The cilia associated respiratory (CAR) bacillus is a respiratory pathogenic bacterium of rats and other species of animal. We determined by enzymelinked immunosorbent assay (ELISA) antibudies t0 CAR bacillus antigen in sera from 20 colonies of rats. Six out of 10 Mycoplasma pulmoms ELISA positive experimental colonies contained CAR bacillus seropositive rats, comprising 26 out of 45 (58 %) samples. CAR bacillus infection was not diagnosed by ELISA in 183 samples from 10 M. pulmonis free SPF-rat breeding colonies

Boot, R

Thuis, H C W

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Suuntl J Lab Amm Sci. No.4 1998,Vol. 25Monitoring of Rat Colonies for Antibodies to CAR Bacillusby H.C‘. W. Thuis & R.BontSection ofLaboratory Animal Micrubmlogy,Diagnostic Laboratory for Infectious Diseases and Perinatal ScreeningNational Institute of Public Health and Lhe Envtronmenl,PO Box 1, NL 3920 Bilthoven, the NetherlandsIntroductionThe cilia associated respiratory (CAR) bacillus is arespiratory pathogenic bacterium of various animalspecies including mice (Griffith et (11.11988), rats(Van sz'eten 1980) and rabbits (Waggie etal.,1987). Guineapigs and hamsters (Matsuxhita eta1. 1989; Shq/‘i-Darkey er al,, 199]) aresusceptible to experimental infection.CAR bacillus infection can be diagnosed byhistology (Ganaway et al., 1985‘), culture (Schoebet aL, 1993)‘ PCR (C'undifir et al..1994) and byvarious serologieal assays such as the indirectimmunofluorcseem antibody assay (IFA)(Mamushita et (11., 1987) and the enzyme—linkedimmunosorbem assay (ELISA) (Ganaway et al‘,1985). We investigated the occurrence of thebacterium by measuring antibodies to CARbacillus antigen in sera from 10 Mpulmnm’sinfected experimental and 10M pulmonis -freebreeding colonies of rats,Materials and MethodsSeraSera were obtained from 20 rat colonies. 73 Semwere from 10 Mycoplasma pulmonis ELISApositive colonies (all NL), which were alsoinfected by one 01‘ more (non)respiral0ry viruses(data provided by Dr. J.T.M. van dcr Logt, ICLASreference centre for rodent viruses, Nijmegen, theNetherlands) As none of these colonies werestrictly barrier-maintained these were consideredconventional. 183 Sera were obtained from 10commercial and institutional, hysterectomy-derived barrier-maintaincd (SPF) breedingcolonies (F, FRG, GB, NL) that were reponed tobe free from infection by Mpulmonis and otherpathogenic microorganisms. (Hist0)pathologieal198examinations for CAR bacillus infection were notincluded in this study.ELISAAntibudies t0 Mpulmom‘s and CAR bacillus weredetermined by ELISA as described forStreptobacillu» monillformis (Boot 22‘ 11/1, 1993)using antigens of Mpulmom's (ICLAS referencecentre for rodent viruses, Nijmegen, theNetherlands) and CAR bacillus (Harlan Olae Ltd..Blackthorn, Bicester, England). The ELISA wasconsidered positive if the optical density (OD) value(extinction) of the l : 50 serum dilution exceededthe mean + 3 S1) 0fthe mean ofthe ODS in the seraofrats from Mpulmam's and CAR bacillus free SPFcolonies. Positive and negative control sera wererun in each test. The CAR bacillus ELISA waspreviously evaluated by comparing ELISA withhistopathology in experimental infections usingCAR bacillus infected tracheal scrapings fromwild Rnorvegicus and in transmission studiesmimicking natural infection in WU rats (Thuis etal., 1998).Statistical analyxisDifferences between groups were evaluated byFischer’s exact lest using the Epistat statisticalprogram run on a personal computer,ResultsAll conventional rat sera were (again) positive bythe Mpulmam‘s ELISA (Table 1). Six of the 10conventional experimental colonies contained ratsshowing antibodies to the CAR bacillus antigen;the 26 CAR bacillus positive samples comprised58 % 0fthc samples from these 6 colonies and 36% of the Mp+ samples, Antibodies to CARbacillus and Mpulmonis were not detected in anyScand 11 Lab. Arum SCI No 4 1993 Vol 25Table / ELISA antibodies to CAR bacillus in conventional M pulmnm’s infected and in SPFcolonies of ratsConventional colonies SPF colonies     antibody ELISA antibody ELISAM pulmanisCAR & CARcolony strain M. pulmonis bacillus colony bacillus1 Wistar 5/5* 1/5 1 1 0/202 LEW/PVG 10/10 8/10 12 0/093 ” 10/10 0/10 13 0/104 Wistar 7/7 3/7 14 0/325 Wistm‘ <1 /4 0/4 1 5 0/406 EN 6/6 1 /6 16 0/287 Wistar 4/4 0/4 17 0/168 Rnnrvegzcus 7/7 6/7 18 0/129 7 10/10 7/10 19 0/0810 Wistar 10/10 0/10 20 0/0873/73 426/73 @253       * positive/examined199 Scuntl J Lab Annn SCI No 4 l998,VoI 250f the 183 samples from the 10 SPF rat coloniesThe percentage of CAR bacillus seropositiveconventional rats was significantly higher than that01' SPF rats (riseher‘s exact test‘ p < 0100])DiscussionThe increased use of SPF animals has decreasedthe occurrence of chronic resp/ralory diseare(CRD) in rats. CRD has been frequently associatedwith l ycoplaxma pulmonis infection (Schoeb eral., 1996) and rather recently also CAR bacillusinfection has been implicated in CRD (Gamzwayet al,,1985).Natural CAR bacillus infection withoutM.pulmonis infection may be rare as Schoeb et a1.(1997) found 15 of 16 primary rat CAR bacillusisolates only to be free from Mpu/mom‘s infection.This finding is relevant to the interpretation ofstudies on the pathogenesis of CRD in rats, as lunglesions observed in experimental infections mayhave been the result of dual infection. Schoeb’sfindings also suggest that Mpulmonis might bepresent as a contaminant in the CAR bacillusantigen used in serological studies. Our study wasnot troubled by such a contamination asMpulmom's positive sera were not invariably CARbacillus positive (Table 1)FELASA recommends to monitor colonies forCAR bacillus infection only when clinical signs orpathological observations suggest infection by thebacterium (Kraft at al., 1994. Rahbt’nder er al.,1996). As no antibodies to CAR bacillus antigen(indicating infection) were detected in any of thesamples from the 10 barrier—maintained breedingcolonies, the recommendations seem reasonable forwell managed SPF colonies of rats. CAR bacillusinfection (as suggested by seropositive animals)may however be present in colonies that should beconsidered as conventional as no specificpreventive measures were in operation andinfections by viruses (data not Shown) andMpulmonis were present. It might be argued thatour results are biased by using Mpu/monisinfection as a criterion for selecting conventionalcolonies but sera from conventional Mpulmonis'uninfected rat colonies were not available.Mpulmam’s infection is not invariablyaccompanied by CAR bacillus infection: the200absence of CAR bacillus infection in 4Mpulmmzix pmitive colonies might partly be dueto differences in the ease of transmission of bothmicroorganisms. Vertical transmission has notbeen documented for CAR bacillus. Verticaltransmission has however been found forAipulmom's (Ganaway e! [111, 1973) but (asexpected) we did not detect the infection in SPFcolonies. Horizontal transmission of CAR bacillusseems to be difficult, but no studies have beenfound comparing the ease of horizontaltransmission of CAR bacillus and Mpulmonisdirectly.In conclusion: our data suggest that CAR bacillusinfection will only rarely be found inhysterectomy-derive¢ barrier-maintained (SPF)colonies of rats but may be encountered in lesswell protected (conventional) colonies. Thepostmortem examination of rats showing signs ofCRD should include a search for (IAR bacillusinfection.SummaryThe eilia associated respiratory (CAR) bacillus is arespiratory pathogenic bacterium of rats and otherspecies of animal. We determined by enzyme-linked immunosorbent assay (ELISA) antibudiest0 CAR bacillus antigen in sera from 20 coloniesof rats. Six out 0f 10 Mycap/asma pulmomsELISA positive experimental colonies containedCAR bacillus seropositive rats, comprising 26 outof 45 (58 %) samples. CAR bacillus infection wasnot diagnosed by ELISA in 183 samples from 10Mpulmonis free SPF-rat breeding colonies,AcknowledgementsDr. J.T.M. van der Logt of the ICLAS referencecentre for rodent viruses is thanked for supplyingserum samples from various rat colonies and thepreparation of Mpu/mom’s antigen.References800/ R‘ RJIG Bak/cm: H Thms, J1. Veenema & Hde Huog. An enzyme-linked immunosorbentassay (ELISA) for monitoring rodent coloniesfor Streptobaeillus monilifortnis antibodies.Laboratory Animals. 1993. 27. 350—357.Cumin?” DD. C Besch-Willéford. RR Hook. CLFranklin & LK Riley Detection of cilia—associated respiratory bacillus by PCR.Journal of Clinical Microbiology. 1994, 32.1930-1934.GanawayJR, .4411/1/[8/1. TD Moore. [U BohnerNatural infection in germfi‘ee rats withMyeoplusrnu pulmonis. Journal of InfectiousDiseases. 1973, 127, 529-537.Ganaway JR, TH Spencer. TD Moore & AMAllen.Isolation, propagation and characterizationofa newly recognized pathogen, ciliaassociated respiratory bacillus of rats, anetiological agent of chronic respiratory disease.Infection & Immunity. 1985, 4.7, 472—479.Grimm JW. W] White, PJ Danneman & CM Lang.Cilia—associated respiratory (CAR) bacillusinfection of obese mice. Veterinary Pathology,1988, 25. 72—76.Kraft V, HM Blanchet. R b’nnt, .4 Denny, AKHansen. A Hem, H van Herc/t. / Kuns/yr'. JRNeedham, W Nzck/as'. A Perrot, C Rehb/nder, YRichard & G de 1002)). Recommendations forhealth monitoring of mouse. rat, hamster.guineapig and rabbit breeding colonies. Labo-ratory Animals.1994, 28, 1-12.Mutsuxhita S, M Kashima & H Juxhima.Serodiugnosis 0f cilia - associatedrespiratory bacillus infection by the indirectimmunofluorescence assay technique.Laboratory Animals. 1987, 21, 356-359.Matsmhz'ta S, H .lm/qz‘ma, T Matsumam & KFu/cutsu. Transmission experiments ofeiliuuesoeiuted respiratory bacillus in mice,rabbits and guineapigs. Laboratory Animals.1989, 23. 96- 102,Rehbinder C. P Baneux, D Forbes, H van Herck, WNicklas. Z Rugaya & (7 H’L'nlder. FELASArecommendations for the health monitoring ofScand. 1.1.311 Annn. Sci. No 4 1998 Vol, 25mouse. rat, hamster, gerbil. guineapig and rabbitexperimental colonies. Laboratory Animals.1996. 30. 193-208.Schoeb TR. K Dybvtg, MK Davidson & JK Davis,Cultivation of Cilia — associated respiratorybacillus in artificial medium and determinationof the 16 S rRNA gene sequence. Journal ofClinical Microbiology. 1993.31.2751-2757.30/1091) TR, JK Davis & JR Lindsey. Murinerespiratory rnyeoplasmosis. rat and mouse. In:International Life Sciences InstituteMonographs on Pathology of LaboratoryAnimals (TC Jones. U Mohr & RD Hunt eds).Berlin. Springer Verlag. 1996, pp 117-128.Schoeb TR, K Dybvz'g, KP Keis/ing, MK Davidmn& JK Davis. Detection of .lillcopluymapulmonis in eilia—assoeiuted respiratorybacillus isolates and in respiratory tracts ofrats by nestedPCR. Journal of Clinical Microbiology. 1997,35, 1667-1670.S'hoji-IMr/cye l" . T [[0/7 & N Kagzyama.Pathogenesis of CAR bacillus in rabbits.guineapigs, Syrian hamsters and mice.Laboratory Animal Science, 1991. 4]. 56"—571.Thul's HCW. JL Veenema, MA Knedam & R Boot.Experimentele CAR bacillus infectie bij ratten.Bioteehniek. 1998, 37: in press (in Dutch).Van sz‘eten MJ, HA Solleveld, JR Lindsey, FG dcGroot, C Zurcher & CF Hollander.Respiratory disease in rats associated with afilamentous bacterium: a preliminary reportLaboratory Animal Science. 1980. 30. 215-221.Waggl'e KS, TH Spencer & AM A/len Ciliasso»ciated respiratory (CAR) bacillus infection inNew Zealand White rabbits. LaboratoryAnimal Science. 1987. 37, 533,20/

Monitoring of Rat Colonies for Antibodies to CAR Bacillus

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Monitoring of Rat Colonies for Antibodies to CAR Bacillus

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