Lyme neuroborreliosis in 2 horses.

Lyme neuroborreliosis--characterized as chronic, necrosuppurative to nonsuppurative, perivascular to diffuse meningoradiculoneuritis--was diagnosed in 2 horses with progressive neurologic disease. In 1 horse, Borrelia burgdorferi sensu stricto was identified by polymerase chain reaction amplification of B burgdorferi sensu stricto-specific gene targets (ospA, ospC, flaB, dbpA, arp). Highest spirochetal burdens were in tissues with inflammation, including spinal cord, muscle, and joint capsule. Sequence analysis of ospA, ospC, and flaB revealed 99.9% sequence identity to the respective genes in B burgdorferi strain 297, an isolate from a human case of neuroborreliosis. In both horses, spirochetes were visualized in affected tissues with Steiner silver impregnation and by immunohistochemistry, predominantly within the dense collagenous tissue of the dura mater and leptomeninges.

Borrelia burgdorferi OspA is an arthropod-specific transmission-blocking Lyme disease vaccine.

Borrelia burgdorferi, the spirochetal agent of Lyme disease, is transmitted by Ixodes ticks. A vaccine based on B. burgdorferi 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. burgdorferi 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.

Protection against antigenically variable Borrelia burgdorferi conferred by recombinant vaccines.

Due to local variation in the antigenicity of the agent of Lyme disease (Borrelia burgdorferi), a vaccine derived from any one isolate of this spirochete may fail to protect against the heterogeneous population of organisms that may be present in an enzootic focus. Accordingly, we determined whether antigenically variable spirochetes delivered by naturally infected ticks, collected from a site where transmission is intense, may fail to infect mice actively immunized with recombinant glutathione transferase outer surface fusion proteins A or B (OspA and OspB). Virtually all mice vaccinated by either immunogen appeared not to become infected, as determined by culture or histopathology of their tissues. We conclude that Osp vaccination of mice effectively prevents infection by the agent of Lyme disease in a simulated natural cycle of transmission.

Vaccination against Lyme disease caused by diverse Borrelia burgdorferi.

Diversity and mutations in the genes for outer surface proteins (Osps) A and B of Borrelia burgdorferi sensu lato (B. burgdorferi), the spirochetal agent of Lyme disease, suggests that a monovalent OspA or OspB vaccine may not provide protection against antigenically variable naturally occurring B. burgdorferi. We now show that OspA or OspB immunizations protect mice from tick-borne infection with heterogeneous B. burgdorferi from different geographic regions. This result is in distinct contrast to in vitro killing analyses and in vivo protection studies using syringe injections of B. burgdorferi as the challenge inoculum. Evaluations of vaccine efficacy against Lyme disease and other vector-borne infections should use the natural mode of transmission and not be predicated on classification systems or assays that do not rely upon the vector to transmit infection.

Protection of mice against the Lyme disease agent by immunizing with recombinant OspA.

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.

Lyme borreliosis in transgenic mice tolerant to Borrelia burgdorferi OspA or B.

The evolution of Lyme borreliosis in transgenic mice tolerant to Borrelia burgdorferi outer surface proteins (Osps) A or B was assessed to investigate the role of immunity to OspA or B in infection and pathogenesis of Lyme disease. Antibodies to OspA or B protect immunocompetent C3H/HeJ or C.B.17 severe combined immunodeficient (scid) mice from challenge with B. burgdorferi. Moreover, arthritis in infected C3H mice resolves with the rise of high titers of B. burgdorferi specific antibodies, including OspA and B, whereas disease persists in scid mice--suggesting that the regression of arthritis may be due to the development of borreliacidal OspA or B antibodies. To evaluate the course of Lyme borreliosis in OspA or B tolerant mice we developed transgenic mice that expressed OspA or B under control of the major histocompatibility complex (MHC) class I promoter. Mice carrying OspA or B transgenes on a C3H/HeJ (C3H, disease-susceptible) or C57BL/6 (B6, disease-resistant) background, immunized with OspA or B, did not mount a humoral or cellular immune response to OspA or B, respectively, but responded normally to other B. burgdorferi antigens. The evolution of Lyme borreliosis, including infection and the development of arthritis and carditis, was similar in transgenic and nontransgenic littermates suggesting that an OspA or B immune response is not singularly involved in either the genesis or regression of Lyme disease in C3H or B6 mice.

Effect of anti-interleukin 12 treatment on murine lyme borreliosis.

The effect of anti-interleukin (IL-12 treatment on Lyme borreliosis in C3H/HeN (C3H) mice was assessed because other studies have implicated CD4+ T cell helper (Th) type 1 responses in the genesis of disease caused by Borrelia burgdorferi. Infection of inbred mice with B. burgdorferi results in varying degrees of arthritis: BALB/c mice develop mild disease and C3H mice develop severe arthritis that is most pronounced 2-4 wk after infection. Since IL-12 is a major inducer of Th1 responses, we blocked this cytokine in vivo in B. burgdorferi infected C3H mice, and evaluated the effects of treatment on the development of arthritis at the peak of acute joint inflammation (14 d) and in the resolution phase (60 d) of disease. As expected, intraperitoneal administration of an anti-IL-12 monoclonal antibody (mAb) to C3H mice resulted in a decrease in both IFN-gamma and B. burgdorferi-specific IgG2a in serum, indicative of diminished Th1 responses. No IL-4 production was detected in serum of anti-IL-12 mAb treated or control mice. IgG1 and IgG2b levels did not increase in B. burgdorferi infected mice treated with anti-IL-12 mAb compared with controls suggesting that Th2 responses were not affected. Nevertheless, CD4+ T cells from both control and anti-IL-12 mAb treated mice had similar in vitro responses to B. burgdorferi antigens. Treatment with anti-IL-12 mAb produced a significant reduction in peak arthritis severity, and an increase in the number of spirochetes in ear tissue. These data show that treatment of B. burgdorferi infected mice with anti-IL-12 mAb results in a reduction of the Th1 and/or innate immune responses in vivo and a reduction in the severity of acute murine Lyme arthritis.

Temporal pattern of Borrelia burgdorferi p21 expression in ticks and the mammalian host.

The temporal synthesis of the P21 protein of Borrelia burgdorferi and the development of the humoral response to this antigen was assessed in infected mice. p21 is a member of the ospE-F gene family and its protein, P21, has been shown to be expressed by B. burgdorferi within infected mice but not by spirochetes cultured in vitro. P21 was not detected on B. burgdorferi in unfed or engorged Ixodes dammini (also known as I. scapularis) ticks, further supporting the postulate that P21 synthesis is specific for the mammalian host. In B. burgdorferi-infected mice, ospE mRNA and OspE antibodies were observed at 7 d, whereas p21 mRNA and P21-specific antibodies were detected at 21-28 d, suggesting that p21 is expressed later than ospE. Moreover, ospA mRNA was not discernible until day 14, indicating that ospA, like p21, is not expressed in the early stages of tick-transmitted murine Lyme borreliosis. Because p21 is expressed during infection in mice, we assessed the human humoral response to P21. 28% (34 of 122) of the patients with either early- or late-stage Lyme disease, and 33% (11 of 33) of the individuals with Lyme arthritis had P21 antibodies, suggesting that a P21 response may serve, at least partially, as a marker of infection. Active immunization with recombinant P21 did not protect C3H mice from tick-borne B. burgdorferi infection, and passive transfer of P21 antiserum to infected mice did not alter the course of disease. These data suggest that the antigenic structure of B. burgdorferi changes during the early stages of murine infection.

Immunization against the agent of human granulocytic ehrlichiosis in a murine model.

The agent of human granulocytic ehrlichiosis (HGE) is a newly recognized tick-borne pathogen that resides within polymorphonuclear leukocytes. C3H/HeN mice can become infected with the agent of HGE (designated aoHGE) by syringe inoculation or tick-borne infection and develop transient neutropenia. They thereby partially mimic human disease and provide a model in which to study immunity to this microorganism. Mice vaccinated with lysates of purified aoHGE, or administered aoHGE antisera, were partially protected from both syringe- and tick-transmitted challenge with aoHGE. These data suggest that antibodies are sufficient to provide substantial, but not complete, immunity against aoHGE.

Lack of infectivity of bovine leukemia (C-type) virus to rats.

Sprague-Dawley rats were given ip injections of bovine culture and sheep cultures of bovine leukemia virus (BLV) and Gross passage-A leukemia virus [MuLV(G)]. Sera were tested to BLV antigens. BLV did not induce tumors in Sprague-Dawley rats, but the rats were susceptible to MuLV(G) at low doses.

Relationship of colonic mucosal background to neoplastic proliferative activity in dimethylhydrazine-treated mice.

Proliferative activity of background and neoplastic colonic mucosa was examined following five months of weekly injections of 1,2-dimethylhydrazine (20 mg/kg) and one or four months of rest to determine whether previously reported changes may result from an acute or chronic effect of dimethylhydrazine and whether differences exist between stages of neoplasia. To determine whether neoplasia is responsive to a proliferative stimulus, 1,2-dimethylhydrazine dihydrochloride-treated mice were inoculated with Citrobacter freundii. The labeling index and the proliferative zone increased in background mucosa after one month; whereas after four months labeling index, proliferative zone and crypt heights increased, but the mitotic index decreased. There was a positive linear correlation between advancing tumor grade and increasing tumor labeling index and mitotic index. Background labeling index, even when elevated by C. freundii inoculation, had no effect upon tumor labeling index. Mitotic index diminished in background and neoplastic mucosa following prolonged rest and increased in both following C. freundii inoculation. These studies show that 1,2-dimethylhydrazine dihydrochloride causes long-term changes in background mucosa that are apart from a reparative response to cytotoxicity. As tumors progress, labeling index and mitotic index increase, suggesting a multistage process of evolution.

Autoradiographic cytokinetics of colonic mucosal hyperplasia in mice.

The cytokinetics of a naturally occurring hyperplastic disease of the colon in mice were determined by autoradiography and compared to kinetic changes seen by others in nonneoplastic and neoplastic colonic disease. Cell cycle parameters were determined using the fraction-labeled mitosis method. Labeling index, labeling pattern, and migration rates were also evaluated. During colonic hyperplasia, there was an increase in variation of DNA synthesis times, resulting in prolongation of the S phase. There also was prolongation of the total cell cycle time and G1 phase. In addition, labeling index was increased 2-fold, the proliferative zone was extended to include the entire crypt column and surface mucosa, and the migration rate was accelerated. These findings parallel the "atypical" cytokinetics found in human and murine neoplastic and preneoplastic disorders of the colon and may be a typical proliferative response of mucosa to a variety of stimuli.

Modification of early dimethylhydrazine carcinogenesis by colonic mucosal hyperplasia.

The interaction of colonic mucosal hyperplasia with early 1,2-dimethylhydrazine (DMH) carcinogenesis was studied in random-bred NIH Swiss mice utilizing hyperplasia-inducing Citrobacter freundii. Mice inoculated with this bacterium developed significantly more DMH focal atypia than did mice without hyperplasia following a single dose of DMH (20 mg/kg). Mice with hyperplasia also developed DMH focal atypia with diminished doses of DMH (10 and 5 mg/kg), while normal mice did not. The effect of C. freundii on early DMH carcinogenesis was shown to be due to the hyperplasia rather than to a direct interaction of the bacterium with DMH. Focal atypia arose in high incidence 1 month after a single dose of DMH (20 mg/kg) but did not appear to progress to later stages of neoplasia, since significantly fewer atypia were present at 2 to 4 months among a randomized population. Colonic focal atypia may represent a reversible preneoplastic or precursor lesion as seen in other tissues, with features more aligned to neoplasia than to hyperplasia.

Morphogenesis of early 1, 2-dimethylhydrazine-induced lesions and latent period reduction of colon carcinogenesis in mice by a variant of Citrobacter freundii.

The morphogenesis of 1, 2-dimethylhydrazine (DMH)-induced lesions in the colon of outbred NIH Swiss mice was determined for up to 5 months of treatment. The effect of hyperplasia on DMH carcinogenesis was also evaluated by introducing a transient hyperplastic stimulus to the colon during the chronic weekly treatment regimen of DMH. The hyperplastic stimulus was a naturally occurring disease of mice, transmissible murine colonic hyperplasia, which is caused by a variant of Citrobacter freundii. In control mice, those not receiving the bacterium, weekly injections of the carcinogen induced neoplastic changes first detectable at two months of treatment in all segments of the colon and in both sexes. The changes increased in frequency and severity with time. Diffuse mucosal hyperpladia and chronic inflammatory and degenerative changes were also associated with DMH after prolonged treatment. The hyperplastic stimulus of C. freundii reduced the latent period for appearance of early DMH tumors, but it had no influence on already established DMH tumors.

Characterization of two genes, p11 and p5, on the Borrelia burgdorferi 49-kilo base linear plasmid.

A putative operon encoding two Borrelia burgdorferi N40 genes, p11 and p5 was cloned and localized to the 49 kilobase linear plasmid. p11 encodes an 11 kDa protein and p5 encodes a 5 kDa protein. The first 88 nucleotides of p11 have 81% identity with orf5 on a circular plasmid from Borrelia afzelii strain Ip21, suggesting that homologues of these genes may be present in different regions of the B. burgdorferi genome.

Graft-versus-host disease and sialodacryoadenitis viral infection in bone marrow transplanted rats.

The effect of a localized viral infection on the occurrence of graft-vs.-host disease (GVHD) was examined in allogeneic rat bone marrow chimeras (ACI/LEW). Animals without clinical evidence of GVHD, 62 days after bone marrow transplant, were infected in salivary and lacrimal glands with sialodacryoadenitis virus (SDAV), and sacrificed 8-25 days postinfection. Using established histologic criteria, GVHD was found more frequently in salivary and lacrimal glands of SDAV-infected chimeras than uninfected chimeras. Skin and oral mucosa, tissues not infected by the virus, showed no differences in occurrence of GVHD, suggesting that the viral infection induced only local and not systemic GVHD. GVHD and SDAV infection, which are histologically similar, were differentiated by examining tissues for SDAV antigen using immunoperoxidase technique. Histologic changes were present for at least 1 week longer than viral antigen, suggesting they represented GVHD rather than viral infection. GVHD and SDAV infection were also differentiated by looking for a histologic feature characteristic of GVHD and not found in SDAV infection (periductal lymphocytic infiltrate). This was found in SDAV-infected chimeras more frequently than uninfected chimeras, suggesting that the viral infection somehow induced GVHD. Results showed a localized increase in the occurrence of GVHD subsequent to localized viral infection.

Response of genetically susceptible and resistant mice to intranasal inoculation with mouse hepatitis virus JHM.

Mouse hepatitis virus (MHV)-JHM infection was studied in genetically susceptible (BALB/cByJ) and resistant (SJL/J) mice following intranasal inoculation at 1, 3, 6 or 12 wk of age. Markers of infection included histology, immunohistochemistry, virus quantification and virus serology. All BALB mice developed severe disseminated disease with high mortality due to encephalitis and hepatitis. Peak MHV titers appeared in brain, liver, spleen and intestine on days 3 or 5. Age at inoculation did not influence virus titers in brain, spleen or intestine, but virus titers in liver were inversely proportional to age at inoculation. In 6-wk-old BALB mice, virus was cleared from spleen, intestine and liver by day 30 and from brain by day 60. In intestine, MHV was localized to lymphoid tissue, without fecal excretion. SJL mice of all ages developed remarkably milder disease with low mortality occurring only among mice inoculated at 1 wk of age. SJL mice inoculated at 1 wk had disseminated infection at day 3, but lesions and antigen were cleared from most organs by day 5. Mice inoculated at 3 and 6 wk of age had minimal or no involvement of peripheral organs, and mice inoculated at 12 wk of age had infections restricted to the nose. At day 5, MHV titers in brain, liver, spleen and intestine were significantly lower or undetectable in SJL mice of all ages compared to age-matched BALB mice. In 6-wk-old mice, MHV was cleared from all organs by day 10. Serum antibody titers to MHV were many-fold higher in BALB mice, compared to SJL mice, which mounted only a modest response.

Sendai viral pneumonia in aged BALB/c mice.

Sendai virus (SV) infection in aged BALB/c mice was evaluated as a natural model for age-associated susceptibility to viral pneumonia. Young (2 month-old) and aged (22-24 month-old) BALB/c mice were inoculated intranasally with 100 median pneumonia doses (PD50) of SV and examined at 6, 10, and 20 days by virus titration, immunohistochemistry, histopathology, and serology. The aged mice had significantly higher virus titers in lung, prolonged infection, delayed development, and resolution of pneumonia and significantly lower serum antibody titers. In a second experiment, the responses of young mice were compared to intermediate-aged mice (11-13 and 17-18 months old). The intermediate-aged mice had some characteristics of young mice and others of aged mice. The results indicate that SV infection can be used to study aging-associated susceptibility to a pneumotropic virus in a natural host, and that susceptibility of mice to viral pneumonia increases gradually during aging.

Characterization of a 30-kDa Borrelia burgdorferi substrate-binding protein homologue.

A Borrelia burgdorferi chromosomal gene encodes a 30-kDa antigen (P30) that has considerable homology with periplasmic substrate-binding proteins of Gram-negative bacteria, and is recognized by antibodies in sera from a subset of patients with Lyme disease and from B. burgdorferi-infected mice. The p30 gene is 801 nucleotides in length and P30 contains 267 amino acids, with a predicted molecular mass of 30 kDa. The P30 amino acid region 36-258 has homology to conserved domains of the oligopeptide permease A of Gram-negative bacteria. Immunofluorescence studies using murine anti-P30 serum suggest that P30 is on the outer surface of B. burgdorferi. P30 expression could be detected in representatives of all 3 subspecies of B. burgdorferi sensu lato, but not in all of the tested strains. Antibodies to P30 were detected in sera of 18 out of 82 patients (22%) with Lyme disease, including individuals with early- or late-stage infection. Although antibodies to P30 are present in the sera of C3H/HeN mice infected with B. burgdorferi for at least 90 days, immunization with recombinant P30 does not protect mice from infection. We conclude that P30 is a putative substrate-binding protein of B. burgdorferi and is immunologically recognized in human and murine Lyme borreliosis.

Intranasally administered alpha/beta interferon prevents extension of mouse hepatitis virus, strain JHM, into the brains of BALB/cByJ mice.

Intranasally administered alpha/beta interferon blocked extension of the coronavirus, mouse hepatitis virus, strain JHM (MHV-JHM), from the nose to the brain of BALB/cByJ mice following intranasal inoculation with the virus. Two hundred units of alpha/beta interferon were administered intranasally to BALB/cByJ mice daily over a five day period. The mice were exposed intranasally to 10(3) median tissue culture infectious doses of MHV-JHM on the third day of interferon treatment. Two days after virus exposure, the proportion of mice with MHV in nasal turbinates was reduced from 10 of 10 in the untreated group to 7 of 10 in the interferon-treated group, and mean titers in virus-containing noses were lower in the interferon-treated group. Five days after virus exposure, the proportion of mice with infectious virus in the brain was significantly lower in the interferon-treated group (1 of 10 mice) than in the untreated group (10 of 10 mice). Systemic infection, as measured by presence and concentration of virus in the spleen, was not affected by intranasal interferon treatment. These results suggest that intranasally administered interferon protects against local extension of MHV-JHM from nose to brain, but not against dissemination of virus to other organs, such as the spleen.