Julie A. Yager

Assessment of the immunogenic potential of Rhodococcus equi virulence associated protein (VapA) in mice.

The development of immunity to Rhodococcus equi, particularly to a virulence-associated protein (VapA) based antigen preparation, was examined in CD1 and BALB/c mice after intraperitoneal vaccination. Immunization with VapA based antigen without adjuvant markedly enhanced organ clearance in CD1 mice but not in BALB/c mice. Delayed type hypersensitivity response and antibody titres in VapA based antigen immunized BALB/c mice were less than in CD1 mice. By contrast also to CD1 mice, sera from immunized BALB/c mice did not react as strongly with VapA in western blots. Use of adjuvants (aluminium hydroxide, iscoms) interfered markedly with the immunogenic properties of the VapA based antigen, in the case of aluminium hydroxide by apparently driving a Th2 type of response. Unexpectedly, iscom adjuvants also impaired immunity and, despite the highest DTH response, produced a low IgG2a response, suggesting that iscomization of the antigen produced a low interferon gamma and high interleukin 2 response. Passive immunization of BALB/c mice with serum from mice immunized with live virulent strain 103+ resulted in only temporary and slight enhancement of organ clearance, supporting the central importance of cellular immunity to R. equi. Immunization with live virulence plasmid- and VapA-positive R. equi strain 103 resulted in marked liver clearance, in marked DTH response and high antibody titres. By contrast, immunization with live virulence plasmid- and VapA-negative strain 103 resulted in slight but variable enhancement of clearance, but insignificant DTH and antibody. The virulence plasmid, and by implication VapA, was thus shown to be critical in determining a highly effective protection to live organisms.

A Prospective Study of the Immunophenotype and Temporal Changes in the Histologic Lesions of Canine Demodicosis

Mural folliculitis is a consistent histologic lesion of canine demodicosis. The objective of this study was to describe the immunophenotype and to evaluate temporal changes in histologic lesions of demodicosis during the course of therapy. Five dogs with demodicosis were examined and biopsied biweekly for up to 14 weeks; three dogs were evaluated once only. Lymphocyte subsets infiltrating the lesions were quantified using immunohistochemistry to detect CD3, CD21, CD4, and CD8 antigens. Lymphocyte subsets in blood were analyzed from four dogs using flow cytometry. Mural folliculitis was always present during clinically active disease. In contrast, following resolution of clinical lesions, perifolliculitis and/or perifollicular granulomas were present but mural folliculitis was absent. Most lymphocytes infiltrating the follicular epithelium in lesions of mural folliculitis were CD3+ and CD8+; the ratio of CD4+ : CD8+ cells in this epithelium was 0.032. In contrast, the perifollicular dermis contained approximately equal numbers of CD4+ cells and CD8+ cells, with slightly fewer CD21+B cells. In peripheral blood, the ratio of CD4+ : CD8+ lymphocytes was reduced and the percentage of CD8+ cells was increased in three of four dogs. These results indicate that mural folliculitis is a consistent lesion of clinically active canine demodicosis and is characterized by infiltration of the follicular epithelium by CD3+ CD8+ T lymphocytes. These lymphocytes are cytotoxic T cells, which may mediate the injury to the follicular epithelium in demodicosis. Alternatively, CD8+ T cells may play a role in resistance to Demodex canis infection or may represent a deleterious immune response in dogs that develop demodicosis.

Bacillary angiomatosis in an immunosuppressed dog

A dog being treated with immunosuppressive doses of prednisone and azathioprine for pancytopenia of unknown origin, developed, over a 2-week period, multiple erythematous nodular lesions in the skin including footpads. Skin samples revealed lesions identical to those of human bacillary angiomatosis (BA). The nodules were composed of multifocal proliferations of capillaries, each lined by protuberant endothelial cells. The capillary clusters were separated by an oedematous connective tissue, lightly infiltrated with degenerate inflammatory cells, including neutrophils and macrophages. Tissue sections stained with Warthin-Starry silver stain revealed large numbers of positively stained bacilli in the stromal tissue, most heavily concentrated around the proliferating capillaries. Lesions of vascular degeneration and inflammation were evident. Bartonella vinsonii subsp. berkhoffii genotype 1 was independently amplified and sequenced from the blood and the skin tissue. The pathognomonic nature of the histological lesions, demonstration of compatible silver-stained bacilli in the tissue, and identification of B. vinsonii subsp. berkhoffii in the blood and tissue indicates that this is most likely the aetiologic agent responsible for the lesions. Antibiotic therapy was successful in resolving the nodules. It would appear that B. vinsonii subsp berkhoffii, like Bartonella henselae and Bartonella quintana, has the rare ability to induce angioproliferative lesions, most likely in association with immunosuppression. The demonstration of lesions identical to those of human BA in this dog is further evidence that the full range of clinical manifestations of human Bartonella infection occurs also in canines.

Immunophenotypic analysis of foal bronchoalveolar lavage lymphocytes

The purpose of this study was to define the normal immunophenotype of equine lymphocytes present within the pulmonary air spaces, and to determine if this changes as foals age from one to ten weeks. Six pairs of mares and foals underwent sequential bronchoalveolar lavage (BAL) between 1 and 10 weeks of age. Data were grouped according to foal age (1, 1-3, 3-6, or 6-10 weeks of age) and were compared to adult control values obtained from the mares. BAL cells were harvested and stained with antibodies to the equine homologues of CD5, CD4, CD8, CD44, MHC I, MHC II and to equine IgG. Data, including percent positive staining and mean fluorescence intensity, were acquired on a flow cytometer gated for viable lymphocytes. All foals had significantly fewer CD5+ lymphocytes than mares, with the largest differences in the youngest animals. The percentage of CD4+ lymphocytes increased as the foals aged, approaching adult levels by 3 weeks of age, while the percentage of CD8+ lymphocytes increased more slowly and approached adult levels by 10 weeks of age. The CD4:CD8 ratio changed from 1.26 at one week of age to 0.78 by 10 weeks of age, compared to an adult value of 0.66. Lymphocytes from foals less than 6 weeks of age expressed MHC II and CD44 at lower levels than adults. The lymphocytic populations within the airways of foals are significantly different from adult animals. This may account for the susceptibility of foals to certain respiratory infections during the first few months of life.

Abnormal sebaceous gland differentiation in 10 kittens ('sebaceous gland dysplasia') associated with generalized hypotrichosis and scaling

A rare congenital dermatosis, characterized by progressive hypotrichosis with variable scaling and crusting, occurred in 10 short-haired kittens in North America and Europe. Lesions appeared at between 4 and 12 weeks of age, commencing on the head and becoming generalized. The tail was spared in two kittens. Generalized scaling was mild to moderate, often with prominent follicular casts. Periocular, perioral, pinnal and ear canal crusting was occasionally severe. The skin was thick and wrinkled in two kittens. Histologically, the main lesion was abnormal sebaceous gland morphology. Instead of regular differentiation from basal cells to mature sebocytes, the glands were composed of a haphazard collection of undifferentiated basaloid cells, some partly vacuolated and a few containing eosinophilic globules. Mitotic figures and apoptotic cells were present in an irregularly thickened follicular isthmus. Lymphocytic mural folliculitis and mild sebaceous adenitis were rare. Orthokeratotic hyperkeratosis and follicular casts were present. Hair follicles were of normal density and were mostly in anagen, but some contained malacic hair shafts. Perforating folliculitis, leading to dermal trichogranuloma formation, occurred occasionally. Further biopsy samples taken at 2 years and at 3 and 4 years, respectively, from two kittens revealed similar but often more severe sebaceous gland lesions. Hair follicles were smaller, with many in telogen. The young age of onset suggests a genetic defect interfering with sebaceous and, possibly, follicular development. These lesions are discussed with reference to studies of mouse mutants in which genetic defects in sebaceous differentiation cause a similar phenotype of hyperkeratosis and progressive alopecia.

Demonstration of equine immunoglobulin in sera from severe combined immunodeficiency/beige mice inoculated with equine lymphocytes.

Peripheral blood lymphocytes were isolated from four normal adult horses on Ficoll-Hypaque density gradients for intraperitoneal inoculation into 16 C.B-17 severe combined immunodeficiency/beige mice in an attempt to create a xenogeneic lymphoid chimera for modeling the equine immune system. The recipient mice had been preconditioned by prior exposure to 200 cGy of gamma irradiation. The mice were monitored for the presence of equine IgG using an ELISA technique. Equine IgG was detected in the sera of 91.7% of murine recipients and in at least one mouse inoculated from each donor horse. The levels of IgG detected in the mice ranged from 2 micrograms ml-1 to over 1000 micrograms ml-1 and showed steady decline from the time of inoculation to the end of the trial. The half-life of equine IgG in the SCID/beige mouse was determined to be 12.1 days by the intravenous injection of 9.1 mg of semi-purified equine IgG into 21 normal SCID/beige mice and taking serial blood samples to obtain the decay curve. The half-life of equine IgG was compared with the extinction curve obtained for the engrafted mice and showed that continuous production within the mice must have been occurring since the slopes of the two lines were different. These results are compared with those achieved in equine PBL-SCID/beige chimeras not preconditioned by irradiation and to those achieved in human and bovine PBL-SCID chimeras.

Establishment of Demodex canis on canine skin engrafted onto scid-beige mice.

A small animal model of canine demodicosis is described. Normal canine skin was engrafted onto scid (severe combined immunodeficient)-beige mice, which lack functional B and T lymphocytes and have reduced natural killer cell activity. The xenografts were later infected with Demodex canis collected from a dog with demodicosis. At 30-112 days following infection, mites were seen histologically in the canine hair follicles of the engrafted skin. Demodex canis adults, nymphs, larvae, and eggs were present in samples macerated in sodium hydroxide. Mite infestations could not be demonstrated in the mouse skin, nor were mites passed from the infected graft to uninfected skin grafts on in-contact mice. This model may be utilized to assess the efficacy of miticidal treatments, to evaluate the importance of specific components of the immune response, and to study the biology of D. canis.