epizootic rabbit enteropathy: experimental transmission...

601Vet. Res. 36 (2005) 601–613© INRA, EDP Sciences, 2005DOI: 10.1051/vetres:2005021

Original article

Epizootic Rabbit Enteropathy: experimental transmission and clinical characterization

Dominique LICOISa*, Monique WYERSb, Pierre COUDERTa

a INRA, UR86 BioAgresseurs, Santé, Environnement, Centre de Tours, 37380 Nouzilly, Franceb Unité d’Anatomie Pathologique, École Nationale Vétérinaire, BP 40706,

44307 Nantes Cedex 03, France

(Received 25 August 2004; accepted 14 December 2004)

Abstract – In late 1996 in France, a severe digestive disease appeared in fattening domestic rabbits.Named the Epizootic Rabbit Enteropathy (ERE), this digestive syndrome has become the maincause of mortality in rabbit farming. The diagnosis in field conditions is difficult because co-infection with other common rabbit pathogens is frequent. By using specific pathogenic free (SPF)rabbits and starting from a field sample of intestinal contents of diseased animals, a virulent material(inoculum) was obtained free of almost all known pathogens but reproduced the symptoms andlesions of ERE. Four hundred and seven SPF rabbits were used in five trials to describe the disease.ERE is characterized by a high contagiousness, 30 to 40% mortality in a few days and about 100%morbidity whatever the dose of the inoculum used. Clinical signs and lesions evolved acutely withthe first sign (rambling noise) appearing one day after inoculation and the disease peaking 4 to6 days later. Growth was strongly lowered from the second day to the end of the second week.Rambling noise and distended abdomen were frequent, mucus excretion and cecal impaction werefrequent but not constant. ERE at necropsy was characterized by the absence of any inflammatoryor congestive lesions on the gut or on other organs but with the typical presence of a stomach and/orduodenum dilated by liquid and gas and by the absence of specific histological lesions. Theetiological agent has not been identified yet, but we demonstrate that the intestinal content wasinfectious as early as the second day. This work constitutes the experimental basis for studies on thisemerging disease within the framework of etiological research led in different Europeanlaboratories working with the infectious material.

epizootic rabbit enteropathy / intestinal pathology / diarrhea / mucoid enteritis

1. INTRODUCTION

In late 1996 and early 1997, an emergentand severe gastrointestinal syndrome appearedin rabbit farms in the west of France. Thispathology is characterized by a distendedabdomen, emission of small quantities ofwatery diarrhea followed by a decrease infeed intake and by high mortality rates (30–

80%) during this period. It spread very rap-idly to other regions of France in 1997 and1998 [11] and in Europe: Spain, Portugal,Hungary, Belgium, The Netherlands, GreatBritain, etc. [9, 17, 20]. Nevertheless, to ourknowledge, this disease has not been reportedin other countries of the world except NorthAfrica (Colin, personal communication). InFrance, it is currently estimated that over

* Corresponding author: [email protected]

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602 D. Licois et al.

95% of farms, whatever the rabbit race andstrain, are or have been affected by thisintestinal affection [19]. Because of therapid spreading of the disease, it was calledEpizootic Rabbit Enteropathy (ERE). Thedisease mainly affects young fattening rab-bits, between six and eight weeks of age.Problems usually occur after weaning buthave also been observed in older rabbits andsometimes in adults or in suckling rabbits,just before weaning. Unlike other epizooticdiseases (myxomatosis, viral haemorrhagicdisease), wild rabbits do not seem to beaffected. Nevertheless, ERE has beenobserved in wild rabbit breeding units [26].

Because of the outbreak of ERE onfarms soon after the delivery of feed by acommercial factory, the feed was initiallysuspected and various hypotheses werestudied (rate and nature of the raw materials,mycotoxins, pesticides, etc.) but all theseassumptions have been eliminated [18].Later it was demonstrated that the feedcould be a passive vector [10, 16]. However,a disease was experimentally reproducedwith the same symptomatology and/orlesions as those of ERE observed in thefield, either with intestinal contents origi-nating from ill or dead animals or by contactbetween animals or also by contact withcontaminated breeding material [26]. Allthese observations associated with thosefrom the field have demonstrated the con-tagious feature of the disease and thereforethat a pathogenic agent is involved in thedevelopment of this intestinal pathology.However, no pathogenic organism has beenidentified and isolated for the moment.

Moreover, although most of ERE clini-cal signs could be reproduced successfully1

[23, 24, 26], we encountered many difficul-ties in obtaining reproducible results, mainlybecause samples originating from the field

are frequently contaminated by opportunis-tic pathogens (coccidia, Escherichia coli,Klebsiella, etc.). In order to reduce this var-iability, different methodologies were triedand several methods of experimental con-tamination were tested: immunosuppres-sion of the animals [25], intubation via theoesophagus, spraying of the inoculum onthe animal or the feed. Different potentiallyinfectious materials were inoculated by theoral route (lung, mesenteric lymph nodes,blood, intestinal contents) without success,except with intestinal contents. This, asso-ciated to the fact that no etiological agenthas yet been identified, led us to use thiskind of biological material in our studies.Thus, an inoculum (TEC) constituted byvirulent intestinal contents without oppor-tunistic pathogens was obtained, as well asother inocula deriving from TEC.

This paper deals with the experimentaltransmission of ERE based on the use ofthese virulent materials and specific patho-gen free (SPF) rabbits. The most constantresults were obtained by contamination ofanimals with these inocula sprayed on thefeed or directly given by the oral route. Thismade it possible to standardize experimen-tal reproduction of ERE and precisely describethe disease.

2. MATERIALS AND METHODS

2.1. Animals and experimental conditions

All the rabbits used were SPF animalsoriginating from the Experimental Unit“Pathologie Aviaire et Parasitologie” (INRA,Nouzilly, France). These SPF rabbits werenotably free of Rotavirus, Eimeria spp.,Passalurus ambiguus, Pasteurella multoc-ida, Clostridium spiroforme and enter-opathogenic Escherichia coli belonging tothe serogroups O2, O15, O26, O49, O85,O103, O109, O128, and O132. They are alsoregularly monitored according to the rec-ommendations of Federation of European

1 Licois D., Lebas F., Coudert P., Le Gall G., Noted’information N° 10 sur les travaux de rechercheconduits sur l'Entérocolite Épizootique du Lapin,[on line], 1999. http://www.tours.inra.fr/urbase/internet/resultats/enterocolite/info10.htm [consultedJanuary 20, 2005].

Epizootic Rabbit Enteropathy 603

Laboratory Animal Science Association(FELASA) [30]. They were reared in highlyprotected facilities (air filtered at 10 µm,specific staff) according to the methods pre-viously described2. At weaning (30 days),they were transferred to experimental facil-ities. The experimental conditions havebeen previously described [6]. Briefly, therooms and material were disinfected twicebefore each experiment (steam under pres-sure followed by gaseous formalin). All theequipment used for water supply was auto-claved before the experiments. The exper-imental rooms were in depression (200 Pa)and the air was filtered (1 µm input–0.3 µmoutput). The animals were distributed with2 or 3 per cage according to their originallitter and weight. They received water andlaboratory feed free of antibiotic and antic-occidial drugs ad libitum (UAR 110, Ville-moison/Orge, France). They were inocu-lated between 33 and 36 days of agedepending on the trial.

2.2. Inocula and inoculation

The first sample used as the inoculum,denominated TEC, was kindly provided byP. Hervouet (Biovac, Beaucouzé, France)and P. Robart (Techna, Coueron, France),in late 1997. It was obtained from rabbitsaffected by ERE on a commercial farm. Thecrude intestinal content (small intestine pluscecum-colon) was diluted to 1/3 in sterilePBS (phosphate buffered saline), filtered(0.5 mm) to eliminate large vegetable par-ticles, then centrifuged at 1000 g for 15 min.This centrifugation does not eliminate bac-teria nor viruses, but does eliminate para-sites (coccidia). The raw supernatant thusobtained constituted the first inoculum,which was stored at –20 °C until use. One

millilitre of this inoculum corresponds toabout 1 mL of fresh intestinal content.Regarding the trials carried out in 2001–2002, different inocula originating fromTEC were used. They were treated asdescribed above except that PBS wasreplaced by sterile water, that was provennot to modify the infectivity of the inoculaand subsequently the response of the ani-mals.

In April 2001, an inoculum (TEC1) wasproduced by mixing several selected sam-ples originating from 5 trials carried out onSPF animals inoculated with TEC. Some ofthese intestinal contents were obtained afterabout 10 successive passages in rabbits.TEC1 came from the intestinal contents of19 sick or dead animals chosen between day3 and day 8 post inoculation (d PI) in orderto cover the acute period of the disease aslargely as possible. A second inoculum(TEC2) was composed of the intestinal con-tents of 27 moribund or dead rabbitsinfected with TEC1, between d3 and d7 PI,and then a third inoculum (TEC3) wasobtained from animals inoculated withTEC2, dead or sick between d2 and d11 PI.TEC3 was stored for over 2 years at –20 °Cwithout losing its infectivity.

Different doses of the inocula expressedin the text for the individual rabbit, weresprayed on 100 g of pelleted feed, consid-ered as a daily ration for one rabbit, then letto dry a dozen hours before being distrib-uted in the mangers.

All the control animals were inoculatedin the same way, with intestinal contentsobtained from healthy SPF rabbits.

2.3. Microbiological characterizationof the inocula

TEC1, TEC2 and TEC3 inocula werepartly characterized at the virological, bac-teriological and parasitical level. Coccidiawere sought for according to the methoddescribed by Coudert et al. [7]. The searchfor rotavirus was performed by an enzymeimmunoassay and PCR according to the

2 Coudert P., Licois D., Besnard J., Establishmentof a Specified Pathogen Free breeding colony(SPF) without hysterectomy and hand-rearing pro-cedures, in: Holdas S. (Ed.), Proc. 4th Congress ofthe World Rabbit Science Association, Budapest,RCPAN, Herceghalom, Hungary, 1988, pp.137–148.


procedures previously described [5]. Cali-civirus was searched for using RT-PCRaccording to the method described byCapucci et al. [3]. The search for otherenterotropic viruses (pestivirus, circovirus,adenovirus, coronavirus, parvovirus) wasperformed by G. Le Gall (AFSSA, Ploufra-gan, France) using PCR or RT-PCR. Thesearch for coliforms and Clostridium spiro-forme was carried out by Professor A. Milon(UMR 960, INRA-ENV Toulouse, France).Detection of C. spiroforme was performedby light microscopic examination on Gramstained smears of 10–1 dilution of the inoc-ula, according to Carman and Borriello [4].The presence of E. coli was evaluated afterculture at 37 °C for 24 h of 10-fold dilutionsof inocula in EMB (Eosine Methylene-blue) agar [21]. The search for otherClostridium and Bacteroides fragilis wascarried out by Doctor M.R. Popoff (CentreNational de Référence des BactériesAnaérobies, Institut Pasteur, Paris, France).PCR detection of toxin genes of C. perfrin-gens (toxins α, β1 β2 and enterotoxin),C. sordelli (toxin LT) and Bacteroides fra-gilis (enterotoxin), was carried out accord-ing to the method previously described [31]after 24 h liquid culture in anaerobic con-ditions, at 37 °C in tryptose-glucose yeast(TGY) extract, followed by DNA extrac-tion (Instagen, Biorad).

2.4. Experimental design (Table I)

The aim of the three trials (A, B and C)was to describe the disease and test the vir-ulence of the inocula at different doses andwith different methods of inoculation. Trial Dwas conducted to analyze the first lesions atautopsy and trial E to detect the earlyappearance of virulent material in the intes-tinal content. All the experiments followedthe guidelines stated in the Guide for theCare and Use of Laboratory Animals [13].

2.4.1. Study of ERE clinical signsand lesions

Trial A: Fifty-five rabbits were distributed2 per cage in 2 different rooms with respec-

tively 23 animals inoculated with 10 mLTEC1 and 32 control animals. They wereobserved for 7 days post inoculation. Inaddition, 8 rabbits were used for routine his-tological examination of the intestinalmucosa. Four inoculated rabbits were suc-cessively slaughtered at d5 and d7 PI.

Trial B: Two groups of 25 and 36 ani-mals were inoculated with 10 mL and 1 mLTEC2, respectively. One group of 12 rab-bits was used as the control. Each group wasin a separate room, with 3 animals per cageobserved for 9 days PI. Thirty additionalrabbits were also used for gross lesionobservation and histological study. Twodiseased animals and one control animalwere slaughtered from d1 to d10 PI.

Trial C: In this third trial, we used 6 batchesof animals (3 per cage) placed in 3 separaterooms: 4 inoculated groups and 2 controlgroups. Three batches of 24 rabbits eachwere inoculated via contaminated feed with10 mL, 1 mL and 0.2 mL inoculum TEC3,respectively. A fourth batch of 24 rabbitswas inoculated by oral route by means of amicropipette with 0.2 mL TEC3. One of thetwo control groups was in a room withoutcontaminated animals. The animals werestudied for 17 days.

Trial D: In this trial, 45 rabbits wereinoculated with 1 mL of TEC2 and 12 wereused as the control. There were 3 rabbits percage in the same room. On days 1, 2 and 3 PI,15 infected and 3 control rabbits per daywere euthanized for autopsy.

2.4.2. Detection of early virulent material in the intestinal content (Trial E)

Five rabbits showing signs of disease(rambling noise for 2 animals and/or weakDWG) and 6 rabbits also with signs of dis-ease (rambling noise for 5 animals, diarrheafor 1 animal and/or weak DWG), originat-ing from inoculated animals of trial D, euth-anized respectively on d1 and d2 PI, wereused. Their intestinal contents were used asfresh material for infecting two groups of15 rabbits (3 per cage). These rabbits were


contaminated (via feed) at their arrival inthe experimental room with 10 mL of thefresh intestinal material. Another group of18 rabbits was used as the control animalsinoculated with fresh samples from SPFanimals.

2.5. Histological examination

Diseased and control rabbits were euth-anized by pentobarbital anesthesia and then

exsanguinated. The abdomens were openedand samples of the intestine (1-cm long) anddifferent organs (5 mm3) were collected andfixed in 10% buffered formalin. In the firstexperiment, 8 inoculated rabbits were usedfor only ileum histological examination. Inthe second experiment, the stomach, duo-denum, jejunum, ileum, cecum, vermiformappendix, proximal colon, lung, mesentericlymph nodes, liver, spleen, kidney and heartwere sampled in 10 control animals and

Table I. Experimental design of the different trials (A, B, C, D and E).

Groups Room No. No. of animals Inoculum Dose (mL of inoculum)

Trial A (7 days)

Control 1 32 HICb 10

Inoculated 2 23 (+ 8a) TEC1 10

Trial B (9 days)

Control 1 12 (+10a) HIC 10

Inoculated, 10 mL 2 25 (+10a) TEC2 10

Inoculated, 1 mL 3 36 (+10a) TEC2 1

Trial C (17 days)

Control 1 1 18 HIC 10

Control 2 3 18 HIC 10

Inoculated, 10 mL 1 24 TEC3 10

Inoculated, 1 mL 1 24 TEC3 1

Inoculated, 0.2 mL 2 24 TEC3 0.2

Inoculated, 0.2 mL (OR)c 2 24 TEC3 0.2

Trial D (3 days)

Control 1 12 HIC 10

Inoculated 1 45 TEC2 10

Trial E (6 days)

Control 2 18 HIC 10

Inoculated IC d1d 2 15 IC d1 10

Inoculated IC d2d 2 15 IC d2 10

a Additional rabbits used for histology.b HIC: Intestinal content from healthy SPF rabbits, administered to animals from a control group byspraying on feed.c Group of rabbits inoculated by the oral route with a pipette. All the other animals in the different trialswere inoculated by spraying the inoculum on feed. Doses are given for the individual animal.d Groups of rabbits inoculated with fresh intestinal content from affected rabbits of trial D, slaughteredon day1 (IC d1) and day 2 (IC d2) post inoculation.


20 inoculated rabbits. In the inoculatedgroup, the animals with the most obviousclinical signs (11 rabbits) were the onlycases submitted to histological examina-tion. The tissue specimens were embeddedin paraffin wax, cut at 4 µm and stained withHematoxylin eosin and Saffran (HES) to beexamined under light microscopy.

2.6. Parameters recorded and statistical analysis

A daily examination of the clinical signswas performed: diarrhea, bloat, rumblingnoise when the animals were grasped andslightly shaken (borborygmus), and cecalimpaction on abdominal palpation. The ani-mals were weighed three times during thefirst week after inoculation and twice later.Weight gains were compared by varianceanalysis of two factors with a mean com-parison by means of the Tukey test using theSystat statistical software package [33] andmortality was analyzed by χ2. All dead rab-bits were autopsied for examination of thedigestive tract and vital organs (lung, liver,heart, kidney, etc.).

3. RESULTS

3.1. Microbiological characteristicsof the inocula

On direct examination, TEC1, TEC2 andTEC3 were controlled free of coliforms(10-1 dilution); the flora was poor and unbal-anced, Gram positive bacteria like Clostrid-ium were dominant; however, Clostridiumspiroforme was not detected. On the con-trary, Clostridium perfringens belonging totypes Alpha or Beta2 were identified. Ineach of the inocula, the search for rotaviruswas negative by an ELISA test but positiveby PCR. The search for other enterotropicviruses (calicivirus, pestivirus, circovirus,adenovirus, coronavirus, parvovirus) wasnegative. No intestinal parasites were de-tected.

3.2. Description of the disease(trials A, B, C)

3.2.1. Mortality and clinical signs

Neither mortality nor clinical sign of dis-ease were observed in the control groups ofany of the three trials, except for the controlanimals of trial C which were located in thesame room as the inoculated rabbits. In thiscontrol group no animal died during the17 days of observation, but clinical signs(rambling noise and/or diarrhea) could bedetected after the 5th day PI in 28% (n = 5/18) of the animals.

After the experimental contamination(day 0) in the three trials A, B and C, mor-tality began on day 3 or 4 with a peak on day5 or 6. For the first cases of mortality, therabbits had no diarrhea. At the end of thesecond week, 30 to 50% of the rabbits weredead or moribund. Regarding morbidity, allthe rabbits showed at least one sign of dis-ease: rambling noise (which was the mostconstant clinical sign, appeared as soon asd2 PI), cecal impaction (which could bedetected as soon as d3 on abdominal palpa-tion), diarrhea, distended abdomen andexcretion of mucus (which could mainly beobserved from d4). On days 4, 5 and 6, mostof these criteria were associated, but noneof them was constant. Diarrhea was veryaqueous and of low intensity at its onset.

3.2.2. Gross lesions

At autopsy, no macroscopic sign ofinflammation or congestion was detectablein any part of the intestine or other organs(liver, spleen, kidney, lung, heart). This par-ticularity concerned all the inoculated rab-bits, whatever the trial. Cecal impactionwhich affected 20 to 30% of the dead ani-mals according to the trial, was either totalor partial (Fig. 1); in the latter case, impac-tion was generally located on the side of theappendix vermiform and the content of theother side was very liquid. The distal colonwas generally empty and free of droppings.


The most constant feature, encountered in50 to 80% according to the trial, was the dil-atation of the stomach filled with liquid andgas (Fig. 2) and the distension of the smallintestine, particularly the duodenum, by alarge amount of liquid and sometimes gas.

Whatever the doses (10 mL, 1 mL or0.2 mL) or the method (feed contaminationor oral route), no differences in mortality,clinical signs or lesions were observed.

3.2.3. Evolution of the daily weight gain

In all trials, the daily weight gain (DWG)of all uninoculated control groups wasaround 40 g/day during the first week(Fig. 3). When the uninoculated controlgroup was placed in the same room as theinoculated animals, a significant decreasein the DWG appeared at the end of the firstweek (P < 0.01).

In all groups inoculated with TEC inoc-ula (trials A, B, C as well as trial D), theDWG began to decrease between d0 and d2PI (Fig. 3). This early DWG decrease wassignificant in each trial (P < 0.01). The low-est DWG in all inoculated groups wasreached 5 to 7 days after inoculation and nodifference was observed according to thedoses used. During this peak of the disease,in each trial (A, B and C), the DWG was sig-nificantly different from that of the uninoc-ulated groups located in a separate room(P < 0.01). Between d7 and d9 (trial B) ord17 (trial C), the surviving animals slowlyrecovered a DWG comparable to that of theuninoculated (and non contaminated) groups.

3.2.4. Histological examination

In trial A, the histological examination(of the ileum) of d5 PI inoculated rabbits(n = 4) did not reveal any degenerativechanges of the epithelium nor inflammatorylesions of the lamina propria. In d7 PI inoc-ulated rabbits (n = 4), a mild and focal infil-tration by a polymorphonuclear heterophilwas observed in the lamina propria of allthe inoculated animals with limited figures

of transepithelial exocytosis. The size andaspect of the villi were normal without anyepithelial changes.

In trial B, none of the 12 control rabbitspresented any histological changes of thedifferent parts of their digestive tract (Fig. 4a),nor of their other examined organs. In the11 inoculated animals examined, histolog-ical lesions were totally absent in 2 rabbitsrespectively sampled at d7 and d10 PI, inspite of marked gross changes observed inboth animals. The stomach as well as theliver, spleen, lung and heart, were devoid oflesions in all the examined rabbits. The dif-ferent parts of the intestinal tract presentedmild to moderate lesions in all the other9 inoculated rabbits. The lesions were char-acterized in 6 of them by a mild to moderateatrophy of the villi with focal images of vil-lous fusion (Fig. 4b). The villi changes wereassociated in two animals with a moderateepithelial exocytosis of heterophil cells. Nochanges in lesion intensity were notedaccording to time PI. In one rabbit slaugh-tered on d2 PI, only a moderate hyperhemialocated in the duodenum was observed. Thejejuno ileal part of the intestine of one rabbitslaughtered at 7d PI and the duodenal partof another rabbit sampled at 5d PI exhibitedfocal limited acute necroticohemorrhagicand ulcerative lesions of the mucosa asso-ciated with numerous bacterial colonies.Moderate diffuse infiltration by polymor-phous inflammatory cells associated with amoderate crypt hyperplasia were the onlylesions observed in one rabbit at d6 PI.Aplasia of the lymphoid follicles of the ver-miform appendix was noted in three ani-mals at d5, d7 and d10 PI.

3.2.5. Detection of early infectious material derived from affected animals (trial E)

In rabbits inoculated with intestinal con-tents coming from animals of trial D euth-anized at d1 PI, no significant differencewas observed between the uninoculated andinoculated groups from d1 to d6: no death,


Figure 2. Six-week-old rabbit, dead 5 days after experimental reproduction of ERE with inoculumTEC3. The stomach and small intestin are distended and filled with liquid and gas and are responsiblefor the bloated abdomen. These lesions associated with the absence of visible inflammation of theintestinal tract can be considered as being pathognomonic of the disease.

Figure 1. Six-week-old rabbit, dead 5 days after experimental reproduction of ERE with inoculumTEC3, showing total caecal impaction, one of the main gross lesions of the disease. There are noinflammation or congestion visible on the caecum wall.


no clinical signs, no decrease in the DWG.Between d0 and d1, the DWG of the unin-oculated group was unusually low andtherefore no conclusion could be drawn.

In the rabbits inoculated with intestinalcontents coming from animals of trial D andeuthanized at d2, a significant lower DWG(P < 0.05) was observed in the inoculatedgroup on day 2 PI (Fig. 5), associated withsigns of rambling noise in 40% of the rab-bits. No mortality was observed until theend of the experiment (D6).

4. DISCUSSION

Under field conditions, very few criteriaenable a precise diagnosis of the ERE andnone of them are specific: increased mor-tality, presence of mucus under the cages,cecal impaction, inefficacy of usual antibi-otics are the most common anatomo-clini-cal signs usually noticed. Associated or pri-mary pathologies generally mask the specificlesions. Moreover, ERE itself apparentlypromoted the development of germs rarely

Figure 3. Description ofthe ERE (Trial B). Dailyweight gain of controlanimals and rabbitsinoculated with 1 mLand 10 mL of inoculumTEC2. The results areexpressed as the mean ±standard deviation.

Figure 4. Histological examination of the jejunum of six-week-old rabbits, slaughtered 10 days afterexperimental reproduction of ERE with inoculum TEC2 (HES x10). (A) healthy non inoculated rab-bit; (B) disease inoculated rabbit. Compared with the control, at the same magnification, the villi ofthe jejunum part of the small intestine appear shortened with irregular shape and size. The superficialepithelium of the villi, the crypts and the lamina propria are normal without any degenerative, hyper-plastic or inflammatory visible changes. The lesions are mild and devoid of etiological specificity.


isolated before 1997 (Klebsiella, E. coli O2,Clostridium, etc.) [1] and even parasiteslike coccidia [8]. In the field, the diagnosisis all the more dubious as the general hygi-enic conditions of the farm are precarious.In the literature, denominations such as“mucoid enteritis”, “mucoid enteropathy”,etc., are misleading because mucus excre-tion as well as cecal impaction in rabbits arephysiopathological reactions common toseveral diseases.

The aim of this work was to demonstratethe transmission of the disease and to iden-tify some specific criteria for diagnosingERE and describing its development. Wedeveloped an experimental model based onthe use of SPF animals and of an inoculumof reference. SPF rabbits are an experimen-tal tool of choice to overcome interferencewith all known pathogens. Starting from afield sample (TEC) of intestinal contentfrom diseased rabbits obtained in 1997 dur-ing the highest period of contagion inFrance, the daughter inocula TEC1, 2 and 3reproduce the same disease. Thus, at leastpart of Koch’s postulate is respected (nev-ertheless isolation of the etiological agent inpure culture is missing). This material is sta-ble throughtout the constitution of the dif-ferent inoculum. In the five trials reported,not only ERE was reproduced on a qualita-

tive level, but the evolution of the diseaseand morbidity were also closely identical.Similar results were obtained with our inoc-ula in more than 2000 conventional rabbits[2, 32]. In the whole trial, morbidity wasclose to 100%. ERE is an acute disease andthe first signs characterized by ramblingnoise begin one day after contamination.The decrease in the DWG was observed asearly as the second day. The peak of the dis-ease was around 5 to 7 days after inocula-tion. Generally, recovery began one weekafter contamination, but the cure was slowto settle. Mortality appeared as soon as day 3with a peak on day 5 and in our experimentalconditions, it varied from 20 to 50%. Theearliest clinical signs were rambling noise,abdominal bloating and cecal impactiondetected on palpation. Diarrhea and thepresence of mucus under the cages were notconstant and observed a little later. ERE iscontagious and when control rabbits areplaced in the same room as the inoculatedanimals they develop clinical signs of thedisease, probably because of contaminationof the environment by the inoculated groupsbut this hypothesis cannot be demonstratedas long as the etiological agent has not beenfound.

At necropsy, the main features were theabsence of obvious gross lesions of themucosa of the different parts of the diges-tive tract, more precisely an acute inflam-matory process or congestion of the smallintestine, cecum and large intestine. Duringthe first 4–5 days, distension of the stomachand/or duodenum by a very liquid and gas-eous content was almost the only pathog-nomonical changes. These main features,associated with the distension of the abdo-men observed on live animals or beforeautopsy, should be taken into considerationfor field diagnosis of ERE. The frequencyof caecal impaction was very variable: gen-erally 20 to 30% of dead animals but it canvary from 0 to 70% [26]. The distal colonwas generally empty. In fact, the digestivechanges observed at necropsy in the inocu-lated rabbits obviously indicate physio-pathological alterations of the digestive

Figure 5. Detection of early virulent material(Trial E). Daily weight gain of control animalsand rabbits inoculated with intestinal content(IC) from affected rabbits slaughtered on day 1(IC d1) and day 2 (IC d2) post inoculation. Theresults are expressed as the mean ± standarddeviation.


functions. Everything occurs as if the intes-tinal transit was stopped for several days,leading to the dilatation of the stomach andsmall intestine filled with large amounts ofliquid and gas and caecal impaction pro-longed paresia of the whole digestive tract.This could also explain the multiplication ofopportunistic germs under field conditions(Escherichia coli O2, Klebsiella, coccidia,etc.).

Histological lesions of the intestinalmucosa, characterized by mild to moderateatrophy and fusion of the villi, associatedwith moderate transepithelial migration ofheterophilic cells and mucosal infiltrationof inflammatory cells, are mostly observedin some inoculated rabbits sampled for his-tological examination. Nevertheless the intes-tinal lesions appear non constant, with someinoculated rabbits being totally devoid ofhistological intestinal changes. Moreover,the lesions always remain moderate andclearly devoid of etiological specificity andmore particularly without lesional kineticswhich usually characterizes the develop-ment of infectious pathogenic agents. Thefocal ulcerative lesions described in twoinoculated animals appeared to be associ-ated with probably secondary bacterialproliferation emphasized by the digestiveparesia. As previously mentioned3, the his-tological lesions of ERE appeared mainlynon repetitively observed in all affectedrabbits and devoid of enough specificaspects to be precisely assigned to an iden-tified pathogenic agent. The macroscopicdigestive changes can be considered as theconsequences of a physiopathological dys-functioning and this fact probably explainsthe obvious discrepancy observed betweenthe gross and histological lesions. Regard-ing the results of our various trials, we can

conclude that gross changes appear as amore reliable tool for diagnostic purposesthan histological findings. Nevertheless, itis noteworthy that only light microscopywas used and further examinations by elec-tron microscopy should be made to com-plete our study.

It has also been shown elsewhere that thedisease does not induce any fever [16].

ERE can be characterized by the absenceof any inflammatory phenomena, whichdistinguishes this affection from the otherusual rabbit intestinal diseases [22]. How-ever, many similarities exist with a diseasepreviously described as mucoid enteritisor mucoid enteropathy [14, 15, 29, 35].According to these authors, this disease hasdecimated large numbers of colonies inGreat Britain, the United States and Hun-gary, the main countries where this diseasehas been described. The absence of macro-scopic inflammation and histological lesions,apart from hyperplasia of mucous cellsthroughout the small intestine, is also a fea-ture emphasized by Van Kruiningen andWilliams [34]. This is why the term mucoidenteropathy was used for mucoid enteritisbecause there was no visible inflammationof the intestine [12]. As for ERE, none ofthe authors mentioned above were able toidentify a pathogen responsible for themucoid enteropathy.

During these trials, we were unable todetect a dose effect in the range of 10 to0.2 mL and further experiments are neededto find the limit of a working dilution. Nev-ertheless, in trial E, we demonstrate that theunknown pathogen is present in the intesti-nal content as early as the second day aftercontamination of animals but we are notstating that it is absent from the intestinalcontent on day 1 PI. In this trial, with theinoculum from day 2, we could observe aclear but moderate disease without any mor-tality. Consequently, early samples may bevery useful. On a practical level, we firsteliminated a part of the flora coming fromintestinal fermentation generated by the

3 Coudert P., Jehl N., Gidenne T., Guittet M.,Larour G., Licois D., Persillon C., De Rocham-beau H., Note d’information N° 15 sur les travauxde recherche conduits sur l’Entérocolite Épizoot-ique du Lapin, [on line] (2003) http://www.tours.inra.fr/urbase/internet/resultats/enterocolite/noteEntero_2003-15.pdf [consulted January 20,2005].


intestinal stasis and then limited the multi-plication of a certain flora introduced withthe inoculum.

The search for a possible pathogenicagent has only revealed the presence of rota-virus and Clostridium perfringens in all ourinocula, but their role remains questionable[5, 9, 27, 28]. Nevertheless, further etiolog-ical investigations are necessary, particu-larly in bacteriology. Several collabora-tions are now underway, especially foretiological research. In this way, furthercharacterization of the early events of thedisease may be very useful for a betterknowledge of the type of agent responsiblefor ERE.

In conclusion, this work constitutes theexperimental basis for studies on this emerg-ing disease within the framework of etio-logical research led in different Europeanlaboratories working with the infectiousmaterial.

ACKNOWLEDGEMENTS

The present study was supported by fundsfrom the Regions Centre and Pays de Loire andby a grant from OFIVAL, managed by a com-mittee ITAVI-INRA-AFSSA. The authors wouldlike to thank A. Niepceron and B. Sewald fortheir helpfull technical assistance. P. Calahorraand D. Pinon are also gratefully acknowledgedfor their collaboration in handling the animalsand Y. Breuzin and A. Francineau for taking careof the SPF rabbit colony.

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