Herpesvirus connection in the expression of autoimmune vitiligo in Smyth line chickens.

The Smyth line (SL) chicken is an animal model for human vitiligo, a common acquired depigmentary disorder affecting about 1-2% of people worldwide. The vitiligo-like depigmentation in SL chickens typically develops when the birds are between 6 and 14 weeks of age and may affect 70-95% of hatch mates. The development of SL vitiligo is considered to depend on two interacting components, namely an inherent melanocyte defect and an autoimmune reaction to melanocytes. Recently, a role for an environmental factor in the expression of vitiligo was suggested by the observation that only 10% of SL chicks imported from the University of Massachusetts (UM) and reared in isolation at biosecurity level 2 (BSL 2) at the University of Arkansas (UA) exhibited vitiligo. Following further assessment of environmental differences between UA and UM SL chickens, three environmental factors that may have influenced the expression of SL vitiligo were identified. Included were housing condition, status of Mycoplasma synoviae infection, and turkey herpesvirus (HVT) vaccination status. Studies were subsequently conducted at UA and UM to assess the role of these environmental factors in the expression of SL vitiligo. M. synoviae infection was not found necessary for vitiligo expression in SL chickens. However, HVT emerged as a strong candidate for an important environmental factor in SL vitiligo. The connection between HVT and SL vitiligo was confirmed for both BSL 2 and conventional housing. Therefore, the observations reported here suggest a strong causative link between HVT infection and SL vitiligo.

Molecular characterization of the Smyth chicken sublines and their parental controls by RFLP and DNA fingerprint analysis.

The Smyth line (SL) chicken, a model for autoimmune human vitiligo, is characterized by a spontaneous posthatch epidermal pigment loss (vitiligo). Even though the immunological and morphological changes accompanying the vitiligo process have been well studied, the genetics of this phenomenon remains elusive. The SL lines have been maintained by nonpedigreed matings since their inception, and therefore, the inbreeding status is unknown. The present study was designed to provide an estimate of the inbreeding coefficients and the molecular genetic profiles of the SL sublines, each homozygous for a different MHC haplotype and their MHC-matched parental control (BL) sublines. The DNA fingerprint analysis revealed that there is a moderate level of inbreeding within the SL and BL parental sublines. Of the two SL sublines studied, SL101 had the highest level of inbreeding (0.948). Similarly, its parental control line (BL101) was more inbred than the parental subline of SL102 (BL102). The very high level of similarity between the SL sublines and their respective parental control lines is shown further by the similarity index (SI) estimates (SI between SL101 and BL101 was 0.949 and that between SL102 and BL102 was 0.932). Restriction fragment length polymorphism (RFLP) analysis of the endogenous viral genes (avian leukosis virus subgroup E, ALVE) showed that five ALVE-related BamH1 fragments were present in the SL101 and four in SL102 sublines, whereas the parental BL101 and BL102 sublines had five and six fragments, respectively. SL101 and SL102 shared two fragments, but the frequencies were different. Similarly, BL101 and BL102 shared two fragments. SL101 and BL101 shared three fragments, and SL102 and BL102 also shared three fragments.

A new recessive ametapodia mutation in the chicken (Gallus domesticus).

An apparently new mutation that is associated with abnormal limb development appeared in a strain of Light Brown Leghorn chickens. Mutants are characterized by the complete absence of the tarsometatarsals, while severely hypoplastic development of the metacarpals is also present. The phenotype of the new mutant (ametapodia-2) closely resembles ametapodia-1, described in 1967, but ametapodia-2 is inherited as an autosomal recessive (AMET*A), while ametapodia-1 was associated with an incompletely dominant gene (MP*A). Only heterozygous ametapodia-1 (MP*N/MP*A) were viable and able to reproduce, while homozygous ametapodia-2 mutants do not normally survive beyond 2-4 days of age. The shankless mutation (SHL*S) also reduces development of the metatarsal and metacarpal bones and has been shown to be associated with a pericentric inversion of chromosome 2. No obvious cytologic abnormality was apparent in ametapodia-2 birds, and offspring of a cross between AMET*A carriers and shankless birds were normal, indicating that the two mutations are not alleles. Ametapodia-1 (MP*A) was found to be linked to the rose comb locus (R) by 16 crossover units. Linkage test matings between AMET*A and (R*R) showed independent segregation, strongly suggesting that the mutation occurred at a relatively distant locus and therefore is probably not allelic to MP*A.

Analysis of the effect of endogenous viral genes in the Smyth line chicken model for autoimmune vitiligo.

The Smyth line (SL) chicken, an animal model for autoimmune human vitiligo, is characterized by a spontaneous posthatch pigment loss, determined to be the result of an autoimmune phenomenon. Because endogenous virus (EV) genes have been reported to be associated with a number of autoimmune diseases of human and animal models, we designed this experiment to investigate the role of EV in the SL vitiligo by using the complete sequence of Rous-associated virus-2 as a probe for EV. An F(2) resource population was developed by the matings of SL and parental control (BL) chickens. Linkage disequilibrium between vitiligo and EV was apparent (16.2-kb SacI fragment, P

Alopecia areata and universalis in the Smyth chicken model for spontaneous autoimmune vitiligo.

The Smyth line (SL) chicken model for spontaneous, postnatal expression of vitiligo may also show varying incidences and degrees of severity ranging from alopecia areata-like to universalis-like integumental changes. Although human vitiligo patients are known to have a four times greater chance of having alopecia areata than do people without vitiligo, in the SL model, feather loss is limited to birds that show some degree of amelanosis of feather and skin tissue. Both the vitiligo and the alopecia have an autoimmune component, as shown by histologic and immunologic studies, including the correctional influences of corticosterone and cyclosporine-A. The major histocompatibility haplotype (MHC) has a major effect on the incidence and expression of the vitiligo, as well as the alopecia that occurs within vitiliginous birds. Three different MHC haplotypes were identified in the original line that was selected for vitiligo, and from these, three sublines were developed, each homozygous for a different haplotype. Of the three sublines (SL101, SL102, and SL103) the vitiligo has a significantly earlier onset and severity in the SL101 than in the other two lines. The incidence of alopecia, however, is significantly lower in the SL101 subline than in the other two. Inheritance of the vitiligo is polygenic with an additional genetic component for the alopecia trait. It is hypothesized, but as yet unproven, that a feather development defect interacts with the SL melanization and immunologic defects to initiate the partial (areata) and complete (universalis) alopecias. The alopecia universalis is rarely seen until adulthood and is characterized by short (<0.5 cm), undeveloped feathers. If feather growth resumes in these birds, the feathers dry up, cease to grow, and often break off.

Development of a chicken Z-chromosome-specific DNA library.

We have developed a chicken (Gallus domesticus) Z-chromosome-specific DNA library in a phage vector by means of chromosome microisolation and microcloning. The chromosomal origin, specificity, and purity was evaluated by fluorescent in situ hybridization (FISH) on chicken metaphases. Heterologous chromosome painting using this Z-chromosome-specific probe on turkey (Meleagris gallopavo) metaphases identified its homologous Z-chromosome, under the same stringent conditions as that used in the chicken, indicating a high degree of Z-chromosome sequence homology among these two species. This chicken Z-chromosome library will facilitate the development of Z-chromosome-specific DNA markers that will be useful for genetic mapping in the domestic chicken and related avian species. The Z-chromosome-specific DNA probe will also be useful for studies pertaining to the sex chromosome evolution in avian species.

Profiles of pulp infiltrating lymphocytes at various times throughout feather regeneration in Smyth line chickens with vitiligo.

Smyth line (SL) chickens develop a spontaneous, autoimmune, posthatch loss of pigment cells (vitiligo) in regenerating feather tissue. Smyth line vitiligo (SLV) is associated with lymphocyte infiltrations prior to and throughout the development of the disorder. It was the purpose of this study to determine the type, relative amounts, and proportions of pulp-infiltrating lymphocytes at various times throughout the growth of regenerating feathers. Feathers were plucked from 8-week-old chickens with and without SLV. Feather pulp cell suspensions were prepared when the regenerating feathers were 2, 3, 4, and 6 weeks of age. Cells were fluorescently labeled using a panel of mouse monoclonal antibodies specific for chicken lymphocytes. Both T and B cells infiltrated the feather pulp of chickens with SLV. T cell levels remained elevated throughout the 6 weeks of feather growth, while B cell levels steadily declined to control levels over the same time. The pulp-infiltrating cells were primarily T cells with an alphabeta T cell receptor expressing the Vbeta1 gene (TCR2+). The ratio between CD4+ and CD8+ cells was 1.42 and 0.75 in 2- and 6-week-old regenerating feathers from chickens with autoimmune SLV, respectively. In non-vitiliginous chickens this ratio was always near 1. These data suggest that TCR2+ T cells play an important role in SLV. CD4+ cells may play a recruiting/activating role, whereas CD8+ cells may have cytotoxic activity specifically directed against melanocytes. Additionally, this is the first report demonstrating the infiltration of B cells into the feather pulp of vitiliginous chickens. These B cells may directly/indirectly contribute to melanocyte destruction in SLV.

Lymphocyte populations in dermal lymphoid aggregates of vitiliginous Smyth line and normally pigmented light brown Leghorn chickens.

Smyth line (SL) chickens develop a spontaneous, autoimmune loss of melanocytes (vitiligo) from the feather. To determine whether lymphocyte infiltration into two-week-old regenerating feathers of vitiliginous chickens was accompanied by lymphocyte infiltration into the skin, skin biopsies were conducted in SL chickens at two-week intervals throughout the development of vitiligo (SLV) when the chicks were 6 to 14 weeks of age. Control skin samples were obtained from age-matched, normally-pigmented Light Brown Leghorn (LBL) chickens. Frozen skin sections were labelled with a panel of mouse mAb to identify various lymphocyte populations. There was no lymphocyte infiltration into the skin of SL or control chickens. However, in both lines of chickens, dermal lymphoid aggregates (DLA), consisting primarily of T cells, were observed. In the DLA of SL chickens, the ratio between CD4+ and CD8+ T cells was lower (P = 0.005) and the percentage of gamma delta T cells higher (P = 0.027) than in the controls. Additionally, in SL chickens the lymphocyte populations in the DLA varied depending on the developmental stage of vitiligo. These data suggest a relationship between the DLA and autoimmune vitiligo in the SL model.

5-azacytidine treatment induces autoimmune vitiligo in parental control strains of the Smyth line chicken model for autoimmune vitiligo.

The effect of 5-Azacytidine (5-AzaC) on melanization was examined in two sublines of the Smyth line (SL) chicken model for autoimmune vitiligo, in two MHC-matched vitiligo-susceptible but normally pigmented controls (BL), and in nonsusceptible controls (LBL). 5-AzaC was administered ip every 3 days from day of hatch to 18 weeks at levels of 1 or 3 mg/kg body wt. Both treatments increased (P < 0.01) the incidence of autoimmune vitiligo in BL controls. In contrast, treatment significantly depressed (P < 0.01) the expected high incidence of vitiligo in one SL subline, but not in the other. There were no apparent pigmentation changes in 5-AzaC-treated LBL controls. 5-AzaC had dose-dependent depressing effects (P < 0.01) on body and lymphoid organ weights. Histological and mitogen assay data suggest negative effects on lymphocyte number and function. The data show that 5-AzaC can initiate autoimmune disease in genetically susceptible but phenotypically normal individuals.

Alterations in blood leukocyte populations in Smyth line chickens with autoimmune vitiligo.

Smyth line (SL) chickens spontaneously develop a posthatch autoimmune loss of pigment cells (vitiligo) in the feather. Concurrent with the development of Smyth line vitiligo (SLV), mononuclear cell infiltration and altered T cell profiles can be observed in the pulp of developing feathers. To determine whether the development of SLV is preceded by or associated with alterations in blood lymphocyte and leukocyte populations, blood leukocyte profiles were established at various times prior to and throughout the spontaneous development of SLV. The proportions among various blood leukocyte populations (percentage of lymphocytes, monocytes, heterophils, eosinophils, and basophils) were determined by immunofluorescence and flow cytometric analyses. Lymphocyte markers included fluorescence-conjugated monoclonal antibodies to identify T cells (CD3), T helper cells (CD4), cytotoxic T cells (CD8), and B cells (IgM). The proportions among blood lymphocyte populations examined did not differ between SL and MCH-matched parental Brown line (BL) control chickens prior to and throughout the development of SLV. Compared to BL controls, SL chickens had, however, increased proportions of inflammatory leukocytes in the blood, particularly at the time when most hatchmates developed SLV. Examination of leukocyte alterations with respect to first observation of SLV revealed that inflammatory leukocyte levels were elevated early in SLV. Although altered leukocyte profiles in the blood were observed during the development of SLV, blood from SL chickens did not reflect alterations in lymphocyte populations known to occur at the site of melanocyte destruction. The role of inflammatory blood leukocytes in the development of SLV needs to be further investigated.

T cells in regenerating feathers of Smyth line chickens with vitiligo.

Smyth line (SL) chickens exhibit a spontaneous, autoimmune, depigmentation (SL vitiligo, SLV) in regenerating feather tissue due to the death of pigment cells (melanocytes). It was the purpose of this study to characterize mononuclear leukocytes which infiltrate the feather as SLV develops. Frozen sections of regenerating features from SL and control chickens were labeled with a panel of mAbs specific for chicken mononuclear leukocytes. SL chickens were found to have significantly greater numbers of T cells in the feather pulp prior to, and throughout, the development of SLV. Initially, both SL and control chickens had a CD4/CD8 ratio near 1.0 in their feather pulp. Following the onset of SLV, the CD4/CD8 ratio in SL chickens dropped below 0.4. Throughout the development of SLV, SL chickens had greater proportions of T cells with alpha/beta T cell receptors than controls. Histological evidence strongly supports an important role for T cells in the pathology of SLV.

Immune response to sheep red blood cells in two Smyth line populations homozygous for different major histocompatibility complex haplotypes.

The Smyth line (SL) chicken is characterized by a high incidence of spontaneous, posthatch, selective destruction of melanocytes, caused by an autoimmune phenomenon. It has been shown that the MHC is associated with the development and severity of the disease. To clarify further the role of MHC haplotypes and other factors leading to an autoimmune response, mean antibody titers to SRBC were determined for 37-wk-old females from 2 SL sublines (SL101 and SL102), each homozygous for a different MHC haplotype, their MHC-matched parental control sublines (BL101 and BL102), and a normally pigmented control, LBL. Although total incidence of amelanosis is approximately the same for both SL sublines, amelanosis occurs earlier and is more severe in SL101 birds. Within sublines, chickens were further classified as to the extent of the feather amelanosis. Neither SL MHC subline had a mean SRBC titer that differed significantly from unrelated LBL controls. Although the secondary response of the two sublines differed from each other (P < .05), neither differed from its MHC-matched parental control; therefore, the differences in immune response appear to be largely MHC-related and not closely related to melanocyte destruction. When SRBC titers were related to amelanotic severity, no differences were found within the SL101 subline, although, SL102 birds that became amelanotic at a later age had a lower primary response to SRBC (P < .05) than the more severely affected group. Birds simultaneously producing both pigmented and amelanotic feather tissue had higher (P < .05) primary and secondary anti-SRBC titers than did the complete amelanotics.

The detection of melanocyte autoantibodies in the Smyth chicken model for vitiligo.

Smyth line (SL) chickens are phenotypically characterized by a posthatch depigmentation (vitiligo) of the feathers. The destruction of melanocytes in the feather follicle as well as in other tissues such as the choroid is genetically determined. Previous studies have shown that bursectomy or treatment with immunosuppressive agents decreases the incidence and severity of SL depigmentation (1). These observations implicate a role for the immune system, specifically the humoral component, in melanocyte destruction. In this study we show that there are circulating melanocyte-specific autoantibodies in the sera of depigmented SL chicks which are not present in sera from Light Brown Leghorn (LBL) control chicks. By immunoblots and by immunoprecipitation of radiolabeled melanocyte proteins, SL autoantibodies were shown to bind to multiple melanocyte proteins between 65 and 80 kDa. These proteins are not detected in SL fibroblasts. By immunoblotting, the incidence of autoantibodies for these 65- to 80-kDa proteins was determined to be 95% in depigmented SL chicks (n = 20), 0% in normally pigmented SL chicks (n = 8), and 5% in LBL chicks (n = 20). Melanocyte autoantibodies are detectable in the sera of affected chicks at or several weeks prior to the expression of depigmentation. This information, plus previously published data, implicate melanocyte autoantibodies in the depigmentary phenomenon of vitiligo observed in Smyth line chickens.

Induction of immunity to spermatozoa in male domestic fowl and effects on fertility.

Male fowl were immunized intravenously (i.v.) or intramuscularly (i.m.) with spermatozoa to assess the effects of immunity to spermatozoa on fertility. Histological and immunofluorescence evaluations of testis and ductus deferens tissues after 24 weeks of immunizations revealed immune cell infiltration and immunoglobulin associated with spermatozoa. The long-term immunization regimen resulted in significant antisperm antibody titres in the immunized groups. When semen from i.v.-immunized males was used to inseminate females, fertility over 7 days was reduced (P less than 0.05). A subsequent experiment using a 10-week i.v. immunization scheme also led to high antisperm titres. Spermatozoa from these males were characterized by lower fertility and duration of fertility than those of controls (P less than 0.05). As in mammals, a reduction in fertility may result from exposure of avian males to sperm antigens.

Albinism in White Leghorn chickens.

The appearance in 1988 of an oculocutaneous albino chick in a Single Comb White Leghorn line suggested a new mutational event. This line was closed in 1949, and has been reproduced each spring since then. Subsequent matings indicated that the mutation occurred at the C pigment locus. A mating of the Wisconsin albino (WIA) to cre/cre (red-eyed white) birds showed the mutation to be incompletely recessive to cre. No segregation was apparent when mated to ca/ca (recessive albinism) birds. These data indicate that the WIA mutation is identical to, or very similar to, the previously described tyrosinase-negative ca mutation at the C locus.

Autosomal albinism affects immunocompetence in the chicken.

Immune responsiveness of three neonatal and juvenile phenotypes, determined by the albinotic C locus, was evaluated. The phenotypes included normally pigmented (C+/-), recessive white (c/ca), and completely amelanotic albinos (ca/ca). No differences in: (1) primary agglutinin levels directed against sheep red blood cells (SRBC) or Brucella abortus (BA), or (2) cell-mediated immunity, as estimated by in vivo mitogen stimulation, were associated with the albino phenotype. The significant suppression of secondary SRBC or BA agglutinins observed in albino chicks was limited and of questionable biological significance. Acquisition of passively transferred maternal BA agglutinins was significantly impaired in albino progeny irrespective of dam genotype. No differences in agglutinin levels were associated with dam genotype. In addition, uptake of yolk sac contents was retarded significantly in albino chicks at hatch. These data suggest that an impaired ability to absorb maternal antibody and not the capacity to mount an active immune response contributes to neonatal mortality in albino chicks.

Effect of eumelanic phenotypes on the expression of amelanosis in the Smyth chicken.

The Smyth line is characterized by an autoimmune loss of melanin in the feather and eye in association with a hypermelanizing melanocyte, which presumably triggers immune system intervention. Inheritance appears to be multigenic. The present study was designed to determine if eumelanin-enhancing modifiers influence the incidence and severity of the line-associated amelanosis. Smyth chicks with dark brown (eb) down had a higher incidence of amelanosis (P less than .01) than did their hatchmates with light brown down. Furthermore, parents with dark down at hatch produced a higher incidence of amelanotic progeny than parents with light down. Reciprocal crosses of the Smyth line to a highly eumelanized (eb/eb) Recessive Black (RB) line produced F1 amelanosis. However, sires from the Smyth line produced significantly more amelanotics than did RB males (P less than .01). The influence of dark down on amelanotic development was also apparent in the Smyth-RBF1. The use of amelanotic F1 parents produced a significantly higher incidence of affected F2 offspring than did the use of unaffected parents. A backcross to the Smyth line produced an incidence of 67.6% amelanosis, whereas only one chick (2.04%) developed amelanosis from an F1 x RB mating. The finding that dark-downed Smyth chicks exhibit, and subsequently produce, a significantly higher incidence of amelanosis supports ultrastructural observations that associate the Smyth line amelanosis with a hyperactive melanocyte. The unusually high expression of amelanosis (22.7%) in the Smyth-RB F1 suggests that the two lines share one or more common eumelanogenic genetic factors.

Ocular pathology in the minimally depigmented subline of the vitiliginous Smyth chicken.

Choroidal melanocytes and the retinal pigmented epithelium (RPE) were studied morphologically and histochemically in the Smyth chicken, an avian model for human vitiligo. The sequence of cytological events occurring in the ocular tissue of minimally depigmented Smyth birds was determined. Abnormalities of melanocytes and the associated inflammation was least severe in peripheral areas of the choroid and most pronounced in the back of the eye at the base of the optic nerve head. In the peripheral choroid, morphologically normal melanocytes and an occasional mononuclear leukocyte were observed. However, some of these morphologically normal melanocytes histochemically demonstrated atypical tyrosinase activity at the trans area of the Golgi apparatus. Toward the back of the eye, the melanocytes first appeared swollen and had retracting dendrites. Ultrastructurally these melanocytes demonstrated an increase in extramelanosomal cytoplasm. Later, melanocytes became spherical and had membrane bound, autophagosome-like compartments of pigment granules. As the melanocyte injury progressed, macrophages invaded the tissue and phagocytized melanocytic dendrites. These were followed by numerous plasma cells. Eventually, the back of the eye contained no pigment and was infiltrated with numerous mononuclear inflammatory cells. The retinal pigment epithelium also demonstrated a gradient in the degree of destruction, related to its topography. These cytological features consisted of the retraction of apical RPE processes, the disappearance of the basal plasma membrane infoldings, and the replacement of Bruch's membrane by collagen-like fibrils. These results demonstrate that the uveitis which develops in vitiligo appears to be a consequence of an inherent choroidal melanocyte defect.

Enhanced integumental and ocular amelanosis following the termination of cyclosporine administration.

The Smyth delayed amelanotic line of chickens display symptoms commonly associated with human vitiligo. Administration of the immunosuppressive compound, cyclosporine, significantly delayed the mean age of onset and incidence of integumental pigment losses in this mutant line of vitiliginous chickens. Associated ocular pathology was also less severe in treated chicks. Termination of cyclosporine administration resulted in enhanced integumental and choroidal amelanosis, choroidal inflammation, and chorioretinal damage beyond that observed in nontreated controls. These results suggest that withdrawal of cyclosporine in treatment of this spontaneous autoimmune disease may exacerbate associated symptoms.

Effect of corticosterone on the incidence of amelanosis in Smyth delayed amelanotic line chickens.

The Smyth Delayed Amelanotic (SDA) line chicken exhibits a postnatal loss of pigment in feathers and choroid presumably due to an autoimmune reaction triggered by a basic pigment cell defect. The present study was designed to determine if the incidence and severity of amelanosis would be affected by administration of corticosterone (CS) in feed. The SDA line chickens were given 0, 20, 30, 40, and 50 ppm CS from 4 to 12 weeks of age. Body weight and incidence of amelanosis were determined weekly and immune response to sheep red blood cells was determined at the end of the trials. Body weight and immune response levels were significantly lower in treated groups than in controls (P less than .001), but no significant differences were found between treated groups on measures of amelanosis or immune response. Incidence of amelanosis in controls (60%) was significantly greater than in treated groups: 20 ppm, 14.2%; 30 ppm, 17.9%; 40 ppm, 12.9%; and 50 ppm, 16.7%. Six weeks posttreatment, treated groups were no longer significantly different from controls in terms of the incidence of amelanosis. It is proposed that the reduction in amelanosis during CS treatment is attributable to suppression of antibody response to the pigment cell antigen by the CS.