The susceptibility of laboratory mice to Borrelia burgdorferi was evaluated for selected genotypes and ages. C3H/He, SWR, C57BL/6, SJL, and BALB/c mice inoculated at age 3 days developed uniformly severe polyarthritis at 30 days after intraperitoneal inoculation. Mice inoculated at age 3 weeks also developed polyarthritis, but severity was influenced by genotype, with C3H/He and SWR mice the most severely affected. Susceptible strains developed higher IgG ELISA antibody titers to B. burgdorferi than did resistant mice. Adult (12 weeks) C3H/He mice were also susceptible, but arthritis was not as severe as in those inoculated at age 3 weeks. SKH (hairless) mice developed polyarthritis but not skin disease when inoculated intradermally. Carditis occurred frequently among C3H/He, BALB/c, and hairless mice and in some SWR mice but not in C57BL/6 or SJL mice. This study demonstrates that severity of Lyme borreliosis is age- and genotype-dependent and that laboratory mice are a potentially valuable model.

Lyme borreliosis is a tick-borne illness caused by Borrelia burgdorferi. The gene for outer surface protein A (OspA) from B. burgdorferi strain N40 was cloned into an expression vector and expressed in Escherichia coli. C3H/HeJ mice actively immunized with live transformed E. coli or purified recombinant OspA protein produced antibodies to OspA and were protected from challenge with several strains of B. burgdorferi. Recombinant OspA is a candidate for a vaccine for Lyme borreliosis.

SummaryBorrelia burgdo~feri, the spirochetal agent of Lyme disease, is transmitted by Ixodes ticks. A vaccine based on B. burgdo~feri outer surface protein (Osp) A protects mice from spirochete infection. Here we report on the expression of OspA on spirochetes inside engorging ticks and relate OspA expression to antispirochetal immunity. Spirochetes in the gut of unfed nymphal ticks were stained by an OspA antibody, whereas in feeding ticks, the majority of spirochetes in the gut and salivary glands did not stain with the antibody. Thus, OspA was not expressed on most spirochetes during transmission from the vector to the vertebrate host. To examine the mechanism of protection afforded by OspA antibody, mice were passively immunized with OspA antibody at different times relative to tick attachment. When OspA antibody was administered to mice before or at the time of tick attachment, spirochetal development events in the vector, such as growth and salivary gland invasion, were blocked and the mice were protected from B. burgdotferi infection. When OspA antibody was administered to mice 48 h after tick attachment, spirochetes persisted in the nymphs and the mice were not protected despite the presence of circulating antibodies in the host as well as in the tick blood meal. Thus, OspA immunity appears to be effective only during a narrow window time at the beginning of the blood meal when antibodies bind to OspA-expressing spirochetes in the tick gut and block transmission from the vector to the host.T he spirochetal agent of Lyme disease, Borrelia burgdooCeri is transmitted by Ixodes ticks. In unfed nymphal ticks, spirochetes are found in the gut (l). During tick feeding, the bacteria disseminate to the salivary glands and infect the vertebrate host (2-4). A vaccine based on B. burgdo~ri outer surface protein (Osp) A which protects mice from infection is currently being tested in human trials (5-7). Here we examine the expression of OspA on spirochetes within engorging nymphs and relate OspA expression to protection afforded by OspA antibody. Materials and MethodsMice. Pathogen-free C3H/HeN (C3H) mice were purchased from the National Institutes of Health (Bethesda, MD). Maintenance and Infection of Ticks. Ixodes dammini (also known as L scapularis)were derived from a colony in its second generation from field-collected adults, and were free of inherited infection. Larvae were allowed to engorge on CD-1 mice that had been infected 2 wk previously by the bites of three to five nymphal I. dammini containing the N40 strain of B. burgdotferi.Engorged larvae were collected, held in mesh-covered plaster of paris containing vials, and were allowed to molt at 95% relative humidity at 21~ The infected nymphs were held under the same conditions before their use in experiments, usually within 2 mo of molting. Antibody Staining of OspA-expressing Spirochetes in NymphalTicks. Guts and salivary glands were dissected out of nymphs and prepared for antibody staining as previously described (2). mAb CIII.78, which binds to a COOH-t...

. After ex pos ure to a varian t of Citro bacter [reundii , suckling and adult mice developed transmissible murine coloni c hyper plasia of the same degree of seve rity . Muco sal hyperpl asia was most severe 2 to 3 weeks after inocu lation and then regressed . Suckling mice had a high morta lity because of seconda ry infla mmat ory and erosive changes . Severe hyper plasia was char acterized by mitotic activity along the ent ire crypt column and surface mucosa .Tra nsmissible m uri ne colo nic hyperplasia is a nat ura lly o ccu rr ing infectious di se ase of mi ce , cha ra cte rize d b y mu co sal hyp e rpl asia of th e d ista l col on . Erosio n a nd inf la m ma tio n ma y ac co m pa ny t he hyp e rpl asia. It is ca use d by a varia nt of Citrobacter [reundii [ I I. In a na tural o u tb re a k ret arded g ro wth , ruffle d fur , soft feces , o ccasio na l rect al pr ol a pse a nd mod erat e mort ality were see n in mic e a t th e lat e s uc kling a nd ea rly we an ling ag es. T he se signs we re se e n rarel y in a d u lts [3].T he ac tua l incid ence of tr an smi ssibl e mu rin e col on ic hyp erpl a sia in mouse co lo nies is difficult to d e termin e , since th e ine xp erien ced o bserve r ma y not not e the vag ue cl in ical signs a nd fre q uent lac k o f o bv io us gross le sion s . A num b e r o f po ssibl y rel at ed synd ro mes of mic e ha ve be en de scri be d b y o t he r la bo ra tories .T wo o f th ese la b orat orie s isol at e d varia nt s of C. fre undii a nd rep ro duce d co lo nic hyp e rpl a sia in experi me nt a lly in o cul a te d mic e [2 , 5 , 6 ]. T he pat ho lo gy de scribe d in th e se re po rts parti all y re sembl ed th at for tr an smissibl e murin e colonic hyp e rpl asia , b ut th ere a re diffe re nc es th at m ak e it di fficult to sta te t he y a re a ll th e sa me di se a se p roce ss . T hese d iffe re nces ma y be re al o r ma y b e ca use d b y vari at io n in co nd it io ns a nd tim e o f sa m p ling . T he ag es of mi ce a nd histo gen e sis o f th e lesion s were not given in re po rt s of ei t her the natu ral o r exp e rime n ta l di se ase . Ot he r possibl y rel at ed le sion s , of un d et e rmin ed ca us e , have bee n d escribe d [3 , 11] . T hese we re cl assi fied as co litis cystica [3 ] a nd ne opl asia [11] a n d had down growth of mu co sal e pi the lium into t he s u b m ucosa . E p ithe lia l d o wn gro wth ha s not bee n see n in tran smi ssible murin e col onic hyp erplasia o r o t he r hyp erpla st ic di se a se s kn o wn to be associa te d wit h C. freu ndii.Ou r stu dy exa mi ne s the path o gen e sis o f t his di se ase in N I H Sw iss m ice a nd co m pa res le sion s to th o se found by oth ers . Becau se of report ed clinical di fferences 223

The effectiveness of antibiotic treatment was examined in a mouse model of Lyme borreliosis. Mice were treated with ceftriaxone or saline solution for 1 month, commencing during the early (3 weeks) or chronic (4 months) stages of infection with Borrelia burgdorferi. Tissues from mice were tested for infection by culture, PCR, xenodiagnosis, and transplantation of allografts at 1 and 3 months after completion of treatment. In addition, tissues were examined for the presence of spirochetes by immunohistochemistry. In contrast to saline solution-treated mice, mice treated with antibiotic were consistently culture negative, but tissues from some of the mice remained PCR positive, and spirochetes could be visualized in collagen-rich tissues. Furthermore, when some of the antibiotic-treated mice were fed on by Ixodes scapularis ticks (xenodiagnosis), spirochetes were acquired by the ticks, as determined based upon PCR results, and ticks from those cohorts transmitted spirochetes to naïve SCID mice, which became PCR positive but culture negative. Results indicated that following antibiotic treatment, mice remained infected with nondividing but infectious spirochetes, particularly when antibiotic treatment was commenced during the chronic stage of infection.