Twenty-one healthy local goats of about eight months old were divided into four groups consisting of six animals

in groups 1, 2 and 3 and three animals in group 4. Goats in groups 1 and 2 were inoculated intratracheally with Mycoplasma arginini. Goats in group 2 were inoculated again with Pasteurella haemolytica A2 six days later. Goats in group 3 were inoculated intratracheally with P. haemolytica A2 alone while goats in group 4 received intratracheal inoculation ofPBS. The goats were euthanised at day 1, 3 and 7 post inoculation with P. haemolytica. Four goats in group 1 and three goats in group 3 had small patches of mild pneumonic lesions. Goats in group 2 had severe lung lesions typical of pneumonic pasteurellosis at the anteroventral region of the lungs. None of the goats in group 4 had pulmonary lesions. P. haemolytica was reisolated from all goats in group 2 and from three goats in group 3 but M. arginini was not reisolated

A. B., Nurida

A., Azri

Abdul Rahman, Sheikh Omar

Saad, Mohd Zamri

Universiti Putra Malaysia Institutional Repository

PertanikaJ. Trap. Agric. Sci. 17(3): 239-242 (1994) ISS : 0126-6128© Universiti Pertanian Malaysia PressExperimental Respiratory Infection of Goats with Mycoplasma arginini andPasteurella haemolytica A2M. ZAMRI-SAAD, A. AZRI1, A.B. NURIDA and A.R. SHEIKH-OMARFaculty of Veterinary Medicine and Animal ScienceUniversiti Pertanian Malaysia43400 UPM Serdang, Selangor Darul Ehsan, Malaysia1Regional Veterinary Diagnostic Laboratory, Persiaran Barat46630 Petaling Jaya, Selangor Darul Ehsan MalaysiaKeywords: respiratory infection, Mycoplasma arginini, Pasteurella haemolytica, goatsABSTRAKDua puluh satu ekor kambing baka tempatan yang berumur kira-kira lapan bulan telah dibahagikan kepadaempat kumpulan yang terdiri daripada enam ekor dalam setiap kumpulan 1, 2 dan 3, dan tiga ekor dalamkumpulan 4. Kambing dalam kumpulan 1 dan 2 telah dijangkiti dengan Mycoplasma arginini secara intra-trakea sebelum kambing dalam kumpulan 2 disuntik Pasteurella haemolytica A2 enam han kemudian.Kambing dalam kumpulan 3 disuntik dengan P. haemolytica sahaja secara intra-trakea sementara kambingdalam kumpulan 4 disuntik dengan PBS. Kambing dibunuh pada han ke 1, 3 dan 7 selepas suntikan P.haemolytica. Empat ekor kambing dalam kumpulan 1 dan tiga ekor kambing dalam kumpulan 3 menunjukkantompok-tompok lesi pneumonia yang kurang teruk. Kambing dalam kumpulan 2 menunjukkan lesi pneumoniapasteurelosis yang teruk di bahagian antero-ventral peparu. Tidak seekor pun kambing dalam kumpulan 4menunjukkan lesi pulmonan. P. haemolytica telah berjaya diasingkan daripada kesemua kambing dalamkumpulan 2 dan daripada tiga ekor kambing dalam kumpulan 3. Walau bagaimanapun, M. arginini gagaldiasingkan.ABSTRACTTwenty-one healthy local goats of about eight months old were divided into four groups consisting of six animalsin groups 1, 2 and 3 and three animals in group 4. Goats in groups 1 and 2 were inoculated intratracheallywith Mycoplasma arginini. Goats in group 2 were inoculated again with Pasteurella haemolytica A2 six dayslater. Goats in group 3 were inoculated intratracheally with P. haemolytica A2 alone while goats in group 4received intratracheal inoculation ofPBS. The goats were euthanised at day 1, 3 and 7 post inoculation with P.haemolytica. Four goats in group 1 and three goats in group 3 had small patches of mild pneumonic lesions.Goats in group 2 had severe lung lesions typical of pneumonic pasteurellosis at the anteroventral region of thelungs. None of the goats in group 4 had pulmonary lesions. P. haemolytica was reisolated from all goats ingroup 2 and from three goats in group 3 but M. arginini was not reisolated.INTRODUCTIONPneumonic pasteurellosis caused by Pasteurellahaemolytica has been recognised as one of the mostcommon diseases of sheep and goats (Gilmour1980). The disease develops following variousstresses on animals, and viral infection in therespiratory tract prior to the infection by P.haemolytica has been shown as one of theimportant causal factors (Davies et al. 1981;BuddIe et al. 1990). While some species ofmycoplasma have been recognised as primarypathogens of pneumonia in animals, the role ofother species in causing lung disease of animalsis still unknown (Jones 1983).Mycoplasma arginini has been isolated fromgoats that died of pneumonic pasteurellosis inMalaysia, but the significance of this organism inthis disease is uncertain (Sheikh-Omar andM.ZAMRI-SAAD, A. AZRI, A.B. NURIDA AND A.R. SHEIKH-OMARMutalib 1985). The present investigation was de-signed to study the role of M. arginini in the de-velopment of pneumonia either alone or incombination with P. haemolytica in healthy goats.MATERIALS AND METHODSAnimalsTwenty-one clinically healthy local goats of about8 months old were selected for the study. Theywere divided into four groups; six goats each ingroups 1, 2 and 3 and three goats in group 4.The groups were kept in separate rooms and feddaily with cut grass. Drinking water was availablead libitum. All goats were healthy; neither P.haemolytica nor M. arginini were isolated from thenasal cavities for a period of 4 weeks prior to theexperimental infection.InoculaThe M. arginini strain used was isolated earlierfrom the lungs of a goat that had died of severefibrinous pneumonia (Sheikh-Omar and Mutalib1985). The organism had been cloned five times.To prepare the inoculum for each goat, 10 colo-nies of M. arginini were cultured in 1 ml myco-plasma broth before they were incubated in car-bon dioxide for 7 d.The P. haemolytica used for the experimentalinoculation in this study had been isolated fromthe pneumonic lung of a goat, and was identifiedas P. haemolytica serotype A2. Prior to the inocu-lation, the organism had been grown in infusionbroth and later diluted to 108 colony forming unit(cfu) per ml.Experimental DesignAll goats in groups 1 and 2 were inoculated with1 ml of the prepared suspension of M. argininiintratracheally by inserting a hypodermic needlebetween the tracheal rings and injecting the in-oculum with a syringe. Six days later, all six goatsin group 2 were reinoculated intratracheallywith1 ml suspension of P. haemolytica A2 accordingto the method described earlier for M. arginini.The goats in group 3 were inoculatedintratracheally with P. haemolytica alone whilegoats in group 4 were the control receiving phos-phate-buffered saline (PBS).The body temperature and clinical signs at-tributable to pneumonia were recorded daily.Two goats each from groups 1, 2 and 3 and onegoat from group 4 were euthanised with saturatedmagnesium sulphate overdose at days 1, 3 and 7post inoculation (pj.) with P. haemolytica. Postmortem examination was carried out immediatelywith a detailed examination on the respiratorytract. Samples of the trachea and the lungs werecollected for bacterial and mycoplasmal isolationand for histological examination.Sample ProcessingIsolations of P. haemolytica from tissue sampleswere made by inoculation onto 5 per cent bloodand McConkey agars, incubated at 37° C for 24 h ,before the colonies suspected of being P.haemolyticawere reinoculated onto blood agar andlater identified (Lenette et al. 1974).Mycoplasmal isolations were performed es-sentiallyas described by Sheikh-Omar and Mutalib( 1985). Samples were cultured on PPLO agar baseplus mycoplasma supplement-G (Oxoid). Theagar plates were incubated in carbon dioxide andexamined daily for the typical 'fried egg' colonies.Samples for histological examination were fixedin 10% buffered formalin for at least 24 h em-bedded in paraffin wax, sectioned at between 4and 6 urn and stained with haematoxylin andeosin.RESULTSClinical ObservationsAll goats were healthy at the time of inoculation,but six goats were coughing for a few secondsduring the intratracheal administration of theinoculum. None of the goats in groups 1, 3 and4 had high body temperature or clinical signs ofrespiratory tract infection. Four goats in group 2were found to be depressed and inactive as earlyas 24 h following inoculation of P. haemolyticaA2.Mter day 3 pj., the goats were found to havemucoid nasal discharges. The body temperatureof all goats, however, remained normal through-out the study period.PathologyGroup 1The lungs of goats killed at day 1 pj. were slightlycongested and moderately oedematous. Four tofive patches ofdark red atelectatic and pneumonicareas of about 0.5 to 2 cm were observed particu-larly in the lung parenchyma near the terminalbronchi and major bronchioles. These lesionswere observed mostly at the anteroventral regionof the lungs. Histologically, there was moderate240 PERTANIKAJ. TROP. AGRIC. SCI. VOL. 17 NO.3, 1994EXPERIMENTAL RESPIRATORY INFECTION OF GOATS WITH MYCROPLASMA ARGININIAND P. HAEMOLITlCA A2bronchiolitis consisting of accumulation of a mix-ture of mononuclear cells and neutrophils in thesubepithelial layer. The bronchiolar associatedlymphoid tissue (BALT) was slightly hyperplasticand oedema fluid was found in most alveoli. Theinteralveolar septa were thickened due tohyperaemia and the presence of neutrophils andmononuclear cells.Grossly, the distribution and size of pulmo-nary lesions observed in goats killed at day 3 pj.were similar to those observed earlier at day 1 pj.but the histological changes were less severe.There was bronchiolitis with marked BALThyperplasia but the oedema fluid was not obvi-ous and the interalveolar septa were not mark-edly thickened. Neutrophils were absent but al-veolar macrophages were obvious in several ar-eas.At day 7 pj. the gross pulmonary lesions wereless extensive. Bronchiolitis and BALT hyperplasiawere absent in most areas. Changes in theinteralveolar septa were similar but milder thanthose observed at day 3 pj.Group 2The lungs of goats killed at day 1 pj. had lesionstypical ofpneumonic pasteurellosis, mostly involv-ing similar lung areas as goats in group 1 (theanteroventral part of the right lung). The lesionsthat appeared dark red and firm were found tovary in size between 1 and 3 cm. The lungs weremoderately oedematous and the distal trachea,bronchi and major bronchioles were markedlycongested. Histologically, there was moderatebronchiolitis with mononuclear cells infiltrationin the subepithelial layer. Interalveolar septa sur-rounding the affected bronchioles were slightlythickened due to congestion and presence of neu-trophils and oedema fluid.The ventral two-thirds of the right apical andintermediate lobes of the lungs ofboth goats killedat day 3 pj. were dark red and firm. Mucoidexudate was observed on the cut surface of theaffected lung parenchyma and from the lumen ofbronchioles. The distal trachea, bronchi andmajor bronchioles were moderately congested.Most alveoli of the affected areas were congestedand their spaces filled with oedema fluid and nu-merous neutrophils. Most bronchioles showedmoderate bronchiolitis with similar neutrophilicexudate in the lumen.The gross pulmonary lesions were most ex-tensive in goats killed at day 7 pj. The lesionsinvolved the whole anterior one-third of the rightlung and the ventral two-thirds of the apical lobesof the left lung; one goat had the right lung par-tially adhered to the thoracic wall. The cut sur-face of the lesions showed patches ofpale necroticareas of about 1 to 3 mm. Most affected alveoliwere filled with a mixture of numerous alveolarmacrophages, neutrophils and fibrin. Some in-terlobular septa were thickened due to the pres-ence of the exudate. A similar exudate was ob-served in the lumen of many bronchioles.Group 3The lungs of goats in group 3 showed gross le-sions similar to those of goats in group 1. Therewere patches of dark red discolouration in theanteroventral areas of the lungs. Histologically,these lesions consisted of slightly thickened in-ter-alveolar septa with moderate accumulationsof neutrophils and macrophages in some alveoli.Group 4No obvious histological lesions were observed inthe lungs of goats in this group except severalfoci of slightly congested areas.MicrobiologyM. arginini was not reisolated from the lungs ofany of the goats. Reisolations of P. haemolyticawere successfully made from the lungs of all goatsin group 2 and of three goats in group 3.DISCUSSIONMost goats developed pneumonic lesions affect-ing the same lung area, but the extent of the le-sions was different. Some of the goats infectedwith M. arginini alone and P. haemolytica alonehad mild, localised pulmonary lesions which re-mained relatively similar in extent throughout theduration of the infection. Combined M. argininiand P. haemolytica infection, however, producedsevere and extensive lesions typical of pneumonicpasteurellosis, and the extent of the lesions in-creased with time ofinfection. Similar synergesticeffects had been described for viruses used inexperimental pneumonic pasteurellosis of goatsand sheep (Davies et al. 1981; Davies et al. 1982;BuddIe et al. 1990).This study also demonstrated that M. argininican produce only mild lesions in the lung ofgoats,and thus this may not lead to a serious disease.The organism appeared to be eliminated early asPERTANlKAJ. TROP. AGRIC. SCI. VOL. 17 NO.3, 1994 241M.ZAMRI-SAAD, A. AZRI, A.B. NURIDA AND A.R. SHEIKH-OMARa result of the inflammatory response followingthe infection. A similar inflammatory responsein pneumonic lungs was observed to eliminatecaprine herpesvirus infection in experimentalpneumonic pasteurellosis of goats (BuddIe et al.1990). Although the mycoplasma was eliminatedearly, the infection was thought to have inducedinitial lung lesions for later establishment of P.haemolytica in lungs. Most intratracheal infectionsin goats with P. haemolytica alone resulted in ei-ther mild pneumonia or the infection being elimi-nated quickly with no obvious pneumonia (BuddIeet al. 1990).ACKNOWLEDGEMENTSThe financial support provided by IRPA grant50332 is gratefully acknowledged.REFERENCESBUDDLE, B.M., A PFEFFER, DJ.W. COLE, H.D. PULFORDand MJ. RALsTON. 1990. Experimental respira-tory infection of goats with caprine herpesvi-rus and Pasteurella haemolytica. N. Z. Vet. J 38:22-27.DAVIES, D.H., M. HERCEG, B.A.H. JONES and D.C.THURLEY. 1981. The pathogenesis of sequentialinfection with parainfluenza virus type 3 andPasteurella haemolytica in sheep. Vet. Microbiol.6: 173-182.DAVIES, D.H., M. HERCEG and D.C. THURLEY. 1982.Experimental infection of lambs with adenovi-rus followed by Pasteurella haemolytica. Vet.Microbiol. 7: 369-381.GILMOUR, NJ.L. 1980. Pasteurella haemolytica infec-tions in sheep. Vet. Quarterly 2: 191-198.JONES, G.E. 1983. Mycoplasmas of sheep and goats:a synopsis. Vet. Rec. 113: 619-620.LENETTE, E.H., E.H. SPAULDING andJ.P. TRUANT. 1974.Manual of Clinical Microbiology, 2nd edn. p.246.Washington: American Society for Mic-robiology.SHEIKH-OMAR, AR. and AR. MUTALIB. 1985. Isolationof Mycoplasma arginini from a goat in Malaysia.Vet. Rec. 116: 330-331.(Received 25 May 1993)242 PERTANlKAJ. TRap. AGRIC. SCI. VOL. 17 NO.3, 1994

Experimental respiratory infection of goats with Mycoplasma arginini and Pasteurella haemolytica A2

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Experimental respiratory infection of goats with Mycoplasma arginini and Pasteurella haemolytica A2

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