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Coronavirus infection in a litterof pupsJ. Vandenberghe a , R. Ducatelle a , P. Debouck a & J.Hoorens aa Faculty of Veterinary Medicine , State University ofGhent , Casinoplein 24, Gent, B9000, BelgiumPublished online: 01 Nov 2011.

To cite this article: J. Vandenberghe , R. Ducatelle , P. Debouck & J. Hoorens (1980)Coronavirus infection in a litter of pups, Veterinary Quarterly, 2:3, 136-141, DOI:10.1080/01652176.1980.9693771

To link to this article: http://dx.doi.org/10.1080/01652176.1980.9693771

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Coronavirus infection in a litter of pupsJ. Vandenberghe, R. Ducatelle, P. Debouck and J. Hoorensi

SUMMARY

An outbreak of a coronavirus infection in a litter of 10-week-old poodle pups froma commercial kennel is described. Coronavirus-like particles were demonstrated byelectron microscopy in the intestinal contents and in the colonic mucosa. Trepone-mas and campylobacters were excreted in great numbers in the faeces. The gastro-intestinal disturbances were complicated by hepatic and renal degeneration andsevere bronchopneumonia.

INTRODUCTION

A coronavirus has been associated withgastro-intestinal disturbances in dogs (1,3). The clinical signs of coronavirus infec-tion are: sudden illness, lethargy, decreaseof appetite, diarrhoea, and vomiting(2). The faeces have a fetid odor and maycontain variable amounts of blood. Mostanimals recover spontaneously within 10days. Symptomatic treatment shortensthe recovery period. Loose stools canpersist for 3 to 4 weeks. Leucopenia isnot a common feature. Most infectionspass subclinically (2).Seventy per cent of dogs of all ages haveseroneutralizing antibodies to T.G.E.-virus, a coronavirus in pigs (17). A possi-ble relation between canine and porcinecoronavirus infections has been menti-oned by Albrecht and Liipcke (1) and byMc Clurkin et al. (13).

MATERIAL AND METHODSThree poodle pups, 10 weeks old, from the samelitter were brought to the veterinary faculty forexamination. Their history was: sudden illness, lackof appetite, and death after a short period of list-lessness and groaning. Profuse watery diarrhoeahad been noticed for one day.

The dogs came from a commercial kennel of aboutfifty salable young dogs of all breeds. About 50 percent of all pups in the kennel developed rhinitis andpneumonia and about 20 per cent developed diar-rhoea, though mortality was low. All kennel dogshad been vaccinated with measles vaccine at the ageof 7 weeks.A visit to the kennel revealed satisfactory hygienicand alimentary conditions.Three further poodle pups of the same litter, suffer-ing from loose stools for one day, were brought tothe laboratory for examination.After clinical observations and routine blood andserum analyses the dogs were euthanized the sameday.

Pathological examination

A detailed post-mortal examination was carried out.Tissue samples were fixed in 10 per cent phosphate-buffered formol saline for histological examina-tion. Tissue of the middle jejunum and cranial partof the colon was fixed in 2.5 per cent glutaraldehydeand 2 per cent paraformaldehyde in 0.1 M cacody-late buffer forelectron-microscopical examinationswith a Zeiss EM 9.

Virological examination

A 20 per cent suspension of the colonic contents wasprepared in phosphate-buffered saline. The super-natant of this suspension, obtained after low-speedcentrifugation, was processed for electron micro-scopical examination as fully described elsewhere(18).

I Faculty of Veterinary Medicine, State University of Ghent, Casinoplein 24, B9000 Gent, Belgium.

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Bacteriological examination

The faeces were examined for Salmonella. Clostri-dium perfringens, Campylobacter jejuni, and Tre-ponema.Routine technics were used for the isolation ofSalmonella, namely enrichment on tetrathionatefluid medium, isolation on Gassner and brilliantgreen media, and the determination on Kligler me-dium.Sheep blood agar plates were inoculated with thecolonic contents for the isolation of Clostridiumperfringens and incubated at 37° C in a BBLanaerobic system with catalyst for 24 hours.For the isolation of Campylobacter fejuni a micro-aerophilic atmosphere was used, as described byLawson and Rowland (12). Samples of the sto-mach, the middle jejunum, the colon, and the faeceswere inoculated on Columbia sheep blood agarplates with 50 pg/ ml actidione, 24 1.U./ ml baci-tracine, 15 pg/ ml cephalotin, 10 U/ ml colistine and5 ug/ ml novobiocine I). Fecal samples of fiveother dogs with loose stools from the same kennelwere examined for Campylobacterjeluni.Rectal samples were inoculated, for the isolation ofTreponema, on Columbia sheep blood agar plateswith 180 1.U./ ml polymycine B sulphate (6) or 400pg/ ml spectinomycine (8). Treponema growth wasinspected after 24, 48, and 72 hours of incubation at42° C in a BBL anaerobic system with catalyst andin a microaerophilic atmosphere (12).

RESULTS

Clinical observationsThe pups had rhinitis and conjunctivitis.They were vomiting mucus with smallamounts of food. Profuse watery diarr-hoea with small amounts of blood wasseen. Two pups were coughing andpneumonia was diagnosed. Blood andserum analysis revealed hyppoproteine-mia (35 g/1 mean value), hypoglycemia(15 mmo1/1), anaemia (3.9 1012 erythro-cytes) and uraemia (3.50 mmo1/1). Thedogs with pneumonia showed lymphocy-tosis.

Pathological findingsThegastro-intestinal wallwasswollenandcongested. The turgor of the intestinalloops was increased. In the stomach therewas abundant mucus. A catarrhal entero-colitis with watery contents was seen.The caecum and colon contained somefibrinous material mixed with a littleblood. The mesenterial lymph nodeswere swollen and congested.Spirochaetes were observed on Giemsastained smears of the gastric mucus fromthe fundus and the pylorus.

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Histologically, the villi of the terminaljejunum were atrophic in only one dog.Some of the crypts of the small intestinewere dilated and filled with cellular de-bris. Deposition of cell debris was alsoobserved in numerous crypts of thecaecum and colon.Two dogs had purulent rhinitis and se-vere catarrhal bronchopneumonia of theventral parts of the apical and cardiallobes. Hepatocytes showed cloudy swel-ling and vacuolar degeneration. Tubul-onephrosis was seen. The spleen wasslightly swollen and depleted, with acti-vation of the germinative centres.Electron-microscopical inspections ofthe middle jejunal epithelium revealed nosignificant lesions. In the colon ultra-structural epithelial cell lesions werefound mainly around the mouth of thecrypts. A number of absorbtive epithelialcells were in a stage of degenerativechange (Fig. 1). The microvilli were shortand irregular, or sometimes even comple-tely absent. The number of free riboso-mes had increased. The cell nuclei wereoften swollen, with a patchy condensa-tion of the chromatin, which was mainlylining the nuclear envelope. Mitochon-dria were swollen. The cytoplasm of theaffected cells had become either clear ordarkened. The intercellular junctions hadsometimes lost contact. In a more ad-vanced stage of degeneration, the cellswere rounded, and subsequently theywere released into the intestinal lumen.In the majority of these degenerating epi-thelial cells, intensely staining coronavirus-like particles were seen (Fig. 2).

They were present in the cytoplasm insidevacuoles. The particles were moderatelypleomorphic, ranging from 50 to 90 nmin diameter. They consisted of a nucleo-capsid of 40 to 80 nm, separated from anouter unit membrane by an interlacingspace of 2 to 6 nm. In the centre, anelectron translucent centre could often beseen with a diameter ranging from 0 to 50nm. The particles were sometimes seenin great numbers inside large cyto-plasmic vesicles, at other times theywere single particles each embeddedin a small vesicle (Fig. 2). Viral particleswere sometimes present between the la-

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mellae of the endoplasmic reticulum. Si-milar particles were sometimes observedinside the Golgi apparatus. Occasionalbud like structures were observed atmembranes of cytoplasmic vacuoles ordilated cysternae of the endoplasmic reti-culum. No budding of viral particlesfrom the cell membrane of the infectedcells was seen. Small, single viral particlesembedded in vesicles were often seen inthe apical part of the cytoplasm, near thecell membrane. Viral particles were alsoseen in large numbers between the micro-villi of heavily infected cells (Fig. 3). Thenucleus did not contain viral particles onany occasion, nor did the goblet cells. Anumber of epithelial cells lining the intes-tinal lumen contained large lipid dropletsand vacuolarly distended endoplasmic re-ticulum (Fig. I). These lipid-containing

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Fig. I. Colonic epithelial celldegeneration. I. Epithelial cellrelease into the intestinal lu-men. 2. Short, irregular mi-crovilli. 3. Patchy codensa-tion of chromatin, lining thenuclear envelope. 4. Clearedcytoplasm. 5. Intracytoplas-mic lipid droplets.EM x 1400, photographic

enlargementx 1.2magnification

cells dit not always show up in viral parti-cles.

Virology

Electron-microscopical examination ofthe intestinal contents of a pup havingdiarrhoea for at least one day revealedthe presence of particles with a typicalcoronavirus morphology (Fig. 4). Theparticles were pleomorphic with a meandiameter of 107 nm including the projec-tions. Rotaviruses or parvoviruses werenot observed.

BacteriologySalmonellae were not isolated. Clostri-dium perfringens was found in threepups. Non-haemolytic treponemata wereisolated on plates incubated in either amicroaerophilic or in an aenaerobic en-

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Fig 2. Entcrocytc, contain-ing single virus-like particlesinside small cytoplasmic va-cuoles (arrows).EM x 9500, photographic

enlargement x 1'2magnification

vironment with and without polymyxine.Campylobacter jejuni was demonstratedin all parts of the intestines of the poodlepups and in the faeces of the five otherdogs with loose stools.

DISCUSSION

The morphological and morphogeneticalproperties of the particles observed in thelarge intestinal contents and in the colo-nic epithelial cells are characteristic ofcoronaviruses (14, 20). The observedvirus particles are morphologically simi-lar to the canine coronavirus describedby Binn et al (3). This virus causes diarr-hoea in dogs (7, 19).Multiplication of canine coronavirus incolonic epithelial cells has not previouslybeen reported. Transmissible gastro-enteritis (TGE) virus, a coronavirus Ofpigs, does not affect the colon (16).

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However, infection of colonic epithelialcells by coronavirus has been demonstra-ted for Neonatal Calf Diarrhoea Corona-virus (NCDCV) (15, 17). In this sense, thepresent coronavirus would be more simi-lar to NCDCV than to TGE, although anumber of authors claim that the caninecoronavirus is serologically related toTGE, if not identical with it (1, 10, 13).The literature (7) as well as research dataseem to suggest that the canine coronavi-rus infection proceeds in the caudal direc-tion over a certain period of time.The presence of virus particles inside theGolgi apparatus has been reported forcanine coronavirus as well as for othercoronaviruses (16, 20). The significanceof this in the process of virus maturationis not clear. The interaction of virus andintestinal cells, and the resulting absorb-tive cell degeneration, suggest that thecanine coronavirus is pathogenic for the

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Fig. 3. Apical part of the cytoplasm of a colonic epithelial cell. I. Short, irregular microvilli. 2. Virus-likeparticles embedded in small cytoplasmic vesicles. 3. Virus-like particles lying free between the microvilli.

enlargementEM x 28000, photographic x 1.2magnification

Fig. 4. Particles with a typical coronavirus morphology as found in the large intestinal contents. Negativestaining PTA 2 per cent.

Fm x 28.000. photographic i enlargementmagnification

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colonic epithelial cells. Destruction ofthese cells means that absorbtion of li-quids will be interfered with. This phe-nomenon may account for the severity of'the clinical symptoms observed in thesepups, especially the rapid dehydration.Clostridium perfringens, Treponema in-nocens (89), Campylobacter jejuni (3)and gastric spirochaetes (21, 22) arecommon findings in dogs. Primary pat-hogenicity of these bacteria in dogs hasnever been proved, but a secondary pat-

hogenic role is not excluded.It is suggested that the present coronavi-rus was the causative agent of the gastro-intestinal disturbances. As aconsequencethe bacterial intestinal balance was dis-turbed and there was an increase of gas-tric ard faecal spirochaetes and spirillaein the colon.This intestinal disfunction was compli-cated by hepatic and renal degenerationand by severe rhinitis, conjunctivitis, andbronchopneumonia.

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