Canine subcutaneous mast cell tumors: cellular proliferation and KIT expression as prognostic indices.

Molecular assays are widely used to prognosticate canine cutaneous mast cell tumors (MCT). There is limited information about these prognostic assays used on MCT that arise in the subcutis. The aims of this study were to evaluate the utility of KIT immunohistochemical labeling pattern, c-KIT mutational status (presence of internal tandem duplications in exon 11), and proliferation markers--including mitotic index, Ki67, and argyrophilic nucleolar organizing regions (AgNOR)--as independent prognostic markers for local recurrence and/or metastasis in canine subcutaneous MCT. A case-control design was used to analyze 60 subcutaneous MCT from 60 dogs, consisting of 24 dogs with subsequent local recurrence and 12 dogs with metastasis, as compared to dogs matched by breed, age, and sex with subcutaneous MCT that did not experience these events. Mitotic index, Ki67, the combination of Ki67 and AgNOR, and KIT cellular localization pattern were significantly associated with local recurrence and metastasis, thereby demonstrating their prognostic value for subcutaneous MCT. No internal tandem duplication mutations were detected in exon 11 of c-KIT in any tumors. Because c-KIT mutations have been demonstrated in only 20 to 30% of cutaneous MCT and primarily in tumors of higher grade, the number of subcutaneous MCT analyzed in this study may be insufficient to draw conclusions on the role c-KIT mutations in these tumors.

Canine subcutaneous mast cell tumor: characterization and prognostic indices.

Histologic grading schemes for canine cutaneous mast cell tumors (MCTs) were not developed for subcutaneous MCTs. Despite this, subcutaneous MCTs are currently categorized by many as grade II or higher. The aim of this investigation was to assess the pathology and clinical outcome for subcutaneous MCTs to provide a more accurate prognosis. Information on clinical outcome for 306 dogs was obtained from veterinarians and correlated with histologic features. Mean and median follow-up was 842 and 891 days, respectively (range, 3-2,305 days). Only 27 (9%) were confirmed as mast cell-related deaths. Metastasis occurred in 13 (4%), and 24 (8%) had local reoccurrence, even though 171 (56%) cases had incomplete surgical margins. Median survival time was not reached, and the estimated 6-month, 1-, 2-, and 5-year survival probabilities were 95%, 93%, 92%, and 86%, respectively. Dogs were euthanized or died as a result of local tumor reoccurrence, additional MCT development distant to the surgical site, or metastasis. Decreased survival time was linked to mitotic index (number of mitotic figures per 10 high-power fields), infiltrative growth pattern, and presence of multinucleation. Both univariable and multivariable analysis showed mitotic index to be strongly predictive of survival, local reoccurrence, and metastasis. The results of the study indicate that the majority of subcutaneous MCTs have a favorable prognosis, with extended survival times and low rates of reoccurrence and metastasis.

Nasal dermoid sinus in an American cocker spaniel.

An American cocker spaniel was presented for a subcutaneous mass and draining tract located between its eyes. Contrast radiography and surgical excision showed communication of the tract with the left frontal sinus and rostral cerebral dura, respectively. A dermoid sinus was diagnosed by a combination of gross and histologic findings.

Primer for non-immunologists on immune-deficient mice and their applications in research.

Studies of immune deficiencies have a history as long as that of immunology. However, reports of two key spontaneous recessive mutations in mice (nude in 1966-1968 and scid in 1983) laid the foundations for widespread application of immune-deficient rodents to a broad range of research topics. More recently, technologies modifying the mouse genome by transgenesis, gene ablation and crossbreeding for lines with multiple immune deficits have provided a large number of new types of immunologically impaired mice. The primary goals of this overview are to help non-immunologists understand key differences between some of the immunodeficient strains, develop an appreciation for the value of information derived from immunodeficient mouse-based research and to encourage expanded, creative use of these specialized research animals. Secondary goals are to promote greater awareness of unexpected outcomes that can arise when working with genetically immune-deficient mice, the need for vigilance in maintaining these research animals, and the care required in interpretation of the data that immune-deficient modeling provides. Two illustrations on developing appropriate immune deficient animal models for a new research application conclude the review.

Role of the 85-kilobase plasmid and plasmid-encoded virulence-associated protein A in intracellular survival and virulence of Rhodococcus equi.

Rhodococcus equi is a facultative intracellular pathogen of macrophages and a cause of pneumonia in young horses (foals) and immunocompromised people. Isolates of R. equi from pneumonic foals typically contain large, 85- or 90-kb plasmids encoding a highly immunogenic virulence-associated protein (VapA). The objective of this study was to determine the role of the 85-kb plasmid and VapA in the intracellular survival and virulence of R. equi. Clinical isolates containing the plasmid and expressing VapA efficiently replicated within mouse macrophages in vitro, while plasmid-cured derivatives of these organisms did not multiply intracellularly. An isolate harboring the large plasmid also replicated in the tissues of experimentally infected mice, whereas its plasmid-cured derivative was rapidly cleared. All foals experimentally infected with a plasmid-containing clinical isolate developed severe bronchopneumonia, whereas the foals infected with its plasmid-cured derivative remained asymptomatic and free of visible lung lesions. By day 14 postinfection, lung bacterial burdens had increased considerably in foals challenged with the plasmid-containing clinical isolate. In contrast, bacteria could no longer be cultured from the lungs of foals challenged with the isogenic plasmid-cured derivative. A recombinant, plasmid-cured derivative expressing wild-type levels of VapA failed to replicate in macrophages and remained avirulent for both mice and foals. These results show that the 85-kb plasmid of R. equi is essential for intracellular replication within macrophages and for development of disease in the native host, the foal. However, expression of VapA alone is not sufficient to restore the virulence phenotype.

Close relationship between equine and human molluscum contagiosum virus demonstrated by in situ hybridisation.

To determine whether the virus responsible for human molluscum contagiosum (MCV) is the causal agent of a similar disease in horses, in situ hybridisations using cloned fragments of human MCV DNA labelled with digoxigenin were carried out on formalin-fixed biopsy sections of lesions from two horses with molluscum contagiosum-like skin lesions. In both instances there was evidence of specific hybridisation of the labelled probe to target DNA in the sections under high stringency conditions, identified by the development of a deep blue-purple stain in the cytoplasm of cells in the stratum spinosum and stratum granulosum of the lesions and the absence of non-specific hybridisation in adjacent non-lesional areas of the epidermis. These results indicate that on the basis of very close homology of their viral DNA sequences, the causative virus of equine molluscum contagiosum is either identical with, or very closely related to, its human equivalent.

Trichophyton dermatophytosis--a disease easily confused with pemphigus erythematosus.

Trichophytosis is a rare diagnosis in dogs in Ontario, but recently 4 dogs with a scaling and crusting, alopecic, facial dermatitis have been so diagnosed. In all cases, the histopathological findings of severe epidermal and follicular interface dermatitis, accompanied by an acantholytic intraepidermal pustular dermatitis suggested an immune-mediated disease.

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.

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.

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.

Use of Rhodococcus equi virulence-associated protein for immunization of foals against R equi pneumonia.

OBJECTIVE: To evaluate use of the virulence-associated protein of Rhodococcus equi in immunizing foals against R equi pneumonia. ANIMALS: Eight (experimental group) and 6 (controls) mares with their foals. PROCEDURE: Virulence-associated protein extracted from R equi was used to prepare an acetone-precipitated. Triton X-extracted (APTX) antigen. After determination of the efficacy of passive immunization, in untreated foals or in foals given plasma from a horse vaccinated with APTX antigen or from a nonvaccinated horse, a field trial was done to evaluate the efficacy of vaccination of 8 mares, twice with APTX before parturition, and of their foals at ages 3 and 5 weeks; 6 mares and their foals served as unvaccinated controls. All 2-day-old foals were given plasma from local donor horses inoculated with a locally produced bacterin. Serum opsonizing activity produced by vaccination with APTX was determined. Passively immunized foals were challenge exposed with an aerosol of virulent R equi. Foals of the field trial were exposed to enzootic R equi infection. RESULTS: Inoculation with APTX resulted in high IgG antibody liters with opsonizing activity. Passive immunization of foals with plasma from an immunized horse enhanced bacterial clearance from the lungs, compared with that in foals not given plasma or given plasma without APTX antibodies. Vaccination of mares and foals exposed to natural infection resulted in development of R equi pneumonia in 4 of 8 vaccinated foals, but in only 1 of 6 unvaccinated foals. CONCLUSIONS: Vaccination with APTX antigen led to high-titer, opsonizing antibody. Plasma from a vaccinated horse appeared to enhance clearance of R equi from the lungs of foals. Paradoxically, vaccination of mares and their foals with APTX antigen did not protect foals and may have enhanced R equi pneumonia in the foals.

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.

Streptococcal toxic shock syndrome in dogs.

OBJECTIVE: To determine the clinical, pathologic, and bacteriologic findings in dogs that developed severe invasive infections with group G streptococci (GGS) over a 6-month period in southern Ontario. DESIGN: Prospective case series. ANIMALS: 7 dogs n southern Ontario with severe streptococcal infection during a 6-month period. PROCEDURE: Using pulsed-field gel electrophoresis, molecular typing of streptococcal isolates was performed. Isolates were examined for the M protein gene emm1.0, pyrogenic exotoxin genes speA, speB, speF, hyaluronic acid synthase genes hasA, hasB, and for C5a peptidase gene scpA by use of DNA probes or polymerase chain reaction. RESULTS: 3 dogs with streptococcal shock without necrotizing fasciitis died or were euthanatized within 48 hours of admission, whereas 4 dogs with streptococcal shock and necrotizing fasciitis survived following surgical debridement, supportive medical treatment, and treatment with antibiotics. Of the 6 Lancefield group G streptococcal isolates available for characterization, 5 were Streptococcus canis and 1 had characteristics of group G streptococcal strains of human origin. Results of molecular typing indicated that isolates were unrelated to each other. Examination of the canine isolates for putative virulence genes found in human group A streptococci resulted in identification of the emm1.0 gene only in 1 of the isolates. The canine isolates otherwise lacked virulence genes associated with human group A streptococcal toxic shock infections. CLINICAL-IMPLICATIONS: The development of severe invasive infection in dogs resulting from GGS indicates that a virulent form of GGS has developed in southern Ontario.

Use of a virulence-associated protein based enzyme-linked immunosorbent assay for Rhodococcus equi serology in horses.

An enzyme-linked immunosorbent assay (ELISA) was developed against Rhodococcus equi using Triton X-114 detergent extracted whole cell material, in which the virulence associated protein (VapA) predominated. Enzymelinked immunosorbent assay titres corresponded to antibody reacting with VapA on Western blots. There was considerable variation in antibody titres of nonimmunised mares and in the time when the colostrally derived antibody of their foals had declined to low or undetectable titres. In general, antibodies in foals declined to their lowest levels at age 4-8 weeks. Seroconversion occurred in foals age 8-10 weeks, but the precise time depended on maternal titre and the month in which the foal was born. Foals reaching age 8 weeks in late summer showed more marked seroconversion than foals born earlier. The ELISA was used to follow the response to immunisation with the same Triton X-114 extracted material. Six mares immunised before parturition with the antigen in aluminium hydroxide adjuvant developed high titres, up to > 102,400 and transferred them to their foals through colostrum. Their foals responded to immunisation with 0.5-1.0 mg antigen 3, 5, 7 and 9 weeks after birth. Antibody titres following immunisation with similar dosage reached up to > 102,400 in a separate group of foals of nonimmunised mares. Nonvaccinated control foals seroconverted at age 6-8 weeks. The VapA based ELISA is useful to follow the course of natural infection with R. equi or immunisation with VapA based antigen.

Role of CD4+, CD8+ and double negative T-cells in the protection of SCID/beige mice against respiratory challenge with Rhodococcus equi.

To evaluate the contributions of T-lymphocyte subsets in pulmonary immunity against Rhodococcus equi, C.B-17 SCID/beige mice were adoptively transferred with splenic lymphocytes from congenic BALB/c mice previously infected with R. equi. Spleen cells were enriched for either CD4+ or CD8+ populations before inoculation, Flow cytometry showed that each enriched population contained less than 0.5% cross contamination. Groups of adoptively transferred SCID/beige mice were sacrificed 6 and 13 d after intranasal infection with R. equi. Bacterial clearance was measured in the lungs, liver and spleen. Lesion development was assessed by gross and histopathological score and the fate of transferred cells assessed by flow cytometry and by immunohistochemistry. SCID/beige mice receiving either CD4+ or CD8+ T-cells were able to clear the infection better than control mice. On d 6 post-infection, bacterial numbers were significantly lower in the lungs of CD4+ transferred mice as compared to CD8+ mice. By d 13, both groups had cleared R. equi from all organs. CD4+ cells were however identified in the lung and spleen of CD8+ recipients at d 13 making conclusions about the role of CD8+ cells in R. equi clearance impossible. By contrast, no significant increases in CD8+ lymphocytes were observed in the organs of CD4+ recipients. All mice developed suppurative bronchopneumonia but lesions were most severe in the CD4+ group. Immunohistochemistry and flow cytometry confirmed that CD4+ and CD8+ cells had migrated to the lungs of adoptively transferred mice. Serum antibody against R, equi was not detected by ELISA in the recipients. SCID/beige mice receiving CD4-CD8- cells were unable to clear R. equi. The study supports the suggestion that CD4+ cells have a central role in R. equi clearance in mice.

Familial cutaneous vasculopathy of German shepherds: clinical, genetic and preliminary pathological and immunological studies.

A genodermatosis affecting the German shepherd breed has been recognized in 26 dogs in Ontario since 1991. Clinical signs, first noted in young puppies,are manifested as pyrexia and lethargy. The main cutaneous lesions are footpad swelling and depigmentation,but there is also crusting and ulceration of ear tips and tail tips, and focal depigmentation of the nasal planum. Affected puppies show no consistent abnormalities in hematological or biochemical parameters, and immunological tests (antinuclear antibody and rheumatoid factor titer,immunoglobulin levels, and CD4+ and CD8+T-lymphocyte percentages) are normal. Bone marrow analysis has shown myeloid hyperplasia in 5 of 7 cases and myelofibrosis has been detected in 1 case. All but 3 of the 19 clinical cases have been strongly positive for platelet factor-3; however, normal puppies routinely develop positive platelet factor-3 tests. Furthermore, affected pups all had normal numbers of platelets on repeat complete blood counts.Light microscopic examination of footpad biopsies reveals a multifocal nodular dermatitis in which neutrophils and mononuclear inflammatory cells surround foci of dermal collagenolysis, and degenerative and inflammatory vessel lesions. Depigmented lesions have a mild, cell-poor, interface dermatitis,characterized by single cell necrosis of the basal cells, in addition to the nodular dermatitis. Similarities and differences between this disease,a condition known as collagen disorder of the footpads of German shepherds and other forms of cutaneous vasculitis in the dog are discussed. The cause and the pathogenesis of the disease are yet to be elucidated;however, pedigree analysis indicates an autosomal recessive inheritance pattern. Hypersensitivity reactions, directed against normal or damaged self-collagen, may be involved. The role of cell-mediated immunity against native or altered collagen is an area worthy of further investigation.

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.

Sensitivity and specificity of bronchoalveolar lavage and protected catheter brush methods for isolating bacteria from foals with experimentally induced pneumonia caused by Klebsiella pneumoniae.

One indication for referral of horses to veterinary hospitals is for diagnosis of the microbiologic cause of pneumonia, particularly when the initial treatment fails. Although endoscopic methods have long been available for microbiologic sample collection, accuracy of these methods under these conditions have not been studied in detail. We compared the bacteria isolated from samples obtained by bronchoalveolar lavage (BAL) with those obtained by protected catheter brush (PCB) from foals with unilateral pneumonia induced by inoculation with Klebsiella pneumoniae. As part of previously described clinical trials, foals were administered antimicrobial therapy IM (n = 15) or vehicle IM (n = 7), and collection of distal airway secretion samples was conducted during the treatment period. Sensitivity and specificity of the sample collection methods were assessed by comparison of the isolates from BAL or PCB samples with isolates from tissue of the inoculated lung lobe, which was the most severely affected lung region. Sensitivity and specificity of BAL for recovery of K pneumoniae (challenge strain) and Streptococcus zooepidemicus (common secondary pathogen) was 90 and 69%, respectively, compared with 76 and 85%, respectively, for the PCB method. Sensitivity was significantly (P = 0.03) higher for BAL (100%) than for PCB (69%) for recovery of K pneumoniae (P = 0.03) from lungs. However, difference in the sensitivity of these methods for recovery of S zooepidemicus was not significant. In conclusion, BAL was a more reliable method for recovery of bacteria from the lungs in chronically infected foals that received antimicrobial treatment.