Transmissible Gastroenteritis in Feeder Swine: Clinical ...

Transmissible Gastroenteritis in Feeder Swine:Clinical, Immunofluorescence andHistopathological Obervations

M. Morin, L. G. Morehouse, R. F. Solorzano and L. D. OIson*

ABSTRACT

Eight feeder swine (four to six months ofage) were inoculated orally with 200,000 to500,000 pig infectious doses (PID) of the Pur-due strain of transmissible gastroenteritis(TGE) virus. Biopsies obtained from theirsmall intestines were examined histopatholog-ically and by fluorescent antibody tissue sec-tion technique at intervals that included 24,48, 72 and 96 hours postexposure, and similarexaminations were carried out at necropsy 168hours postexposure. Evidence of virus infec-tion was demonstrated in all segments of thesmall intestine except the upper duodenum andthe viral antigen was found only in the cyto-plasm of the absorptive cells covering the villi.Although six of the eight pigs failed to showclinical signs of TGE, typical microscopic le-sions of villous atrophy with replacement ofcolumnar absorptive cells by cuboidal cellswere observed in seven pigs, and TGE virusantigen was demonstrated in the intestinalcells of four of eight pigs during the first weekpostexposure. The infection was usually mildto moderate and focal in the pigs withoutclinical signs of the disease and more severeand extensive in the pigs with clinical signs of

*Departments of Pathology (Morin, Morehouse and Ol-son) and Microbiology (Solorzano), School of VeterinaryMedicine, University of Missouri-Columbia, Columbia,Missouri.

Contribution from the Missouri Agricultural ExperimentStation, Journal Series No. 6519. Supported in part bya fellowship from the Canadian Medical Research Coun-cil, National Research Bldg. M-58, Montreal Road, Otta-wa, Canada, and from the National Pork ProducersCouncil, 4715 Grand Avenue, Des Moines, Iowa.

Present address of M. Morin: Departement de Pathologieet microbiologie, Ecole de Medecine Vet6rinaire de l'Uni-versite de Montreal, St-Hyacinthe, Quebec, Canada.

This paper in substance forms part of a thesis submittedby M. Morin to the University of Missouri in partialfulfillment of the requirements for the Ph.D. degree inPathology.

Submitted October 6, 1972.

the disease variable in severity. It was con-cluded that TGE virus probably replicated inall feeder swine exposed, and that the presenceor absence of clinical signs of TGE in thesepigs was related to the severity and extent ofthe villous atrophy and columnar cell replace-ment induced in their small intestines.

RESUME

On a administre a huit porcs a l'engraisse-ment (ages de quatre 'a six mois), par la voiebuccale, de 200,000 'a 500,000 doses infectieusesporcines (DIP) de la souche "Purdue" du virusde la gastro-enterite transmissible (GET). Onpreleva des biopsies de leur intestin grele eton les examina au moyen de l'histopathologieet de l'immunofluorescence, a intervalles de 24,48, 72 et 96 heures apres l'infection; on proce-da 'a des examens similaires au moment de lanecropsie, c'est-'a-dire 168 heures apres l'in-fection. On demontra l'evidence de l'infectiondans tous les segments de l'intestin grele, saufdans le duodenum proximal, et on ne decelal'antigene viral que dans le cytoplasme des cel-lules absorbantes bordant les villosites. En de-pit du fait que six des huit porcs ne manifes-terent aucun signe clinique de GET, on observaquand meme des lesions microscopiques carac-teristiques d'atrophie des villosites, avec rem-placement des cellules cylindriques absorbantespar ces cellules cubiques, chez sept sujets; ondecela aussi l'antigene viral de la GET dansles cellules de l'intestin de quatre des huitporcs, au cours de la premiJere semaine apresl'infection. En general, cette infection se tra-duisit par des foyers ou les lesions etaient be-nignes ou moderees, chez les porcs qui nemanifesterent pas de signes cliniques de la ma-ladie; elle s'avera cependant plus grave et plus

Vol. 37 - July, 1973 239

diffuse chez ceux qui presenterent des signescliniques plus ou moins prononces. On conclutque le virus de la GET se multiplia probable-ment chez tous les porcs ii l'engraissementexposes ia la maladie et que la presence oul'absence de signes cliniques de GET chez cessujets fut reliee 'a la gravite et it l'etendue deI'atrophie des villosites et du remplacement descellules cylindriques provoquees dans leur in-testin grele.

INTRODUCTION

Transmissible gastroenteritis (TGE) isan enteric disease of swine caused by avirus and characterized by vomition, pro-fuse diarrhea, dehydration, and a highmortality in piglets under two weeks of age(5). The TGE virus replicates through allsegments of the small intestine of baby pigswith the highest titer in the jejunum (11).Using immunofluorescence and the electronmicroscope to study the small intestinal mu-cosa of baby pigs infected with TGE virus,it appeared that the virus replication oc-curred only in the cytoplasm of the absorp-tive cells on the villi, but not in the undif-ferentiated cells of the crypts of Leiber-kuhn (16, 17). The infection caused a rapiddestruction of the villous epithelium whichled to a villous atrophy (10, 12, 13, 18).

Clinical signs of TGE are frequently ob-served in feeder swine on the same farmhaving problems with the disease in babypigs. In epizootiological studies, it appearedthat the disease often started in feederpigs and then spread to baby pigs and theirdams on the same farm (3, 6, 7). The dis-ease was also observed to affect entire herdsof feeder swine when baby pigs were notpresent on the farm (5). In older pigs diar-rhea can be profuse and severe, but this isusually transitory and the mortality is low.These pigs have been suspected to be a re-servoir of TGE infection and to play an im-portant role in the virus survival duringsummer (8). The disease has been repro-duced experimentally in five to six monthold swine (14).The study reported herein was under-

taken to make clinical observations and im-munofluorescence and histopathologicalstudies of the intestinal tracts of individualfeeder pigs following oral exposure to theTGE virus.

MATERIALS AND METHODS

VIRUS

Two inocula were prepared with the Pur-due strain of TGE virus.1 The first inocu-lum was a homogenate of the small intes-tine and its content from a five day old pig48 hours following oral exposure to thevirus. This 20% homogenate in phosphatebuffered saline (PBS) was centrifuged at1,000 X g for 30 minutes and the superna-tant fluid was frozen in screw-capped vialsat -70°C. The second inoculum was a poolof undiluted intestinal content collectedfrom two baby pigs 48 hours after oral ex-posure to the virus. These two inocula con-tained approximately 105 pig infectiousdoses (PID) per mililiter as determined bytitration in baby pigs.

PIGS

Nine pigs (eight infected, one control)approximately four to six months old wereused in the study. Eight pigs were from asecondary SPF herd maintained by the De-partment of Veterinary Pathology at theUniversity of Missouri, and one pig (No.7) was purchased from a private farm withno history of TGE. Two pigs received ap-proximately 200,000 PID of the first ino-culum orally, and six pigs received approx-imately 500,000 PID of the second inoculumby the same route (Table I). The studywas conducted with one pig at a time.Clinical signs of TGE were recorded. Se-quential biopsies were taken from the jeju-num of these pigs through a laparotomy in-cision in the left flank. The schedule ofthe biopsies performed on each pig is shown(Table I). Surgical anesthesia was obtainedin the following manner: a tranquilizer,(phencyclidine hydrochloride)2 was injectedintramuscularly at a dosage of 0.8 mg perkg of body weight. The pig was immobilizedwithin five to eight minutes after injection.Surgical anesthesia was then induced withether.3 After the laparotomy incision was

'Obtained from Dr. E. 0. Haelterman, Department of

Veterinary Microbiology, Pathology and Public Health,School of Veterinary Science and Medicine, Purdue Uni-versity, Lafayette, Indiana.

2Sernylan'RI, Bio-Ceutic Laboratories, Inc., St. Joseph.Missouri.

3Ether Squibb, E. R. Squibb and Sons, New York, NewYork.

Can. J. comp. Med.240

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Vol. 37 - July, 1973

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241

made, a loop of jejunum approximately 4.0inches long was exposed and a biopsy wasobtained with curved scissors midway be-tween the mesenteric and the antimesentericborders of the gut. The surgical procedurewas the same as one described for simpleenterotomy in the dog (1). The gut sampleobtained was divided immediately for thefluorescent antibody tissue section tech-nique (FAT) and histopathological exam-inations.At the time of necropsy (Table I), sam-

ples were obtained from the upper duode-num, lower duodenum, upper jejunum, mid-jejunum, lower jejunum, ileum, tonsil.pancreas, spleen and mesenteric lymphnodes for the same purpose as listed forthe biopsies. The gut samples were obtainedfrom the wall of the small intestine in asimilar manner to that described for thebiopsy. The villi were immediately placedin contact with formalin for rapid fixation.

Specimens for the FAT were placed inscrew-capped vials and rapidly frozen in amixture of dry ice and alcohol and storedat -70°C. At the time of sectioning, theywere embedded with OCT embedding me-dium4 on the cryostat specimen holder, andthe gut samples were oriented to obtainlongitudinal sections of the villi. Sectionswere cut 8.0 ,u thick in a cryostat at -28°C.The slides obtained were allowed to dry for15 minutes at 37°C and were then fixed inacetone at -15°C for 15 minutes. After fixa-tion, they were dried for ten minutes at37°C and then stored in a sealed box at-15°C until ready for immunofluorescentstaining.

Specimens collected for histopathologywere fixed in 10%c neutral buffered forma-lin and processed for paraffin tissue sec-tions according to conventional methods.Sections were cut 6.0 to 8.0 pu thick andstained with hematoxylin and eosin.

FLUORESCENT ANTIBODY

The conjugate was prepared from theserum of a 12 week old pig hyperimmunizedwith the Purdue strain of TGE virus. Thepig was first exposed orally at six weeksof age with 2.0 ml of a 20%,( intestinal ho-mogenate containing the virus and devel-oped a severe diarrhea. The inoculum fortwo subsequent doses of virus was preparedin the following manner: 70.0 ml of thevirus-containing intestinal homogenate was

4Tissue-Tek, Ames Co., Elkhart, Indiana.

* W* E{ a

Fig. 1. Normal jejunal mucosa of a four month old pig.Notice the elongated villi covered by columnar epithelialcells. H & E X450.

centrifuged at 14,500 X g for one hour at5°C. The supernatant fluid obtained (50.0ml) was passed through a 0.22 mp. filterand then centrifuged at 105,500 X g for fivehours at 5°C. The supernatant fluid wasdiscarded and the pellets obtained were re-constituted with 13.0 ml of a sterile PBSsolution, at pH 7.3. Twenty-one days afterthe initial oral exposure, 2.0 ml of thisinoculum were injected intramuscularly. Asecond injection was given subcutaneouslyfourteen days later with 2.0 ml of the ino-culum mixed with 2 ml of Freund's adju-vant. Fourteen days later, the pig was ex-sanguinated and its serum collected. Theantibody titer was estimated by using aserum neutralization test performed incell culture using a cytopathogenic effect ofthe Miller strain of TGE virus5 in a swinetestis cell line6 as the indicator system. Theserum had the ability to neutralize 1,000

5Obtained from Dr. E. H. Bohl, Department of VeterinaryScience, The Ohio Agricultural Research and Develop-ment Center, Wooster, Ohio.

60btained from Dr. A. W. McClurkin, National AnimalDisease Laboratory, Ames, Iowa.

Can. J. comp. Med.242

tissue culture infectious doses (TCID) ofcytopathogenic virus at a dilution of 1:1600.The methods used for the extraction of thegamma globulin fraction and the labellingof this fraction with fluorescein isothio-cyanate7 were those used to prepare conju-gate for hog cholera (20). The staining pro-cedures used were those routinely employedfor the detection of hog cholera virus (2)by the FAT. Specificity of the fluorescencewas tested in the following manner: frozensections from the small intestines of unin-fected pigs, when stained with the fluores-cent antibody preparation, failed to showfluorescence similar to that seen in the sec-tions from pigs exposed to TGE virus.Specificity was also demonstrated by usingone-step and two-step inhibition tests; theanti-TGE antiserum used as inhibitor inthese tests was that from which the conju-gate was prepared. Finally, gut sectionsfrom TGE-infected pigs were stained withanti-hog cholera, anti-distemper, and anti-

7B.B.L., Division of Bioquest, Cockeysville, Maryland.

bovine virus diarrhea virus conjugates;fluorescence similar to that produced bythe anti-TGE conjugate was not observed.

RESULTS

CLINICAL SIGNS

All pigs exposed to the TGE virus weredepressed ten to 24 hours following expos-ure; however, only pig No. 5 had softstools 48 hours after exposure and pig No.9 had diarrhea at 24 hours, which persistedfor three days (Table I).

GROSS LESIONS

During surgery, grossly visible lesionsin the gut were seen only in pig No. 5, 48hours postexposure. The lesion in the seg-ment of small intestine, which was exam-

TABLE II. Summary Comparing Results of the Immunofluorescence and HistopathologicalStudies

Pigs l1 2 3 4 5 6 7 8 9

Immunofluorescencea - + - - + + + - -

Histological lesionsb - + + + - + + +

&Demonstration of TGE virus by immunofluorescence- = Negative+ = Viral antigen was demonstratedbMicroscopic lesions of TGE- = Microscopic lesions were not demonstrated+ Microscopic lesions were demonstratedoPig No. 1 was an uninfected control

TABLE III. Correlation of Clinical Signs of TGE with Severity of Small Intestinal Infection asEstimated from Results of the Immunofluorescence and/or Histopathological Studies

Pigs 1e 2 3 4 5 6 7 8 9

Clinical signsa t - - - --

Intestinal Infectionb -

-Clinical signs of TGE- = No signs of enteric disorders+ = Soft stool

++ = DiarrheabSmall intestinal infection by TGE virus

- = No evidence of intestinal infection+ = Infection mild, very focal in nature++ = Infection moderate but still focal

+++ = More severe and extensive infectionePig No. 1 was an uninfected control

-+ + rn+r + ++ ++ ++

Vol. 37 - July, 1973 243

ined, consisted of a thin appearance of thewall and distention of this segment byyellow fluid. On postmortem examination,similar lesions were observed in some seg-ments of the small intestine of pig No. 9.

IMMUNOFLUORESCENCE ANDHISTOPATHOLOGICAL STUDIES

TGE antigen was detected by the FATin four of eight pigs that had been exposedto the virus and microscopic lesions usuallyassociated with the disease were observed inseven of the eight pigs (Tables I and II).Neither lesions nor positive fluorescencewere seen in the noninfected control pig(Figs. 1 and 2).The TGE viral antigen was detected only

in the cytoplasm of the absorptive epithelialcells covering the villi of the small intestineand was not seen in the epithelial cells lin-ing the crypts of Lieberkuhn. Specific fluor-

Fig. 3. Biopsy obtained from the jejunum of pig No. 5, 48hours after exposure to TGE virus. Notice villi shorterthan normal and blunted. Cells covering their upperpart are undergoing degeneration. H & E X420.

_w_.-v_ ~~~~~~~~~N.

Fig. 2. Normal jejunal villi stained with fluorescentantibody. Notice the bright nonspecific fluorescence pro-duced by eosinophils infiltrated in the villous core. X250.

escence was usually bright, apple green incolor, granular and often more intense inthe apical cytoplasm of the cell (Figs. 4 and9). This was in contrast to the dull greencolor of noninfected epithelial cells (Fig.2).The following microscopic lesions were

observed in the biopsy and postmortem sam-ples obtained from the small intestine of theinfected pigs: 1) villous atrophy and blunt-ing variable in severity (Figs. 5 and 8);2) replacement of the absorptive columnarepithelial cells by flat to cuboidal immatureepithelial cells (Figs. 5, 6 and 8); and 3)thickening of the intercryptal lamina pro-pria, and elongation of the crypts of Lieber-kuihn (Figs. 5 and 8). Edema occasionallycould explain the thickening of the laminapropria (Fig. 5), but in most instances,this change was not present.

Lesions were observed in the small in-testine of all the pigs exposed to the virus(Table III). In pigs No. 2, 3, 4, 6, 7 and 8,it was usually mild to moderate and focalin nature. Lesions in pig No. 2 were repre-sentative of the group (Table I). In thebiopsy obtained at 24 hours postexposure(PE), no viral antigen was detected by theFAT but a number of foci of villous atrophy

Can. J. comp. Med.244

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and replacement of absorptive columnarcells by cuboidal cells were present. In thebiopsy obtained 96 hours PE, TGE antigen .was detected in a few of the absorptive epi-thelial cells (Fig. 9). Lesions of TGE weremoderate in severity and focal. At the time Kof necropsy, one week after exposure, viral /.. Vantigen was still present in all segments 'of the smal intestine with exception of the , M *4'upper duodenum. Usually, only a few cellswere involved, but in the section obtained 2from the lower jejunum, almost all the epi- 00

thelial cells covering the villi had viral anti-gen in their cytoplasm. Microscopic lesions Xwere also present in some segments, butl .'they were focal, and a number of normalvilli were present in the sections (Fig. 8). . V tTGE antigen was not demonstrated by theFAT in pigs No. 3, 4, and 8 but typical mi- Fig. 5. Biopsy obtained from pig No. 5, 96 hours post-croscopic lesions of villous atrophy were exposure. Notice the severe villous atrophy, thickeningcroscpiclsionsof vilous troph were of the lamina propria and elongation of the crypts. Hdetected. It should be pointed out here that & E X155.TGE antigen was demonstrated by the FAT

-8= /: in the gut of two pigs (Nos. 2 and 7), thathad never shown clinical signs of enteric dis-order, one week after their exposure to thevirus. Signs of severe and extensive intes-tinal infection were observed in pigs No. 5and 9. In the biopsy obtained from pig No.5 at 48 hours PE, almost all the absorptivecells covering the villi had viral antigen in

A.-] 1S_ ^- their cytoplasm (Fig. 4). Several of thesevilli were blunted, moderately shorter thannormal, and several of their absorptive cellswere undergoing degeneration and desqua-mation (Fig. 3). In the biopsy obtained at96 hours PE, severe and extensive lesions ofvillous atrophy and replacement of colum-nar cells by cuboidal immature cells werepresent (Figs. 5 and 6). Some of the imma-ture cells covering the villi had viral anti-gen in their cytoplasm, but the number ofpositive cells had decreased significantly(Fig. 7). At the time of necropsy, threedays later, severe lesions of TGE were stillpresent in the jejunum but there were signsof villous regeneration. In several areas,villous atrophy was not as severe and sev-eral villi were covered by epithelial cellswhich were almost columnar. Viral antigenwas not detected by the FAT in those cells.Similar histopathological observations weremade in pig No. 9 but viral antigen was notdetected by the FAT.

Fig. 4. Fluorescent antibody stained section from the Viral antigen was not detected in the ton-biopsy in Figure 3. All the epithelial cells covering the sil, mesenteric lymph nodes, spleen or pan-viUi have a large amount of viral antigen in their cYto- creas of any of theplasm. X550. cesoanoftepigs.

Vol. 37 - July, 1973 245

DISCUSSION

From the results of this study, it is sug-gested that the site of replication of theTGE virus in feeder swine is the same as N-that observed in baby pigs (11, 16). Evi-dence of virus infection was seen in all,segments of the small intestine except theupper duodenum and the viral antigen was -found only in the cytoplasm of the absorp-tive cells covering the villi. The crypt cellswere not involved. The constant presence ofeosinophils in the villous core and the in-tercryptal lamina propria resulted in vary- Fig- 7- Fluorescent antibody stained section from the

biopsy in Figures 5 and 6. Notice the presence of fewing degrees of nonspecific fluorescence; cells w ith viral antigen in their cytoplasm. X730.however, they were easily identified be-cause of their location and their characteris-tic appearance in the presence of incandes-cent light. The microscopic lesions observed 4 pin this study were very similar to those that.have been described for TGE infeCtion inbaby pigs (12, 13, 18) and feeder swine(14). Pensaert et al (16), were unable to _demonstrate TGE viral activitv by immuno-fluorescence either in intact baby pigs or -_in isolated intestinal loops seven days PE. -,According to those workers, villi in the in- F'testines of the baby pigs that had survived _

Fig. 8. Section from the jejunum of pig No. 2, one weekafter exposure to TGE virus. A focal area of villousat-ophy can be seen on the right. Notice the presence ofnormal villi on the left. H & E X100.

the disease had almost reached their orig-&V- inal length after seven days. In the study

reported herein, severe lesions of villousatrophy with evidence of a beginning ofvillous regeneration were observed in twoof the pigs killed seven days PE. Viral anti-

- gen was also detected by the FAT in the je-; junal cells of two of the pigs killed seven

_i e-ji days PE. Thus, there appears to be a dis-_%7-e tinct chronological difference in the de-

velopment of TGE lesions in the differenti _ i 2t 5 age groups of pigs.

In this study, all the pigs exposed to TGE-_G _ ++p virus had evidence of the infection in their

"Ys , small intestines despite the fact that only;- i3i s < ; X two of them had clinical signs of enteric dis-

orders. The infection was mild to moderateand focal in the pigs without clinical signs

5ame hiopsy 5N+k0 of TGE and more severe and extensive inthe pigs which had a diarrhea or soft stool.

Fig. 6. Same biopsy as that shown in Figure a Notie From these observations, it is suggestedthe flat to cuboidal cells lining the atrophic villi. H & E that the presence or absence of clinical signs

Can. J. comp. Med.

.Abuu.

246

Ar

Fig. 9. Fluorescent antibody stained section from thebiopsy obtained from pig No. 2, 96 hours postexposure.Notice the presence of a few cells with specific fluores-cence in their cytoplasm. X900.

of TGE in feeder swine exposed to the virusis related to the severity and extent of thevillous atrophy and cell replacement inducedby the virus. In baby pigs infected withTGE virus, the immature cells covering theatrophic villi have an abnormal microvillousborder (12, 13, 19) and because of this,these cells are probably poor in several im-portant hydrolytic enzymes (3, 13, 19) thatare normally present in this region. Thesechanges, combined with the villous atrophy,cause a maldigestion and malabsorption ofnutrients because of a loss of the digestive-absorptive surface of the small intestine(4, 9, 10, 13, 19). In feeder swine with fo-cal lesions, the loss of digestive-absorptivesurface is probably not severe enough to in-duce visible signs of diarrhea or soft stool.However, in those instances where moreextensive lesions develop, the surface lossis too severe and this leads to visible clin-ical signs of enteric disorders such as softstool or diarrhea variable in severity.

In pigs No. 3, 4, and 8, the lesions weresuggestive of a less severe infection. Fail-ure to demonstrate viral antigen in thosepigs may have been simply a problem ofsampling. Pig No. 9 was already in theatrophic stage of TGE when the first biop-sy was obtained at 48 hours PE, and it hasbeen noted in baby pigs (15) and in thisstudy that the amount of viral antigen de-tectable by the FAT decreases significantlyin this stage. It should be mentioned, how-ever, that the TGE antigen was demon-strated in one pig (No. 6) which had shownneither lesions of TGE nor clinical signs

of enteric disorders. This would suggestthat a combination of immunofluorescenceand histopathological examinations on spe-cimens obtained from the jejunum may bethe best approach for the confirmation of adiagnosis of TGE in feeder swine.

It appeared that TGE virus probably re-plicated in all susceptible feeder swine thatwere exposed. Apparently, some of thesepigs had only a focal, mild to moderate in-fection and, likewise, suffered no apparentdiarrhea or soft stool. Other pigs of thesame age exposed to a similar amount ofvirus, had a more severe and extensive smallintestinal involvement and displayed clin-ical signs of enteric disorders variable inseverity. In this study, feeder swine weresingly exposed to the virus and this mayhave accounted for the low incidence of se-vere infections. If several feeder swine inclose contact are exposed to TGE virus andsome of them (e.g. pigs No. 5 and 9) suffera more severe intestinal infection, theyprobably will shed larger amounts of virusin their feces. This would contribute to anincrease in the amount of virus to whichthe other pigs are exposed which mightraise the incidence of severe infections andsigns of enteric disorders.Some authors (3, 8) feel that feeder

swine may play an important role in theepizootiology of TGE. Results of this studysuggesting that some feeder swine can sup-port a focal growth of TGE virus in theirsmall intestine without clinical signs of thedisease, for at least one week after theirexposure, stresses the importance for fur-ther studies in this area. Experimental andfield studies are presently underway in ourlaboratory with the principal goal of fur-ther defining the role of feeder swine asa reservoir of TGE infection for baby pigs.

ACKNOWLEDGMENTS

The technical assistance of Mr. LeonardRifas is greatly appreciated.

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20. U.S.D.A. Diagnostic Services: National Animal Dis-ease Laboratory, Ames, Iowa. Suggested componentsand procedures for utilizing the tissue culture fluor-escent antibody technique (FA) in the diagnosis ofhog cholera. 1965.

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