Respiratory reovirus 1/L induction of intraluminal fibrosis, a model of bronchiolitis obliterans organizing pneumonia, is dependent on T lymphocytes

Bronchiolitis obliterans organizing pneumonia (BOOP) is a clinical syndrome characterized by perivascular/peribronchiolar leukocyte infiltration leading to the development of intraalveolar fibrosis. We have developed an animal model of BOOP where CBA/J mice infected with 1 x 10(6) plaque-forming units (PFU) reovirus 1/L develop follicular bronchiolitis and intraalveolar fibrosis similar to human BOOP. In this report, we demonstrate a role for T cells in the development of intraluminal fibrosis associated with BOOP. Corticosteroid treatment of reovirus 1/L-infected mice both inhibited the development of fibrotic lesions when administered early in the time-course and promoted the resolution of fibrotic lesions when corticosteroid administration was delayed. Further, the depletion of either CD4(+) or CD8(+) T cells before reovirus 1/L infection also inhibited fibrotic lesion development. Both corticosteroid treatment and depletion of CD4(+) or CD8(+) T cells also resulted in decreased expression of the proinflammatory and profibrotic cytokines, interferon (IFN)-gamma and monocyte chemoattractant protein-1 (MCP-1). Further, treatment of mice with a neutralizing monoclonal antibody to IFN-gamma also significantly inhibited the development of fibrosis. Taken together, these results suggest a significant role for T cells in the development of reovirus 1/L-induced BOOP fibrotic lesions in CBA/J mice and suggests that T(H)1-derived cytokines, especially IFN-gamma, may play a key role in fibrotic lesion development.     

Respiratory Reovirus 1/L Induction of Diffuse Alveolar Damage: Pulmonary Fibrosis Is Not Modulated by Corticosteroids in Acute Respiratory Distress Syndrome in Mice

Acute respiratory distress syndrome (ARDS) is a clinical syndrome characterized by diffuse alveolar damage (DAD) secondary to an intense host inflammatory response of the lung to a pulmonary or extrapulmonary infectious or noninfectious insult. We have previously described a unique animal model in which CBA/J mice infected with reovirus 1/L develop ARDS. This model recapitulates the histopathological changes observed in human ARDS, which consist of the overlapping phases of exudation, including the formation of hyaline membranes, regeneration, and healing via repair with fibrosis. In this report, we show that the development of DAD in the acute phase of the disease and intraalveolar fibrosis in the late phase of the disease was not modulated by treatment with methylprednisolone (MPS). In the presence or absence of MPS, the majority of cells infiltrating the lungs after reovirus 1/L infection were polymorphonuclear leukocytes and macrophages. A number of key proinflammatory and anti-inflammatory cytokines/chemokines that are observed in the BAL fluid of ARDS patients were also found in the lungs of mice after reovirus 1/L infection and were not modulated by MPS. These include interferon-γ, interleukin-10, and monocyte chemoattractant protein. The histopathology, cytokine/chemokine expression, and response to corticosterids in reovirus 1/L-induced ARDS are similar to what is observed in human patients, making this a clinically relevant model.     

Respiratory reovirus 1/L induction of diffuse alveolar damage: a model of acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a clinical syndrome that is characterized by diffuse alveolar damage usually secondary to an intense host inflammatory response of the lung to a pulmonary or extrapulmonary infectious or noninfectious insult. In this report we describe a unique animal model in which CBA/J mice infected with reovirus serotype 1, strain Lang develop ARDS. This model recapitulates the histopathological changes observed in human ARDS, which consists of the overlapping phases of exudation including the formation of hyaline membranes, regeneration, and healing via resolution and/or repair with fibrosis. While the consequences of a number of infectious and noninfectious insults in various animal systems have been developed as models of human ARDS, they are models of acute lung injury and are of short-term duration. Therefore, they do not recapitulate all of the clinical and pathological phases observed in human ARDS. Thus, study of the cellular and molecular factors involved in these distinct phases of the disease have been limited. Reovirus 1/L infection of CBA/J mice will allow investigations of the pathophysiology of ARDS as it progresses from the initial stages of edema and neutrophilia to fibrotic lesion development in late stages.     

Respiratory reovirus 1/L induction of intraluminal fibrosis. A model for the study of bronchiolitis obliterans organizing pneumonia.

Bronchiolitis obliterans organizing pneumonia (BOOP) is a term that was first applied in 1985 to describe a long-observed but unclassified pattern of acute lung injury. BOOP lesions are characterized by fibrous extensions into the alveolar spaces in association with a peribronchiolar organizing pneumonia. Since 1985, an increasing number of reports of BOOP have appeared in the clinical literature, and it is now accepted that BOOP is a significant pulmonary syndrome. Although BOOP can be associated with a number of documented pulmonary insults, many cases are not associated with known causes and are thus classified as idiopathic. The lack of an appropriate small animal model that closely mimics the generation of BOOP lesions has been an impediment to basic studies of the pathogenic mechanisms responsible for the generation of BOOP in humans. In this report, we describe an animal model for BOOP in which CBA/J mice infected with reovirus serotype 1/strain Lang develop BOOP lesions. These lesions closely resemble those seen in humans and occur in a well defined temporal sequence that proceeds from initial peribronchiolar inflammatory lesions to characteristic, fibrotic cellular BOOP lesions over a 3-week time course.     

Degradation of Serum Amyloid A in Amyloid-Susceptible and Amyloid-Resistant Mouse Strains

Degradation of serum amyloid A (apoSAA) by resident peritoneal cells (RPCS) and conditioned medium (CDM), prepared with RPCS, from amyloid-susceptible CBA/J mice, amyloid-resistant CE/J mice and their amyloid-resistant CBA/J × CE/J F₁ progeny was investigated in vitro . Serum amyloid A was derived from murine acute phase (AP) plasma and associated with high density lipoprotein (HDL). Degradation of apoSAA by RPCS and CDM from CBA/J mice was complete while degradation by RPCS and CDM from CE/J mice did not occur. Degradation of apoSAA by RPCS and CDM from CBA/J × CE/J F₁ hybrid mice was indistinguishable from that by RPCS and CDM from the CBA/J parent. Intermediate fragments were not detected with either RPCS or CDM from CBA/J mice or CBA/J × CE/J F₁ hybrid mice. Degradation of apoSAA was inhibited by phenylmethanylsulfonyl fluoride (PMSF) indicating that the enzyme, secreted into the fluid phase, was a serine esterase. Unlike apoSAA, HDL-associated apoA-1 remained intact. It was thus concluded that while selective degradation of HDL-associated apoSAA (apoSAA-HDL) by RPCS from the CBA/J and CE/J mice was significantly different, the genetic study did not support the hypothesis that there was direct linkage between impaired degradation of apoSAA-HDL in the CE/J mouse strain and protection against amyloid fibril formation. As amyloid resistance in CBA/J × CE/J F₁ hybrid mice is not attributable to failure to express the amyloidogenic isoform apoSAA₂, the study supports the original hypothesis that amyloid resistance may be linked to expression of apoSAA_cej     

Genetic variation influences the B-cell response to immunization with a pneumococcal polysaccharide conjugate vaccine

CBA/J mice immunized with pneumococcal 23F-CRM(197) vaccine produce significantly lower titers of 23F-specific antibodies and fewer 23F-specific antibody-secreting cells (ASC) than did BALB/c or (CBA/J x BALB/c)F(1) (CCBAF(1)) mice. The reduced 23F-specific titers of CBA/J versus BALB/c or CCBAF(1) mice are presumably related to lower frequencies of 23F-specific ASC influenced by genetic variation.     

The effect of Rauscher and Moloney leukaemia virus on amyloid development in casein-treated CBA mice.

Infection of adult CBA mice with Rauscher or Moloney leukaemia virus concomitantly with caseination significantly accelerated spleen amyloid development in irradiated, bone-marrow protected mice, but had no effect on untreated, adult thymectomized or thymectomized irradiated mice. Spleen tissue of mice infected with Moloney virus had the highest titre in the mice with accelerated amyloid development.     

Tumour metastasis in mice with reduced immune reactivity. IV. Restoration of relative resistance to metastasis formation in thymectomized sub-lethally irradiated mice by spleen cells.

Adult thymectomy and/or sub-lethal irradiation increased spontaneous metastasis formation in several syngeneic murine systems (MCA-induced sarcomas MCG101 in C57BL/6J mice, MCG12 in CBA mice and MCA-induced epidermoid carcinoma EpCa1 in CBA mice). In order to differentiate between immunological and non-immunological effects of these treatments on tumour spread the effects of presumed selective restoration of immune competence by transfer of syngeneic spleen cells from normal donors shortly after irradiation (1.5 months before tumour transplantation) was perfomed. This procedure brought the resistance of thymectomized irradiated mice to tumour spread back to the level seen in untreated mice, providing further evidence for the conclusion that the indrease of tumour spread in this experimental model to a major part was immune-mediated. In thymectomized irradiated mice adoptive transfer of concomitant immunity by spleen cells from tumour-bearing mice soon after tumour transplantation completely inhibited the spread of the highly antigenic MCG101 and reduced that of the weakly antigenic MCG12 to control level. In contrast, adoptive transfer after tumour resection had no significant effect of the spread from either to the tumours.     

Regulation of interleukin-6 fetoplacental levels could be involved in the protective effect of low-molecular weight heparin treatment on murine spontaneous abortion

PROBLEM: CBA/J x DBA/2 abortion rate could be the consequence of a deficient local production of T helper (Th2) cytokines, which cause fetal wastage via fgl2 prothrombinase. Heparin reduces significantly the abortion rate in mice and recurrent spontaneous abortion (RSA) patients. We proposed to determine the effect of enoxaparin on the levels of local interleukin (IL)-6 during murine pregnancy. METHOD OF STUDY: Recombinant human IL-6 (rhIL-6) or enoxaparin were inoculated in CBA/J x DBA/2 pregnant mice on days 6.5-12.5. IL-6 levels in sera as well as in culture supernatants of day 9.5 fetoplacental units of CBA/J x BALB/c control mice or CBA/J x DBA/2 abortion combination were determined by enzyme-linked immunosorbent assay (ELISA) test. RESULTS: CBA/J x DBA/2 fetoplacental units secreted significantly lower levels of IL-6 with regard to CBA/J x BALB/c normal units. rhIL-6h and enoxaparin treatments decreased the resorption rate and regulated IL-6 fetoplacental levels. CONCLUSION: This study suggests that regulation of IL-6 fetoplacental levels could be involved in heparin-mediated anticoagulation protection against abortion.     

In vivo elimination of T cells expressing specific T-cell receptor V beta chains in mice susceptible to collagen-induced arthritis.

Type II collagen-induced arthritis (CIA) is believed to be dependent on T cells expressing a limited number of V beta chains. Two different methods were used to selectively eliminate T cells expressing a certain T-cell receptor (TcR) V beta chain in mouse strains susceptible to CIA. In vivo treatment with monoclonal anti-V beta 6 or anti-V beta 8.1,2 antibodies did not alter CIA, despite a reduction of the major part of the V beta 6+ or V beta 8.1,2+ lymph node cells (LNC), as measured by flow cytometric (FACS) analyses. The reduction was not due to complete elimination of V beta 6+ or V beta 8.1,2+ cells, since part of the V beta 6 and V beta 8.1,2 expressing cells returned later, even in mice that had been thymectomized first to prevent maturation of new T cells. In contrast, treatment with antibodies against CD4 efficiently abrogated development of CIA. In the (CBA x DBA/1J)F1 and the (BALB/c x DBA/1J)F1 mice, where M1s1a was combined with expression of I-E, the V beta 6+ LNC were deleted. In spite of the deletion, both F1 strains were highly susceptible to CIA.     

Pathogenesis of acute myocardial necrosis in inbred mice infected with coxsackievirus B3

The pathogenesis of myocardial necrosis due to CB3W infection was studied in BALB/c and C3H/HeJ mice. BALB/c mice infected with 5 x 10(4) pfu were found to die of massive hepatic coagulative necrosis before myocardial changes occurred. Reducing the inoculum size to 5 x 10(2) pfu resulted in sublethal hepatic involvement and multifocal myocardial coagulative necrosis by day 7 p.i. In contrast, C3H/HeJ mice survived infection and developed multifocal myocardial coagulative necrosis, but not liver disease following inoculation with as much as 5 x 10(6) pfu of CB3W. As with BALB/c mice infected with 5 x 10(2) pfu, myocardial lesions became apparent in C3H/HeJ mice a few days after peak cardiac virus titer was attained. Minimal inflammatory infiltrate was seen following development of cellular necrosis and was restricted to the areas of virus-induced pathologic change. However, no evidence was found for virus-specific cytotoxic T cell activity or for delayed type hypersensitivity responses. Furthermore, myocardial necrosis in CB3W-infected, T cell-depleted C3H/HeJ mice was as severe as in CB3W-infected, immunocompetent mice. These data have led us to conclude that cardiac lesions were due to virus-induced cytopathology rather than immunopathogenic mechanisms.     

B1 cells contribution to susceptibility in experimental paracoccidioidomycosis: immunoglobulin isotypes and repertoire determination.

Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America. The experimental murine model has been used to approach the disease, with susceptible and resistant mice strains that reproduce most of the main human immunological features. Since the hypergammaglobulinemia observed in susceptible mice and humans might have an influence on B1 cells, we investigated its role during the experimental infection with Paracoccidiodes brasiliensis. CBA/Nxid mice, deficient in B1 cells, and CBA/Nxid reconstituted with B1 cells isolated from the non-mutant CBA/J strain were infected with 106 yeast forms of P. brasiliensis. At the 8th and 22nd week post infection the DTH response of CBA/Nxid mice was significantly higher after 24 h of P. brasiliensis antigens inoculation and the specific humoral response was reduced, in comparison to CBA/J or recCBA/Nxid. Production of NAbs is a hallmark of the B1 subset. Higher Ig productions to auto antigens such as DNA, MBP and RBC were observed in CBA/J infected mice or recCBA/Nxid. Anti P. brasiliensis IgG2a was produced by CBA/Nxid mice early in infection, while CBA/J or recCBA/Nxid presented increased levels of this isotype only after the 8th week of infection. Furthermore, western blotting analysis showed that CBA/Nxid mice expanded less clones against P. brasiliensis antigens, with weakly detectable anti-gp43 antibodies while CBA/J mice produce IgM anti-gp43 at the 2nd week of infection and IgG anti-gp43 at the 2nd and 8th week. On the other hand, recognition of gp70, a fungal antigen that, as gp43, inhibits macrophage activation was not compromised in B1 deficient mice. These results suggest that B1 cells might have influence in the kinetic of production of protective isotypes of immunoglobulins and their repertoire that could contribute to an early drive towards a Th2 response, affecting the cellular response in susceptible mice during experimental paracoccidiodomycosis.     

Effects of anti-I-A and anti-I-E monoclonal antibodies on the induction and expression of experimental autoimmune thyroiditis in mice

Susceptibility to experimental autoimmune thyroiditis (EAT) in mice is linked to the I-A subregion of the major histocompatibility complex (MHC). The present study was undertaken to assess the effectiveness of anti-I-Ak monoclonal antibody (MAb) 10-2.16 in preventing or arresting the development of EAT. Spleen cells from CBA/J or (CBA/J x Balb/c) F1 mice given 10-2.16 prior to sensitization with mouse thyroglobulin (MTg) and adjuvant could not transfer EAT to normal recipients, and cells from these mice did not proliferate in vitro to MTg. Donor CBA/J mice given 10-2.16 before immunization and recipients of cells from such mice produced little MTg-specific IgG1 or IgG2b antibody but did produce nearly as much IgG2a as controls. The effects of in vivo treatment with 10-2.16 appear to be due to elimination of Ia + cells rather than to modulation of Ia or induction of suppressor T cells. When 10-2.16 was added to in vitro cultures it also prevented the proliferation and activation of sensitized CBA/J or F1 effector cell precursors. Other mAb specific for MHC class II gene products, but not associated with disease susceptibility, expressed by CBA/J (I-Ek) or F1 (I-Ad) mice (14-4-4S or MK-D6 respectively), also prevented in vivo sensitization, but did not block in vitro activation. Anti-I-Ak was also effective in preventing EAT if multiple injections of mAb were given to recipients of sensitized EAT effector cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemokine expression during development of fibrosis versus resolution in a murine model of granulomatous experimental autoimmune thyroiditis

Severe granulomatous experimental autoimmune thyroiditis (G-EAT) in DBA/1 or CBA/J wild type (WT) mice at day 19 progresses to fibrosis by day 35, but severe G-EAT in DBA/1 interferon (IFN)-gamma-/- mice or less-severe G-EAT at day 19 in WT mice resolves by day 35. To study the role of chemokines in autoimmune diseases and fibrosis, profiles of chemokines and chemokine receptors were analyzed in DBA/1 WT versus IFN-gamma-/- and CBA/J thyroids, which have distinct outcomes of autoimmune inflammation. Gene expression of CXC chemokine ligand 1 (CXCL1) and CXC chemokine receptor 2 (CXCR2) paralleled neutrophil infiltration and thyrocyte destruction in DBA/1 WT or CBA/J thyroids, and gene expression of CC chemokine ligand 11 (CCL11), CCL8, and CC chemokine receptor 3 paralleled eosinophil infiltration in IFN-gamma-/- thyroids. Gene and protein expression of CXCL10, CXCL9, and CXCR3 was significantly lower in IFN-gamma-/- compared with DBA/1 WT thyroids. Moreover, immunostaining showed that CXCL10 was expressed by thyrocytes and inflammatory cells, and strong expression of CXCL10 by thyrocytes was as early as day 7. High expression of CCL2 was only observed in severely destroyed DBA/1 WT or CBA/J thyroids, which would develop fibrosis. Thus, the differential expression of chemokines may direct distinct cellular populations in DBA/1 WT versus IFN-gamma-/- thyroids. Up-regulation of CXCL10 by thyrocytes suggests its role in regulating the recruitment of specific subsets of activated lymphocytes to the thyroid during autoimmune inflammation. The early expression of CXCL1, CXCL10, and CCL2 may suggest their involvement in the initiation and perpetuation of disease in severe G-EAT thyroids, which progress to fibrosis.     

Effects of monoclonal antibodies to adhesion molecules on eosinophilic myocarditis in Toxocara canis-infected CBA/J mice

Eosinophilic myocarditis followed by fibrosis of the cardiac muscle was observed in addition to peripheral blood eosinophilia in CBA/J mice infected with Toxocara canis. The infected mice were used as an experimental model of eosinophilic endomyocarditis associated with hypereosinophilic syndrome. Effects of in vivo treatment with MoAbs to adhesion molecules on eosinophilic myocarditis were examined using this experimental model. Expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells of capillaries in myocardium were increased 1 and 2 weeks after infection. Infiltration of very late antigen (VLA)-4⁺ and/or CD11a⁺ cells into the cardiac muscles was also observed 1 and 2 weeks after infection. Infiltration of eosinophils into the heart was significantly suppressed by anti-CD18 MoAb and anti-VLA-4 MoAb, and focal fibrosis of the cardiac muscle was also significantly suppressed by combined administration of anti-CD18 and anti-ICAM-1 MoAbs. These results indicate that adhesion molecules may play important roles in eosinophilic myocarditis, and that blockade of interaction between adhesion molecules and their ligands may help to control it.     

Intra-alveolar fibrosis of idiopathic bronchiolitis obliterans-organizing pneumonia. Cell-matrix patterns.

Idiopathic bronchiolitis obliterans-organizing pneumonia (BOOP) is characterized by air space fibrosis of unknown origin. Clinical resolution under steroid treatment suggests the removal of the fibrotic lesion. Open lung biopsies of four patients with idiopathic BOOP were studied by immunochemistry and electron microscopy. Three distinct cell-matrix patterns of intra-alveolar bud were found to represent the sequential evolution of the fibrotic process: fibrinoid inflammatory cell clusters in which immunoglobulins and procoagulant factors (fibrinogen, factors VII and X) were identified; fibroinflammatory buds in which desmin-containing fibroblasts were observed migrating, proliferating, and secreting matrix proteins; fibrotic buds in which myofibroblasts organized a loose connective matrix predominantly composed of fibronectin and type III collagen. Extending forms of fibrotic buds may join contiguous alveoli. Fibrotic bud remodeling ability is correlated to the nature and organization of the matrix components but the factors permitting intra-alveolar matrix degradation must be characterized.     

Vaccination with Toxoplasma gondii SAG-1 protein is protective against congenital toxoplasmosis in BALB/c mice but not in CBA/J mice

We evaluated the effect of vaccination with the SAG1 protein of Toxoplasma gondii against congenital toxoplasmosis in mice with different genetic backgrounds. In BALB/c mice (H-2(d)), vaccination reduced the number of infected fetuses by 50% and was associated with a mixed type 1 and type 2 immunity. In CBA/J mice (H-2(k)), vaccination increased the number of infected fetuses by 50% and was associated with a predominant type 2 response. Our results indicate that the effect of vaccination with SAG1 is controlled by the genetic background of the mouse.     

Strain-dependent protective effect of adult thymectomy on murine infection by Mycobacterium lepraemurium.

C57BL/6, DBA/2, BALB/c and CBA mice were thymectomized as adults, or sham-thymectomized, and infected subcutaneously with 10(6) MLM. The number of MLM in the spleen and in the inoculated footpad was measured after 1 year of infection as well as the DTH reactions and the IgM and IgG antibody levels to MLM. Non-thymectomized mice exhibited a broad spectrum of resistance to MLM infection and of T cell mediated immunity grading from the highly resistant C57BL/6 strain to the highly susceptible CBA strain. In between, DBA/2 was found more resistant than BALB/c mice. Adult thymectomy reduced by 100 times the MLM number in the spleen of infected DBA/2 mice, without affecting that measured in the inoculated footpad, and significantly decreased DTH reaction in the same strain. No effect of adult thymectomy was observed in any other strain, except for an increase of anti MLM antibodies in BALB/c mice. These results may suggest that the medium-resistant DBA/2 strain develops after MLM infection suppressor T cells which favour MLM dissemination and are sensitive to adult.     

Defective immunoglobulin M responses to vaccination or infection with Schistosoma mansoni in xid mice.

Mice vaccinated with irradiated Schistosoma mansoni cercariae develop a persistent immunoglobulin M (IgM) antischistosomulum antibody response. To investigate the possible role of antilarval IgM antibodies in the effector mechanism of vaccine-induced immunity, CBA/N mice, which have an X-linked genetic defect resulting in impaired IgM antibody responses to certain antigens, were analyzed for their resistance to a challenge infection. When either infected with unattenuated parasites or vaccinated with irradiated cercariae, mice of this inbred strain failed to produce detectable IgM antibodies to schistosomulum surface membrane and soluble worm antigens. To analyze the effect of this IgM deficiency on immunity, F1 hybrids were constructed between CBA/N females and nondefective C57BL/6J males. As expected, vaccinated (CBA/N X C57BL/6J)F1 females, as well as (CBA/J X C57BL/6J)F1 males and females, produced normal IgM antibodies to both surface antigens and worm antigen extracts. However, such antibodies were not produced by (CBA/N X C57BL/6J)F1 males (hemizygous for xid). Nevertheless, (CBA/N + C57BL/6J)F1 males displayed the same high levels of immunity to challenge infection as (CBA/N X C57BL/6J)F1 females and (CBA/J X C57BL/6J)F1 males and females. These results indicate that vaccine-induced immunity is not dependent on an IgM response to schistosome antigens.     

Quantitation of virus-specific classes of antibodies following immunization of mice with attenuated equine herpesvirus 1 and viral glycoprotein D

The antibody responses of CBA/J mice infected intranasally (i.n.) with either the attenuated KyA strain or the pathogenic RacL11 strain of equine herpesvirus 1 (EHV-1) or immunized with recombinant glycoprotein D (rgD) were investigated using the ELISPOT assay to measure EHV-1-specific antibody-secreting cells (ASC) in the regional lymphoid tissue of the respiratory tract. IgG, IgA, and IgM ASC specific for EHV-1 were detected in the mediastinal lymph nodes (MLN) and lungs 2 weeks after i.n. infection with EHV-1 strain KyA or RacL11, or immunization with heat-killed KyA or rgD. EHV-1-specific ASC were present in the MLN and lungs at 4 and 8 weeks, but declined in frequency by fivefold in the lung at 8 weeks. However, i.n. immunized (2 x 10(6) pfu KyA or 50 microgram rgD/mouse) mice infected at 8 weeks with pathogenic EHV-1 RacL11 resisted challenge and showed eight- and tenfold increases in MLN ASC and lung ASC, respectively, by 3 days after challenge. In contrast to the intranasal route of immunization, intraperitoneal immunization yielded ASC frequencies in the MLN and lungs that were only slightly above those of nonimmunized control mice. These data indicate that immunization with infectious or heat-killed EHV-1 KyA, or rgD, induces significant levels of virus-specific ASC both in the MLN and lungs, a specific memory B-cell response, and long-term protective immunity. The finding that the numbers of ASC induced by the pathogenic strain versus the attenuated strain of EHV-1, which were virtually identical, indicated that the ability to generate a B-cell response is independent of and does not contribute to EHV-1 virulence.