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Isolation of a porcine respiratory,non‐enteric coronavirus relatedto transmissible gastroenteritisM. Pensaert a , P. Callebaut a & J. Vergote aa Laboratory of Virology, Faculty of Veterinary Medicine ,State University of Gent , Casinoplein 24, Gent, 9000,BelgiumPublished online: 01 Nov 2011.

To cite this article: M. Pensaert , P. Callebaut & J. Vergote (1986) Isolation of a porcinerespiratory, non‐enteric coronavirus related to transmissible gastroenteritis, VeterinaryQuarterly, 8:3, 257-261, DOI: 10.1080/01652176.1986.9694050

To link to this article: http://dx.doi.org/10.1080/01652176.1986.9694050

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Isolation of a porcine respiratory, non-entericcoronavirus related to transmissiblegastroenteritis

M. Pensaert, P. Callebaut, and J. Vergote1

SUMMARY A porcine respiratory, non-enteric virus which is related to the coronavirus transmissiblegastroenteritus virus (TGEV) has been isolated in pigs and in cell culture. The isolate was designatedTLM 83.It has become very widespread and enzootic among the swine population in Belgium and in other swineraising countries. It causes an infection of the lungs andappears to spread by aerogenic route. It doesnot replicate in the enteric tract. The experimental infection in conventional and gnotobiotic pigs inisolation remains subclinical. The infection, either experimental or in the field, results in the formationof antibodies which neutralise the classical enteric TGEV. Based on this relationship, this virus isassumed to be a new TGEV-related porcine respiratory coronavirus or TGEV itself which has totallylost its tropism for the enteric tract.

INTRODUCTION

Transmissible gastroenteritis (TGE) inswine is caused by a coronavirus, calledtransmissible gastroenteritis virus (TGEV),which was first isolated in 1946 (1). Thevirus induces diarrhoea in swine of all ages,and pigs infected during the first week ofage usually die from dehydration. TGEVhas a worldwide prevalence. The pathoge-nesis of TGE has been studied in detail andthe virus replicates primarily in absorptiveepithelial cells covering the small intestinalvilli. Virus replication in these cells resultsin degeneration, villous shortening, anddiarrhoea. TGEV is, therefore, a specificenteropathogenic virus (4). It has, how-ever, also been reported to be able to re-plicate in the tonsils and in the respiratorytract (3, 6). This respiratory infection withfield strains of TGEV appears to be second-ary to the enteric infection and does notresult in respiratory disorders. Field strainsof TGEV which replicate in the respiratorytract always infect the enteric tract in thesame host.

In the authors' laboratory, serological sur-veys have been carried out at regular timeintervals between 1968 and 1984 on sowsera, collected in slaughterhouses, for thepurpose of determining the prevalence ofthe TGEV in Belgium. The percentage ofsow sera with neutralising antibodies var-ied between 12 and 24%, and a certaindegree of correlation was present with thenumber of clinical TGE outbreaks report-ed and diagnosed during the winter pre-ceeding the survey.In May 1984, however, such correlationwas not seen in another survey in which68% of total of 265 sow sera were found tohave seroneutralising (SN) antibodiesagainst TGEV, even though the number ofoutbreaks in the previous winter had notbeen higher than usual. Vaccinationagainst TGE is not performed in Belgium.This high incidence of antibodies amongthe Belgian swine population in the ab-sence of clinical TGE outbreaks suggestedto us that a change in the virus or in itsepizootiologic behaviour had taken place.

I Laboratory of Virology, Faculty of Veterinary Medicine, State University of Gent, Casinoplein 24, 9000Gent, Belgium.

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Similar observations of a high incidence ofTGE antibodies in the swine populationwithout disease have been made in othercountries such as the Netherlands (8) andDenmark (2) but no explantation was avail-able.Research was conducted to elucinate thisproblem and the results will be reportedhere chronologically.

SEROLOGICAL EXAMINATIONS ON CLOSEDBREEDING FARMS

Sera were collected in October 1984 fromprimiparous and multiparous sows on 11closed breeding farms which had not experi-enced a clinical TGE outbreak for at least 4years. All the sows on 10 to 11 farms hadSN antibodies against TGEV (Purduestrain) in the microtitre neutralisation test(7). This finding was surprising, consider-.ing the absence of a history of TGE. En-zootic TGE was not likely to be present,since feeder pigs and fattening swine onseveral of these farms were devoid of TGEantibodies at the time of the examination.It could, however, not entirely be excluded,even though evidence existed that TGEVor a TGEV-related virus had caused infec-tion without disease on these farms but haddisappeared again, leaving the youngerswine with no antibodies. Moreover the1 1 th farm, which was negative for TGEantibodies in .0ctober, had become posi-tive in January and no diarrhoea had beenobserved.A thorough serological examination andfollow-up was subsequently carried out on5 of these farms in an attempt to determinethe nature of this TGEV-like infection. Thenumber of sows on these 5 farms variedfrom 45 to 200. The examination was start-ed in February 1985 and was continueduntil February 1986. A total of 49 groupsof pigs (5-6 pigs per group) were serolo-gically followed monthly, starting at thetime of weaning (age 4 weeks) until the endof the fattening period.Seroconversion against TGEV occurredon all 5 monitored farms in the pigs weanedduring the months of February to April1985. Pigs weaned between May and No-vember, however, dit not seroconvert on 4of the 5 farms (nrs 1, 2, 3,3). These 4 farms

35)Y

had temporarily become free of the TGE-like agent but they were reinfected again inDecember 1985. No abnormal diarrhoealproblems were observed throughout theyear on these farms.On one farm (nr 4), the infection remainedpresent throughout the year 1985. Thisfarm was followed more closely to deter-mine the age of the pigs at which the infec-tion took place.The age until which the maternal anti-bodies persisted in pigs from TGE-seropositive mothers was first determined in thefeeder pigs on the four farms on which theinfection had disappeared. The results ofthis serological study showed that the anti-bodies of maternal origin were detectablein 100% of the pigs at the age of 3 to 6weeks (geometric mean SN titre-GMT of35,5), in 84% at 7 to 10 weeks (GMT of7,9), in 29% at 11 to 14 weeks (GMT of 4,4)and in 1,5% at 15 to 18 weeks. It was thusconcluded that maternal antibodies remain-ed detectable at the most until 16 weeks ofage and that the mean half-life was 12,04days. Any deviation from this rate of de-cline was considered a result of infection.To determine at what age the infectiontook place in pigs on farm 4, the antibodytitres were followed in 10 groups of pigs. Adeviation of the normal maternal antibodydecline and formation of active antibodytitres occurred in all the groups. Mater-nally derived antibodies in the blood gra-dually changed into actively formed an-tibodies, proving that an infection with theTGE-like virus had taken place. From theslopes of the curves, it was concluded that 8of the 10 groups had been infected prior tothe age of 3 monts, 4 of the 8 had beeninfected within one month after weaning.All the animals had antibodies at the end ofthe fattening period (5 months) and the SNtitres varied from 2 to 192. It was conclud-ed from these results that the TGE-likevirus was enzootic in -farm 4.A preliminary identification attempt ofthis TGE-like agent was made by the exam-ining of 26 sow sera from the farms men-tioned above. In these sera, the SN titre wasdetermined not only against TGEV butalso against 2 other coronaviruses: felineinfectious peritonitis virus (FIPV) and ca-nine enteric coronavirus (CCV), which are

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known to be related to TGEV (5). It wasfound that the SN titres against FIPV andCCV were respectively 5 and 3 times lowerthan those against TGEV. These resultsindicated that the agent was most closelyrelated to TGEV and that it was neither aFIPV nor a CCV which had possibly adapt-ed to swine.

ATTEMPTS TO DEMONSTRATE THE TGEV ORTHE TGE-LIKE VIRUS IN FAECES

In order to exclude an enzootic entericTGE viral infection, pooled faecal sampleswere collected from each of the 49 groupsof pigs which were followed serologicallyon the 5 farms mentioned above. Thesamples were collected monthly and storedat 25° C. Only those samples which hadbeen collected from groups of pigs thatafterwards proved to have seroconvertedagainst TGEV, and particularly those col-lected 1 and 2 months prior to seroconver-sion, were examined for the presence ofTGE viral antigens. These amounted to atotal of 173 pooled samples. Additionally,275 faecal samples were collected from in-dividual animals which had loose stools ordiarrhoea during the period of experimen-tation on the farms.All faeces samples were subjected to anELISA test which is routinely applied fordemonstrating TGEV in faecal material.The technique of this ELISA will be report-ed later but it is essentially a blocking testwhich has been proved to be specific, sus-ceptible, and very reliable for the detectionof the classical enteric TGEV in faeces ofexperimentally and field infected swine ofall ages. None of the faecal samples examin-ed from the groups or from the individualanimals on the 5 farms were positive forTGE viral antigens.Pooled ELISA-negative faecal samplesfrom several groups of pigs of farm 4,

which had been collected one and twomonths prior to seroconversion, were oral-ly inoculated in 4 pigs which had no an-tibodies against TGEV and which werekept in individual isolation. None of thepigs became seropositive for TGEV, con-firming that TGEV or a TGE-like viruswas not present in the faeces.These results excluded the presence of anenzootic infection with the enteric TGEV

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and pointed towards the existence of aTGEV-related virus or a TGE-like viruswithout tropism for the enteric tract.

DEMONSTRATION OF THE TGE-LIKE VIRUSIN SENTINEL PIGS ON FARM 4

Eleven conventional 6 week-old sentinelpigs, without SN antibodies against TGEV,were introduced on farm 4, on which it hadbeen shown that the infection with theTGE-like virus persisted throughout theyear. They were intermingled with 30 fresh-ly weaned pigs that had been born on thefarm. Seven sentinel pigs were killed, oneeach at respectively 3,5,7,9,11,14, and 17

days after inoculation and tonsils, trachea,lungs, and small intestine with its contentswere collected for further examination.The 5 remaining pigs were used for serolo-gical follow-up. Also, faecal samples werecollected every two days from the sentinelpigs.The surviving pigs had built up SN anti-bodies against TGEV at 14 days after theintroduction (titres of 3 to 24), proving thatinfection with the TGE-like virus had oc-curred in the sentinel pigs soon after theirintroduction. No clinical disease was ob-served. All 45 faecal samples collected werenegative in respect of TGE viral antigens inELISA, and no fluorescence was observed,with a TGEV conjugate, in the intestines ofthe sacrificed pigs. These results allowedthe conclusion that infection with the TGE-like virus had occurred in the sentinel pigsand confirmed that it was non-enteric.

VIRUS ISOLATION IN PIGS AND IN CELLCULTURES

A pool was made of suspensions (25%) ofthe respiratory tract tissues from sentinelpigs which had been sacrified at 3 and 5days and at 7 and 9 days after introductionrespectively. These suspensions were oro-nasally inoculated into two 10-day-old pigswhich were devoid of antibodies againstTGEV and which were kept in individualisolation. The pigs remained healthy andbuilt up SN antibodies against TGEV(titres 192 and 96) within 14 days after theinoculation. Another pig which was orallyinoculated with pooled intestinal contents

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of the sentinel pigs killed at 5 and 7 daysremained seronegative for TGEV.A second virus isolation experiment wasperformed in a colostrum-deprived hyster-ectomy-derived piglet (nr 1490) inoculatedat the age of 17 days with respiratory tractsuspension of the sentinel pig which hadbeen sacrified 5 days after the introduc-tion on the farm. The pig 1490 remainedhealthy but seroconverted against TGEVand had a SN titre of 256 at 21 days afterinoculation. From these isolation experi-ments in pigs, it was clear that the TGE-like virus was present in the respiratorytract of the sentinel pigs killed between 3and 7 days after the introduction on thefarm. Tonsillar swabs were collected dailyfrom pig 1490 and were used for virus isola-tion in cell cultures.

Virus isolation attempts were made in pri-mary pig kidney cells from the respiratorytract tissue suspensions of the same senti-nel pig (killed 5 days) and from the tonsillarswabs of pig 1490. The attempts yielded acytopathic (CP)agent. The CP agent wasdesignated TLM 83. With this CP agent,preliminary identification studies havebeen performed both in cell cultures and incolostrum-deprived piglets.Infected cell cultures show fluorescencewith a conjugate prepared against TGEVbut not against the other 2 porcine corona-viruses, namely hemagglutinating encepha-litis virus and porcine epizootic diarrhoeavirus. The isolate TLM 83 was neutralisedby sera collected from experimental pigswhich were inoculated with respiratory tis-sue suspensions and which seroconvertedagainst TGEV. It was also isolated fromthe tonsillar swabs collected from pig 1490between 3 and 7 days after inoculation.

The cytopathic effect in primary pig kidneycells is characterised by clustering of round-ed up cells and becomes visible at 2 daysafter inoculation. Even though it progres-ses, it does not involve the entire mono-layer and remains limited to a number offoci of degenerated cells. The CP virus doesnot have any haemagglutinating or haem-adsorbing capacity. Up to 6 serial pass-ages have been made successfully and a titreof 103'5 TCID5, per 0.5 ml of tissue culturefluid can be obtained.

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The CP virus, TLM 83, induces a sub-clinical infection upon oronasal inocula-tion of colostrum-deprived piglets. Thesepigs build up antibodies which neutraliseTGEV. Pigs inoculated with the CP agentTLM 83 and sacrificed 3 or 4 days latershow immunofluorescence with a TGEVconjugate in the lungs but not in the smallintestine. With the intestinal contents ofthese latter sacrificed pigs, the infectioncan be reproduced neither in other pigs norin cell cultures. It is, therefore, clear thatthe isolate TLM 83 is the respiratory TGE-like virus.

GENERAL CONCLUSIONS

Based on the experiments described, it canbe concluded that the isolate, TLM 83, is arespiratory but non-enteric virus which isrelated to the classical enteric coronavirus,TGEV. It is not pathogenic for convention-al or gnotobiotic pigs in isolation. It causesa cytopathic effect in primary pig kidneycells. The respiratory infection can be re-produced with the CP isolate.TLM 83 may be a new porcine respiratorycoronavirus which is related to TGEV or itmay be the classical TGEV which has to-tally lost its tropism for the enteric tract. Itis very widespread among the swine popu-lation and has become enzootic.

ACKNOWLEDGEMENTS

This research work was funded by the Institute forScientific Research in Agriculture and Industry, Brus-sels, Belgium. The technical assistance of Miss LievcSys and Miss M. Carpentier is gratefully acknowledg-ed.

REFERENCES

I. Doyle, L. P. and Hutchings, L. M. A transmissiblegastroenteritis in pigs. J. Am. Vet. Med. Assoc.1946; 108: 257-9.

2. Eskildsen, M. and Havg, P. State Veterinary Insti-tute for Virus Research, Denmark. Personal com-munication, 1986.

3. Furuuchi, S., Shimizy, Y., and Kumagai. T. Multi-plication of low and high cell culture passagedstrains of transmissible gastroenteritis virus in or-gans of newborn piglets. Vet. Microbiology 1978/1979; 3: 169-78.

4. Haelterman, E. 0. On the pathogenesis of trans-missible gastroenteritis of swine. J. Am. Vet. Med.Assoc. 1972; 160: 534-40.

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5. Horzinek, M. C., Lutz, H., and Pedersen, N. C.Antigenic relationship among homologous struc-tural polypeptides of porcine, feline, and caninecoronaviruses. Infection and Immunity 1982; 37:1148-55.

6. Kemeny, L. J., Wiltsey, V. L., and Riley, J. L.Upper respiratory infection of lactating sows withtransmissible gastroenteritis virus following con-tact -exposure to infected piglets. Cornell Vet.1975; 65: 352-62.

7. Voets, M. Th., Pensaert, M., and Rondhuis, P. R.Vaccination of pregnant sows against transmis-sible gastroenteritis using 2 attenuated virusstrains and different inoculation routes. The Vet.Quarterly 1980; 2: 211-9.

8. Voets, M. Th. Gezondheidsdienst voor Dieren,Boxtel, the Netherlands. Personal communica-tion, 1985.

Theileria taurotragi in ZambiaF. Jongejan2, F. L. Musisi, P. D. S. Moorhouse, M. Snacken3,G. Uilenberg4

SUMMARY Theileria sp. (Bwengwa) of low virulence was isolated by feeding R. appendiculatus tickscollected from the field on a susceptible calf and subsequently transmitted between cattle by R.appendiculatus ticks- Theileria sp. (Bwengwa) was shown to be T. taurotragi on parasitological,clinical and serological grounds. T. taurotragi is the fourth Theileria sp. shown to be present inZambia.

INTRODUCTION

Three species of Theileria in cattle havebeen reported from Zambia. The most im-portant one, Theileria parva, transmittedby the tick Rhipicephalus appendiculatus,was first recorded in 1922 in the NorthernProvince (1).T. parva parva-type parasites have been iso-lated from the field, whereas the presenceof bovis- and/or lawrencei-types of T. parvaremains to be confirmed (F. L. Musisi, un-published). Theileria velifera was the se-cond species reported from Zambia andwas found throughout the country (8). Athird species, Theileria mutans, was re-cently isolated and transmitted betweencattle by the tick Amblyomma variegatum(7).During studies on theileriosis in cattle inZambia occasional mild strains of theile-ria were encountered. Mild theileria strainswhich are transmissible by R. appendicula-

tus were identified as Theileria taurotragiin East Africa (5) and southern Africa (9).In this paper, we report the isolation of amildly pathogenic Theileria sp. from cattlein Zambia and its subsequent identifica-tion as Theileria taurotragi.

MATERIALS AND METHODS

IsolationBrown ear ticks were collected from cattle at Bwengwa,Monze District, Southern Province of Zambia andsubsequently identified in the laboratory. Isolation oftheilerial parasites was attempted by feeding unen-gorged adult R. appendiculatus ticks from this collec-tion in earbags on experimental calf no. 132. Labora-tory strain of R. appendiculatus.A strain of R. appendiculatus was used which orig-inated from a colony established at the former EastAfrican Veterinary Research Organisatidn, Muguga,Kenya. It was maintained by feeding on the ears ofrabbits and cattle. Oviposition, hatching and moult-ing were performed at 28°C and 85 per cent relativehumidity (rh), quiescent stages being kept at 20°C and

I UNDP/FAO Animal Disease Control Project P.O. Box 30563, Lusaka, Zambia.I Present address and address for correspondence: Department of Tropical Veterinary Medicine and Proto-

zoology, University of Utrecht, P.O. Box 80.172, 3508 TD Utrecht, The Netherlands.2 Present adress: IBT-Salsbury, Groupe Solvay Sante Animale, B.P. 1311 37013 Tours Cedex, France.3 Department of Tropical Veterinary Medicine and Protozoology, University of Utrecht, P.O. Box 80.172,

3508 TD Utrecht, The Netherlands.

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