia coli sero-types associated with diarrheal disease. Center forDisease Control Laboratory Manual, National Com-municable Disease Center, Atlanta, Ga.4. Ewing, W. H., H. W. Tatum, B. R. Davis, and R. W.Reavis. 1956. Studies on the serology of the Esche-richia coli group. Center for Disease Control Publica-tion, Communicable Disease Center, Atlanta, Ga.APPL. MICROBIOL.5. Jones, J. E. T., K. C. Sellers, and H. W. Smith. 1962.The evaluation of live and dead Escherichia colivaccines administered to the pregnant female in theprevention of scouring (Diarrhea) in piglets. Vet. Rec.74:202-203.6. Kohler, E. M., R. B. Moore, and S. Smith. 1968.Serologic response of swine to Escherichia coli anti-gens. Amer. J. Vet. Res. 29:1419-1428.7. Kunin, C. M., and M. V. Beard. 1963. Serologicalstudies of 0 antigens of Escherichia coli by means ofthe hemagglutination test. J. Bacteriol. 85:541-548.8. Lemcke, R. M., and A. Hurst. 1961. Antibody content ofcolostrum and piglet serum following vaccination ofthe sow. J. Comp. Pathol. Ther. 71:268-278.9. Neter, E., E. A. Gorzynski, N. J. Zalewski, R. Rachman,and R. M. Gino. 1954. Studies on bacterial hemag-glutination. Amer. J. Publ. Health 44:49-54.10. Neter, E., N. J. Zalewski, and W. W. Ferguson. 1953.Escherichia coli hemagglutinin response of adult vol-unteers to ingested E. coli 055: B5. Proc. Soc. Exp.Biol. Med. 82:215-219.11. Sharpe, H. B. A. 1965. A study of antibody levels in theyoung pig. Res. Vet. Sci. 6:490-497.12. Svendsen, J., and M. R. Wilson. 1971. Immunity toEscherichia Coli in pigs: effect of feeding colostrum orserum from vaccinated sows to Escherichia coli-infected gnotobiotic pigs. Amer. J. Vet. Res.32:899-904.13. Tennant, B. 1971. Neonatal enteric infections caused byEscherichia coli. Ann. N. Y. Acad. Sci. 176:9-135.14. Vosti, K. L., A. S. Moto, J. J. Older, and L. A. Rantz.1964. The serologic specificity of crude and purifiedantigen extracts of Escherichia coli in hemagglutina-tion reactions with rabbit and human antisera. J.Immunol. 93:199-204.15. Westphal, O., 0. Luderitz, and F. Bister. 1952. Uber dieExtraktion von Bakterien mit Phenollwasser. Z. Na-turforschg. 7 (b):148-155.16. Wilson, M. R., and J. Svendsen. 1971. Immunity toEscherichia coli in pigs. the role of milk in protectiveimmunity to E. coli enteritis. Can. J. Comp. Med.35:239-243.

Comparison of the Direct Agglutination and Indirect Hemagglutination Tests in the Determination of Blood Serum Titers to \u3ci\u3eEscherichia coli\u3c/i\u3e Organisms

https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1106&amp;context=virologypub

Comparison of the Direct Agglutination and Indirect Hemagglutination Tests in the Determination of Blood Serum Titers to \u3ci\u3eEscherichia coli\u3c/i\u3e Organisms

Abstract

Similar works

Full text

Available Versions

DigitalCommons@University of Nebraska