Experimental Edema Disease of Swine(E. coli Enterotoxemia)

III. Pathology and PathogenesisR. E. Clugston, N. 0. Nielsen and D. L. T. Smith*

ABSTRACT RESUME

Experimental colibacillary (Escherichia coli)enterotoxemia as described in this report mi-mics natural edema disease both clinicallyand in gross pathology. The histopathologyis characterized by accumulations of non-inflammatory edema and by arteriopathy. Thesmaller arterial and arteriolar changes re-corded here are similar to those described innatural edema disease. The vascular changesdescribed in recovered cases of experimentalcolibacillary enterotoxemia concur with thosereported in so-called subacute and chronicedema disease. The arteriolar changes thatoccur in colibacillary enterotoxemia of swineare comparable to those associated withhypertension.

Thin sections of cerebral cortex from fourpigs with acute experimental edema diseasewere examined by electron microscopy in anattempt to demonstrate brain edema. Sectionsfrom one pig taken during the convulsivephase of disease revealed dilatation of peri-vascular glial processes. However, examina-tion of sections taken from three other pigsduring an earlier phase of the neurologicaldisturbance revealed no significant lesions.We were unable to ascertain the role of brainedema in the pathogenesis of the nervous sys-tem disturbance in these experiments.

*Department of Veterinary Pathology, Weste-n Collegeof Veterinary Medicine. University of Saskatchewan,Saskatoon, Saskatchewan.

This work is taken from the Ph.D. thesis of Dr. Clug-ston, University of Saskatchewan, 1971.

The experiments were supported by a National ResearchCouncil of Canada Grant, A2901, and were do'ne whileDr. Clugston was a Fellow of the Medical ResearchCouncil of Canada.

Present address of senior author: Veterinary ServicesDivision, Alberta Department of Agriculture, O. S.Longman Buildiing, P. O. Box 8070, Edmonton, Al-berta T6H 4P2.

Submitted April 3, 1973.

L'enterotoxemie experimentale ia Escherichiacoli, telle qu'on la decrit dans cet article, si-mule la maladie naturelle de l'oedeme, tantpar les signes cliniques que par les lesionsmacroscopiques. Les lesions microscopiques setraduisent par des accumulations d'oedemenon-inflammatoire et de l'arteriopathie. Leslesions arterielles et arteriolaires mineuresque nous rapportons ici ressemblent 'a cellesqu'on a decrites dans la maladie naturelle. Leschangements vasculaires decrits chez les porcsayant survecu ia l'enterotoxemie colibacillaireexperimentale correspondent 'a ceux qu'on arapporte's dans les cas presumement subaiguset chroniques de la maladie de l'oedeme. Leschangements arteriolaires qui se produisentdans l'enterotoxemie colibacillaire porcine res-semblent 'a ceux qui accompagnent l'hyper-tension.On a examine au microscope electronique

des coupes minces du cortex cerebral de quatreporcs souffrant de la maladie experimentalede l'oedeme, dans le but de deceler la presenced'oedeme cerebral. Les coupes du cerveau d'unde ces porcs, preleve au cours de la phaseconvulsive, ont revele la presence d'une dila-tation des processus gliaux perivasculaires.Toutefois, l'examen des coupes du cerveau destrois autres porcs, 'a une phase anterieure dutrouble neurologique, n'a pas revele la presencede lesions significatives. Les auteurs n'ont pasreussi ia confirmer le role de l'oedeme dans lapathogenese du trouble du systeme nerveuxcentral, au cours de leurs experiences.

INTRODUCTION

Colibacillary (Escherichia coli) entero-toxemia or edema disease of swine is anacute disease characterized clinically by

Can. J. comp. Med.34

Fig. 1. Eye and adnexa. Note the protrusion of con-junctiva due to accumulation of edema; pig killed 25hours after intravenous EDP.

neurological signs and pathologically byedema at specific sites (1, 6, 9, 10, 12, 17,19, 20, 22, 23, 24, 25).The characteristic lesion, edema, focused

attention on the cardiovascular system. Ar-terial and arteriolar lesions including muraledema, hyaline degeneration and fibrinoidnecrosis have been described in naturaledema disease (7, 16, 18, 21). Kurtz et al(8) described the pathology in pigs killedfour days to six weeks following recoveryfrom acute clinical edema disease. Theydescribed fibrinoid necrosis, medial necro-sis and adventitial proliferation of smallarteries and arterioles in most body sys-tems. In addition to arteriopathy they ob-served malacic lesions in the brain stemof some pigs. These workers introducedthe terms subacute and chronic edema dis-ease to refer to their recovered cases andstated that this syndrome was character-ized by panarteritis.

Neurological signs including ataxia, con-vulsions and paralysis characterize clinicaledema disease. It has been suggested thatthese may be due to cerebral edema. In thiscondition clinical signs are first manifest-ed by irritation followed by the develop-ment of paralysis and their severity varieswith the rate of development of the edema(1'.Gruner (5), based on his ultrastructural

examinations of perifocal inflammatoryedema obtained by biopsy from human sub-jects, reported that brain swelling appearedto result from three phenomena; intercel-lular fluid accumulation, bubbles in myelinsheaths and swelling of astrocytic process-es. He believed astrocytic swelling, especi-

ally that of perivascular glial processes, tobe the most important process in brainedema. In general, it appears that corticaledema is primarily swelling of the astro-cytes, whereas edema of white matter isan accumulation of edema fluid in inter-cellular spaces between myelin sheaths(5, 26).Using pigs with experimental edema dis-

ease, an attempt was made to test thehypothesis that the neurological signs weredue to central nervous system (CNS)edema by examining suitably prepared sec-tions of cerebral cortex in the electron mi-croscope. Thorotrast was used as an elec-tron-dense marker to evaluate vascularpermeability.

This paper is a report of the gross andhistological findings from selected cases ofexperimental colibacillary enterotoxemia inpigs examined during the acute phase andfollowing recovery periods of varying dura-tion and results from electron microscopicexaminations of cerebral cortex from acutecases.

Fig. 2. Cross-section through cardial gland region andesophageal cardia of stomach. Note accumulation ofnon-inflammatory edema in submucosa of cardial glandregion; pig killed 25 hours after intravenous EDP.

Vol. 38 - January, 1974 35

9/9 8/85/9 5/88/8 8/86/8 1/85/8 0/8

TABLE II. Gross Necropsy Findings in Experi-mental Edema Disease

Lesion Group A Group B

Edema- Subcutis ...... 8/9 0/8-Lung......... 2/9 0/8- Cardial sub-

mucosa ..... 9/9 0/8- Mesentery of

spiral colon 4/9 0/8-Meninges . .7/9 0/8

Lymph nodes 4/9 0/8Ulceration esopha-

geal cardia . .... ..1/9 3/8Cornification eso-

phageal cardia. 4/9 2/8

TABLE III. Arteriopathy in ExperimentalEdema Disease

Lesion Group A Group B

Mural edema.......Hyaline change.....Medial necrosis.....Adventitial proli-

feration..........

6/97/94/90/9

MATERIALS AND METHODS

ANIMALS

Edema disease was produced experimen-tally in pigs by the intravenous inoculationof edema disease principle (EDP) (3).Seventeen pigs of mixed breeding (Yorkand Landrace) weighing from 15 to 46kg were selected from a large series ofexperimental cases (3) for gross and his-tological examinations. Cases were selectedthat developed a distinctive edema diseasesyndrome with minimal signs of endotoxinshock. These were divided into two groups.Group A, consisting of nine pigs, was usedto study the pathology of acute experimen-tal colibacillary enterotoxemia. Group B,consisting of eight pigs, was used to studythe pathology of experimental colibacillaryenterotoxemia following recovery periodsof varying duration after the acute syn-drome. Two uninoculated pigs served toprovide control tissues. An additional sixpigs were used for ultrastructural studiesof brain. Animals were kept in pens withslatted floors and fed commercial 16% hoggrower and water ad libitum. Pigs weremaintained in experimental quarters forat least one week prior to inoculation.

INOCULATIONS0/85/8 An effective dose (0.10 to 0.40 mg pro-8/8 tein per kg body weight) of EDP prepared

6/8 from E. coli 0139 (3) was given i.v. to

TABLE IV. Distribution of Histological Lesions in Experimental Edema Disease

Group A Group B

Site Edema Arteriopathy Edema Arteriopathy

Cardial submucosa.............Fundus submucosa.Pylorus subrnucosa.Small intestinal submucosa.Large intestinal submucosa.Urocoele submucosa............Lung.........................Lymph node..................Muscle.......................Adrenal gland.................Spinal cord....................Cerebral cortex................Medulla......................Brain stem....................Cerebellum ...................

Can. J. comp. Med.

TABLE I. Clinical Findings During AcutePhase of Experimental Edema Disease

Sign Group A Group B

Subcutaneous edemaAltered voice.......Ataxia.............Convulsions........Paralysis...........

6/71/80/81/85/82/61/84/80/80/85/96/97/99/95/9

6/82/81/82/85/85/60/87/90/82/81/92/90/92/90/9

2/40/40/40/40/40/40/40/40/40/41/80/80/81/80/8

4/43/42/44/44/44/42/44/42/43/46/87/87/85/85./8

36

each pig used for gross and histopatholog-ical studies. EDP was dissolved in M/15phosphate buffer, pH 8.2. In some casesclinically recovered animals received fur-ther effective doses of EDP. The EDPused for ultrastructural studies was di-alyzed through an XMJ0O Diaflo mem-brane and was not treated with sodiumdeoxycholate. For the ultrastructural stud-ies two control pigs were given M/15 phos-phate buffer, pH 8.2, i.v. Three experimen-tal and both control animals receivedthorotrast,' 0.1 mg/kg body weight 30 to80 minutes prior to euthanasia and the re-maining pig received EDP alone.

GROSS AND HISTOPATHOLOGY

Complete necropsy examinations wereconducted on all animals at post-inoculationtimes varying from 25 to 75 hours in theacute disease group (Group A) and fromten to 55 days in the recovered group(Group B). Prior to necropsy the animal;

1Te3tagar Company Inc.

either died from acute disease or were eu-thanized by the intravenous administrationof barbiturates or by brain perfusion withformaldehyde solution under general halo-thane anesthesia.

Various tissues were fixed in 10% neu-tral buffered formalin, embedded in paraf-fin, sectioned at 6 ,u, and stained withhemotoxylin and eosin.

ULTRASTRUCTURAL STUDIES

Twenty-eight to 49 hours after inocula-tion with EDP during the acute phase ofedema disease, experimental animals wereanesthetized with halothane. The skin wasincised along the ventral midline of theneck from the level of the mandibularangles to the anterior sternum. Both caro-tid ar'eries were exposed by blunt dissec-tion and polyethylene catheters were in-serted. Following transection of both in-ternal and external jugular veins, 1 litreeach of paraformaldehyde, 4.0% in phos-phate buffer (pH 7.2 to 7.3, final osmolar-ity - 782 milliosmols); and gluteralde-

Fig. 3. Submucosa, cardial gland region of stomach: left control, right submucosal edema, pig killed 58 hoursafte; intravenous ETP- Note edema fluid contains very few cells. H & E. X136.

Vol. 38- January, 1974 37

hyde, 2.5% in phosphate buffer (pH 7.2 to7.3 - 782 milliosmols), were perfused se-quentially. Perfusion times varied from 20to 30 minutes.

Following removal of the calvarium andexcision of the perfused brain, slices 2 to 3mm thick were removed from the motorcortex (superior longitudinal gyrus). Cubesless than 1 mm square and 3 to 4 mm inlength were removed from these slices andimmersed in 2.5% gluteraldehyde solutionat room temperature for a total fixationtime of two hours. Following a wash periodof one hour in phosphate buffer (pH 7.2 to7.3, 320 to 327 milliosmols), the cubes werefixed in cold osmic acid, 1% in phosphatebuffer for one to one and one-half hours.Tissues were dehydrated in alcohol, embed-ded in epoxy resin, Epon2, or Spurr3, andthin sections cut with a diamond knife in aPorter Blum MT II ultra-microtome. Thetissue sections were placed on 200 mesh cop-per grids and stained with uranyl acetate,

2Epon-Epoxy 812-Fisher Scientific

3Spurr-Polysciences Inc.

1% in water for 30 minutes at roonm tem-perature, and Reynolds' lead citrate for twominutes at 10°C. The prepared sectionswere examined and photographed in anelectron microscope, Zeiss E.M. 9A.

RESULTS

Clinical findings are summarized inTable I. All pigs in both groups developedtypical clinical signs of edema disease in-cluding subcutaneous edema and neurolog-ical disturbances. The syndrome was de-layed in onset (17 to 57 hours after inocu-lation) and progressed in severity. Clinicalsigns of edema disease had disappeared inthe pigs of Group B at the time they wereeuthanized.

GROSS PATHOLOGY

Gross necropsy examinations of all pigsin Group A revealed accumulations of non-

Fig. 4. Brain sten; left control, right perivascular edema, pig dead 70 hours after intravenous EDP. H & E.X540.

Can. J. comp. Med.38

inflammatory edema in sites typical ofedema disease (Table II, Figs. 1 and 2).No edematous lesions were observed in thepigs in Group B. A few pigs in both groupshad cornification and, in some cases, ul-ceration of the esophageal cardia region ofthe stomach.

HISTOPATHOLOGY

The pathological histology of experimen-tal colibacillary enterotoxemia was charac-terized by accumulations of non-inflam-matory edema (Table IV, Figs. 3 to 5) andarteriopathy (Tables III and IV, Figs. 6 to8).The primary arterial changes in acu'e

cases were mural edema and hyaline degen-eration. In recovered cases medial necrosismanifested by nuclear pyknosis and kary-orrhexis in the muscle cells of the arteri-oles was the most prominent change ob-served. Hyaline change and adventitial pro-liferaticn were also common in recoveredcases (Table III).

The distribution of lesions is recordedin Table IV. Main sites of non-inflamma-

tory fluid accumulation in Group A werethe submucosa of the cardial gland areaof the stomach, the submucosa of thececum, lymph nodes and the central nervoussystem. Edema was an uncommon findingin tissues from pigs in Group B. Arteriolarchanges in acute cases were most commonlyobserved in the submucosa of the large in-testine, the urinary bladder and in lymphnodes. Arteriopathy was common in bothgroups but was more frequent and moreadvanced in tissues examined from animalsin Group B (Table III).

ULTRASTRUCTURAL STUDIES

The first of the four pigs given EDPdeveloped a severe central nervous systemdisturbance and was killed 28 hours afterinoculation during the convulsive phase and30 minutes after the intravenous adminis-tration of thorotrast. Gross lesions in thispig were typical of edema disease, withedema in the subcutaneous tissues of theforehead, in the submucosa of the cardialgland region of the stomach and in the

Fig. 5. Cerebral sulcus; left control, right meningeal edema, pig dead 45 hours after intravenous EDP. H & E.X136.

Vol. 38 - January, 1974 39

mesentery of the spiral colon. Meningealedema was also observed grossly. Electronmicrographs from the cerebral cortex ofthis pig revealed extensive accumulationsof electron-dense particles, apparently with-in perivascular glial processes. Electron-dense particles were also observed insmooth muscle cells of arterioles. The peri-vascular glial processes appeared to beexpanded (Fig. 9).

The two additional pigs given thorotrasti.v. during the acute phase of experimentaledema disease had typical edema accumula-tions and were killed during the early phaseof neurological disturbance. Ataxia was theonly clinical sign referable to the centralnervous system at the time of euthanasia.Electron-dense particles were observed inthe perivascular glial processes in the cere-bral cortex of these pigs, but the glial pro-cesses appeared normal.The fourth EDP inoculated animal in

this series was not given thorotrast. Thispig developed typical accumulations ofedema and was killed when ataxic duringthe early neurological phase of the disease.

Electron micrographs from the cerebralcortex of this animal contained no recog-nizable lesion.No clinical signs or significant gross le-

sions were observed in the two control pigsgiven phosphate buffer i.v. and subsequent-ly, thorotrast i.v. 50 and 60 minutes priorto brain perfusion. Some electron micro-graphs from one of these controls containedelectron-dense granules in the perivascularglial processes. Electron micrographs fromthe other control pig did not have recog-nizable thorotrast in extravascular tissues.

DISCUSSION

The diagnosis and selection of experi-mental cases of colibacillary enterotoxemia(edema disease) for this study were madeon the basis of clinical signs, most prom-inent of which were subcutaneous edemaand neurological disturbances. Gross le-

Fig. 6. Arterioles in submucosa of cardial gland region of stomach; left control, right mural edema, pig dead 47hours after intravenous EDP. H & E. X540.

40 Can. J. comp. Med.

sions, particularly the accumulation ofedema at specific sites, confirmed the diag-nosis in those pigs which died or werekilled during the acute stage (Group A).In the recovered group (Group B) thediagnosis of colibacillary enterotoxemia wasmade solely on the basis of the clinicalsigns of edema disease observed during theacute stage. There were no characteristicgross lesions in Group B.The cornification, erosion and ulceration

of the esophageal cardia, in some cases withassociated inflammatory edema in the sub-mucosa of adjacent areas of the cardialgland region, were considered to be inci-dental findings.

In experimental colibacillary enterotox-emia, where there were no lesions in theesophageal cardia, the edema accumulationswere non-inflammatory in nature. The fluidappeared low in protein and contained vir-tually no inflammatory cells. This type offluid accumulation could have resultedfrom a relatively mild increase in vascularpermeability, with an increased permeabil-ity to solutes but of insufficient degree to

permit excess loss of colloids from the in-travascular compartment.The vascular lesions, restricted to small

arteries and arterioles, appeared to haveprogressed from mural edema to hyalinedegeneration followed by necrosis and ad-ventitial proliferation. There are two theo-retical possibilities for the nature of arteri-olar hyaline. One is that it derives in s-itufrom degeneration of medial smooth musesthe other that it has a hematogenous origin(13). The morphological changes reportedhere implicate the arterioles and small ar-teries as the site of increased net outwardmovement of fluid leading to the accumu-lation of non-inflammatory edema. If thishypothesis is correct, the hyaline materialin vessels in experimental colibacillary en-terotoxemia probably is hematogenous inorgin. The subsequent necrosis and reac-tion are incited by the presence of fluidand hyaline material in the vessel wall.

Arteriosclerosis including hyaline arteri-olar sclerosis, hyperplastic arteriolarsclerosis and fibrinoid necrosis of arteri-oles is classically associated with hyperten-

Fig. 7. Arterioles in submucosa of urinary bladder, Ieft control, right hyaline change, pig killed 58 hours after-intravenous EDP. H & E. X540.

Vol. 38 - January, 1974 41

Fig. 8. Arterioles in submucosa of cardial gland region of stomach; left control, right proliferative arteriopathy,pig killed 17 days alter intravenous EDP. H & E. X540.

sioIn in human pathology (14). Dustin (4)pointed out the non-specific nature of fi-brinoid arteriolar necrosis and stated thatthis type of arteriolar change could occurin a variety of different clinical or experi-mental situations.The role of brain edema in the patho-

genesis of the central nervous system dis-turbance in experimental edema diseasewas not established. In most brain histol-ogy sections from Group A animals, lesionsof perivascular and/or meningeal edemawere present. Ultrastructural evidence ofdilatation of perivascular glial processeswas present in cerebral cortex samples fromone pig killed during the convulsive phaseof disease. However similar sections fromthe other three EDP inoculated pigs, killedduring a less severe stage of neurologicaldisturbance did not contain recognizablelesions. These latter animals may have beenexamined at a stage of disease prior to thedevelopment of a morphological lesion. Ad-ditionally, correlation of light microscopicand electron microscopic examination werenot made. Such correlation might have fa-cilitated the detection of lesions.

Attempts to determine the permeabilitycharacteristics of cerebral vessels, usingthorotrast as an electron-dense marker,during the acute stage of edema diseasewere inconclusive. Electron-dense granuleswere observed in extravascular sites in

Fig. 9. Cerebral cortex pig killed 28 hours after intra-venous EDP. Electron dense particles in smooth musclecell and in perivascular glial processes. Uranyl acetateand lead citrate. X27,000.s - smooth muscle cellg - glial process- Thorotrast particles

Can. J. comp. Med.42

cerebral cortex samples from all pigs inoc-ulated with EDP, and also in some electronmicrographs from one of the controls.Rowley (15) reported that the intravane-ous administration of thorotrast to rats re-sulted in mast cell damage with subsequentincreased vascular permeability and criti-cized the use of this material as a perme-ability marker. Lampert and Carpenter(11) were unable to detect extravascularthorotrast in normal rats and believed thismaterial satisfactory to test vascular per-meability. The artifactitious movement ofmorphological tracers into cells, possiblydue to rupture of cell membranes duringfixation, is a common problem (2). Eithermast cell damage with release of perme-ability factors or direct cell membrane rup-ture may explain the extravascular locationof thorotrast particles in our pigs. Moreextensive ultrastructural studies, using ad-ditional morphological tracers and correla-tion of light and electron microscopic ex-aminations, should help to elucidate thepathogenesis of the central nervous systemdisturbance.

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