key: cord-0722695-6jczwxo6
authors: Loa, Chien Chang; Lin, Tsang Long; Wu, Ching Ching; Bryan, Thomas; Hooper, Tom; Schrader, Donna
title: The effect of immunosuppression on protective immunity of turkey poults against infection with turkey coronavirus
date: 2001-11-06
journal: Comp Immunol Microbiol Infect Dis
DOI: 10.1016/s0147-9571(01)00033-9
sha: 8a541d5535412574cecb9a7bf5489e569896d15c
doc_id: 722695
cord_uid: 6jczwxo6

The objective of the present study was to evaluate the protective effect of humoral and cellular immunities on turkeys infected with turkey coronavirus (TCV). Two trials were conducted with two separate hatches of turkey poults. Turkeys were experimentally immunosuppressed with cyclosporin A (CsA) or cyclophosphamide (CY) and infected with TCV. Prior to infection, treatment with CsA selectively suppressed T cell activity as revealed by 2–3 fold decreased (p<0.1) lymphocyte proliferation responses to a T cell mitogen, concanavalin A (Con A). Treatment with CY mainly induced B cell deficiency as indicated by significant reductions (p<0.05) in antibody responses to sheep erythrocytes 7 days after injection. Body weight gain of turkeys treated with CY was significantly lower (p<0.05) than that of untreated turkeys at 9 days post-infection (PI). Turkeys treated with CY had 1–2 fold higher immunofluorescent antibody assay (IFA) scores for TCV antigens (p<0.05) in the intestine than untreated turkeys at 9 or 14 days PI. These results suggested that humoral immunity against TCV infection may be important in turkeys.

Turkey coronaviral enteritis is an acute and highly infectious disease, which has contributed to signi®cant economic losses in the US turkey industry, particularly in Indiana and North Carolina, for the last decade [1, 2] . The clinical signs (diarrhea, decreased body weight gain, inappetence, and ruf¯ed feathers) of the disease usually appear in turkey poults at 7±28 days of age. The morbidity is usually high and the mortality varies. In an epidemiological study, turkey coronavirus (TCV) was identi®ed in 63% of poult enteritis mortality syndrome (PEMS)-affected¯ocks in North Carolina (Guy and Barnes, unpublished data). The coronavirus isolated from affected turkey¯ocks was found to be antigenically cross-reactive with antibodies speci®c to infectious bronchitis virus (IBV), a chicken coronavirus [3] .

Once coronaviral enteritis is introduced into areas with high concentrations of turkeys, it is not easily eliminated and is encountered frequently in turkey¯ocks [4, 5] . Treatments of the disease are often unsuccessful and there are currently no effective vaccines or medications to prevent viral infection. It is very likely that the disease will continue to negatively impact the turkey industry until effective strategies for prevention and control of the disease are established.

Infections of host animals with other coronaviruses, bovine coronavirus (BCV) [6] , transmissible gastroenteritis virus (TGEV) [7] , or IBV [8] , stimulate protection of animals against subsequent challenge with the same virus. Although the detailed mechanisms for such protection are not clear, the protective immunity induced by the corresponding coronavirus during the primary infection is essential. Both serum IgG and fecal IgA antibody responses increased following secondary exposure with BCV. The virus-neutralizing antibody responses in serum were also increased following secondary challenge with BCV [6, 9] . The presence of high numbers of antibody-secreting cells and high lymphocyte proliferation activity was correlated with protection against subsequent challenge of TGEV [7] . Antibody responses to IBV were detected in serum, tracheal washes, and tears of chicken infected with IBV. Vaccination studies on IBV generally correlated neutralizing serum antibody responses to protection [10] .

Turkey¯ocks recovered from coronaviral enteritis (bluecomb disease) may develop life-long immunity against challenge infection [11] . However, the nature of such resistance is not completely understood. Serum from recovered turkeys could neutralize the infectivity of TCV-containing intestinal contents [11, 12] . Serum antibodies to TCV were detectable by immuno¯uorescent antibody assays (IFA) from 9 to 160 days post-infection (PI) [13] . Antibodies to TCV in the intestine and bile were detectable by immunodiffusion test [14] or a sandwich IFA [15] up to 6 months PI. The lymphocyte proliferation responses to mitogen was reported to be enhanced in turkeys infected with TCV [16] . In addition, sequential kinetic studies of humoral and cellular immune responses of turkey poults infected with TCV isolated in our laboratory have shown that activation of speci®c immune responses (humoral and cellular) are associated with the decrease of TCV antigens in turkey intestines (unpublished data). However, which immunity, humoral or cellular, provides the necessary protective immunity for turkey poults against infection with TCV is not clear. Such information is critical in developing strategies for the diagnosis, control, and prevention of turkey coronaviral infection in turkey¯ocks.

The purpose of the present study was to determine the protective immunity against turkey coronaviral infection in turkeys infected with TCV. Turkey poults were treated with selective T cell immunosuppressant (cyclosporin A, CsA) or B cell immunosuppressant (cyclophosphamide, CY) and subsequently challenged with TCV. The severity of infection was evaluated and compared.

One-day-old turkey poults (British United Turkey of American, BUTA) of both sexes were obtained from Perdue Farm (Washington, IN). They were free of recognized pathogens for turkeys, including TCV. They were housed in Horsfall±Bauer isolation units under negative-pressure ventilation. Food and water were provided ad libitum.

TCV was isolated from intestines of turkey poults with outbreaks of acute enteritis. Affected intestines were homogenized with 5-fold volume of phosphate-buffered saline (PBS) solution, clari®ed by centrifugation at 3000 £ g for 10 min, and ®ltered through 0.45 and 0.22 mm membrane ®lters (Millipore, Bedford, MA 01730). Twenty-two day-old embryonated turkey eggs were inoculated with 200 ml of the ®ltrate via amniotic sac route. Embryo intestines were harvested in 3 days. Intestines of serially propagated TCV-infected turkey embryos were homogenized with 5-fold volume of PBS buffer. The mixture was centrifuged at 3000 £ g for 20 min. The supernatant was used as the inoculum for experimental infection of turkey poults. The inoculum further examined by electron microscopy (EM) and immuno-EM contained TCV but not other pathogens.

Two trials with two separate hatches of turkey poults were conducted. The experimental design of trials 1 and 2 is outlined in Table 1 . In trial 1, 110 turkey poults were randomly separated into four groups. Thirty turkey poults in group I were treated with CsA at 7 days old and every third day thereafter. Thirty turkey poults in group II were treated with CY at 1, 2, and 3 days old. Thirty turkey poults in group III were not treated with any immunosuppressant. At 10 days old, ®ve turkey poults from each of these groups were used for evaluation of humoral antibody responses to sheep red blood cells (SRBC). Another ®ve turkey poults from each of these groups were used for evaluation of cellular immunocompetence by in vitro lymphocyte proliferation assay. The remaining turkey poults in each of the above groups were infected with TCV at 10 days old. Twenty turkey poults in group IV were neither treated with immunosuppressant nor infected with TCV and served as normal control group. Five poults from each of these four groups were weighted at 10 days of age (prior to infection). The same ®ve poults were weighted at 19 days of age (termination of the experiment). The body weight gains of individual poults were calculated. Five poults were randomly selected from each group and necropsied at 10 h, 3, and 9 days PI. Intestines were collected from each bird and the duration of TCV in the intestine was examined by IFA.

In trial 2, 75 turkey poults were randomly separated into three groups. Twenty-®ve turkey poults in group I were treated with CsA at 4 days old and every third day thereafter. Twenty-®ve turkey poults in group II were treated with CY at 1, 2, and 3 days old. Twenty-®ve turkey poults in group III were not treated with any immunosuppressant. At 10 days old, ®ve turkey poults from each group were used for evaluation of humoral antibody responses to SRBC. Another ®ve turkey poults from each group were used for evaluation of cellular immunocompetence by in vitro lymphocyte proliferation assay. The remaining Table 1  Experimental design   Treatment  Trial  Days of age  Days PI   1  2  3  4  7  10  10 h  3  6  9  12 14

x x a Turkey poults were treated with CsA, 100 mg per kg body weight, at every third day as indicated. b Turkey poults were treated with CY, 5 mg per turkey poult per day on the ®rst 3 days post-hatching. c Turkey poults were intramuscularly injected with SRBC at 10 days of age. Antibody titers to SRBC were determined by hemagglutination at 7 days after injection. d Lymphocyte proliferation responses to Con A. e Turkey poults were infected with TCV at 10 days of age. f Body weight (BW) gain was determined from 10 (prior to infection) to 19 days of age. g IFA for TCV antigen in the intestine.

turkey poults in each group were infected with TCV at 10 days old. Five poults from each of these four groups were weighted at 10 days of age (prior to infection). The same ®ve poults were weighted at 19 days of age (termination of the experiment) and the body weight gains of individual poults were calculated. Five poults were randomly selected from each group and necropsied at 3 and 14 days PI. Intestines were collected from each bird and the duration of TCV in the intestine was examined by IFA.

CsA (BIOMOL, Plymouth Meeting, PA) was dissolved in mineral oil and administered intramuscularly every 3 days as previously described [17] . The dose was 100 mg per kg of body weight. Turkey poults were given the ®rst dose at 7 days old (3 days prior to challenge infection) in trial 1 and at 4 days old (6 days prior to challenge infection) in trial 2 and received subsequent doses every third day thereafter throughout the experimental period.

CY (Sigma, St. Louis, MO) was administered intraperitoneally at a daily dose of 5 mg per turkey poults for the ®rst 3 days post-hatching as previously described [18] .

Ten-day-old turkey poults from groups treated with CsA or CY and untreated groups were injected intravenously with 1 ml of 7% saline suspension of SRBC (Sigma) prior to infection with TCV. Blood samples were collected at 7 days after injection. Anti-SRBC antibody titers were quanti®ed by hemagglutination assay [19] . The titer was expressed as the log 2 of the reciprocal of the highest dilution in which visible agglutination was observed.

Blood was obtained from 10-day-old turkey poults from groups treated with CsA or CY and untreated groups prior to infection with TCV. Heparinized blood was diluted at 1:40 and dispensed in a 96-well plate. To each well of three sequential triplicate cultures were added 100 ml of RPMI medium without mitogen or containing 40 mg/ml of concanavalin A (Con A) (Sigma). Cell cultures were incubated at 41 8C for 48 h in a humidifying air atmosphere at 5% CO 2 . Cultures were pulsed with 1 mCi of 3 H-thymidine in 50 ml of RPMI medium per well for the ®nal 5 h of incubation. Cells were harvested onto glass ®lter papers with an automatic cell harvester (Packard, Downers Grove, IL). The counts per minute (CPM) values of individual cell samples were measured in a Packard TopCount Scintillation Counter (Model B9904, Packard). The CPM values of the triplicate cultures within each sample were averaged. Stimulation index (SI) was calculated by dividing the average CPM value in mitogen-stimulated cultures by the average CPM value in unstimulated culture.

The jejunum and ileum distal to the Merckel's diverticulum from each turkey poult at each time interval was obtained for IFA. The IFA used for detection of TCV was described previously [13] . Brie¯y, intestines were frozen immediately at 220 8C and embedded in Tissue-Tek O.C.T. compound (Miles Laboratories, Elkhart, IN). Intestinal sections were cut serially with a cryostat into 6 mm thickness pieces and ®xed in absolute acetone for 10 min. Tissue sections were incubated with turkey antisera speci®c for TCV [2, 3] at a dilution of 1:40 in dilution buffer containing 150 mM phosphate buffer, 0.85% NaCl, 1% BSA, and 0.02% Tween-20 in a humidifying chamber at room temperature for 30 min. After washing three times with PBS buffer, intestinal sections were incubated with uorescein isothiocyanate (FITC) conjugated goat anti-turkey IgG (H 1 L) antibodies (Kirkegaard and Perry Laboratories, Gaithersburg, MD) at a dilution of 1:40 in dilution buffer at room temperature for 30 min. Slides were coversliped and examined on a¯uorescent microscope (Nikon, Tokyo, Japan). The IFA staining positive for TCV antigen appeared in the cytoplasm of enterocytes. The intensity of IFA staining was scored on a scale of 1±4 with 4 being the most intense staining. The distribution of area with positive staining within section was categorized into diffuse, multifocal, and focal and scored as 3, 2, and 1, respectively. The scores of intensity and area distribution were added. The ®nal value indicated the overall amount of staining in a section, with higher value indicating more positive staining. The consistency of IFA is assured by inclusion of positive controls with intestinal sections from turkey embryos infected with TCV and negative controls with intestinal sections from normal turkey embryos in the assay.

Calculated means from each group were compared by one-way analysis of variance (ANOVA). A pooled t-test was used to determine signi®cant difference at p , 0:05 or p , 0:1:

The effects of treatments with CsA or CY on antibody response to SRBC and lymphocyte proliferation response of whole blood cells to Con A in 10-day-old turkey poults prior to infection with TCV are shown in Table 2 . Turkeys treated with CY had signi®cantly lower p , 0:05 antibody response to SRBC than untreated controls in both trials. The differences in antibody response to SRBC between turkeys treated with CsA and untreated controls are not statistically signi®cant in both trials. There were considerable variations in the lymphocyte proliferation responses. Nevertheless, the lymphocyte proliferation responses of turkeys treated with CsA were signi®cantly lower p , 0:1 than that of untreated controls in both trials. The lymphocyte proliferation responses of turkeys treated with CY were signi®cantly lower p , 0:1 than that of untreated controls in trial 1 but not in trial 2.

Effects of CsA-or CY-induced immunosuppression on body weight gains of turkeys experimentally infected with TCV at 10-day-old are shown in Table 3 . The body weight gains of all treated or untreated groups were signi®cantly decreased p , 0:05 after infection with TCV as compared with that of turkeys neither treated with any immunosuppressant nor infected with TCV. The body weight gain of turkeys treated with CY was signi®cantly lower p , 0:05 than that of untreated controls after experimental infection with TCV in both trials. The differences in body weight gain after experimental infection with TCV between turkeys treated with CsA and untreated controls were not statistically signi®cant in both trials.

Effects of CsA-or CY-induced immunosuppression on the severity of TCV infection in the intestine of turkey poults are shown in Table 4 . At 10 h PI, the scores on IFA staining for TCV antigen were below 3.2 in all infected groups and the differences among groups treated or untreated with immunosuppressant were not signi®cant. At 3 days PI, the scores on IFA staining for TCV antigen were above 5.6 in all infected groups and the differences among groups treated or untreated with immunosuppressant were not signi®cant. At 9 (trial 1) and 14 (trial 2) days PI, turkeys treated with CY had signi®cantly higher p , 0:05 scores on IFA staining for TCV antigen than untreated turkeys and the differences of scores between turkeys treated with CsA and untreated controls were not statistically signi®cant. Non-infected control turkeys remained negative for TCV antigen in intestines by IFA throughout the entire experimental period.

CsA is a selective T cell immunosuppressant [17, 20, 21] . Activation of T cells by mitogen or by mixed lymphocyte reaction is inhibited by CsA. This compound exerts Table 2 Effect of CsA and CY on humoral and cellular immune responses of turkeys (* the difference between groups treated with CsA or CY and untreated group is signi®cant p , 0:1; ** the difference between groups treated with CsA or CY and untreated group is signi®cant p , 0:05) its effect by acting as a prodrug, only becoming active when complexed to endogenous, intracellular receptor, and the resulting complex blocks a late stage in the signalling pathway initiated by the T cell receptor [20] . The blocking effect leads to a failure of transcriptional activation of T cell genes, such as those encoding the cytokines, particularly interleukin 2 (IL-2). As a consequence, all cellular immune responses driven by IL-2 are in¯uenced. As expected, treatment of turkeys with CsA caused a de®ciency in lymphocyte proliferation response to Con A while it had a limited effect on antibody response to SRBC. The effect of treatment with CsA on infection of turkey poults with TCV could not be determined in the present study. However, the results could not rule out the involvement of cellular immunity against TCV infection. The T cell function was not completely inhibited by CsA as revealed by considerable lymphocyte proliferation responses to Con A after the treatment. It is possible that the protection effect provided by T cells was very ef®cient and not affected by partial inhibition, or the effect of partial inhibition of T cell function by CsA on infection of turkey poults with TCV was not detectable by the evaluation methods in the present study.

The considerable variations in the lymphocyte proliferation responses between individual birds may be caused by variation in the number of lymphocytes in blood samples at the time of sample collection from different birds or resulted from the genetic diversity. The ability of chicken lymphocytes to respond to Con A was under genetic control [22] . Lymphocytes from chickens with genetic difference had different proliferative response to Con A. In addition, the genetic selection of turkeys for increased body weight or egg production affected the mitogenic responses of turkey lymphocytes to Con A [23, 24] . The turkeys in the present study were obtained from commercial turkey¯ocks originate from outbred breeder stocks and had diverse genetic backgrounds. Similar Table 3 Effect of CsA and CY on body weight gain of turkey poults experimentally infected with TCV mechanism(s) of genetic control as demonstrated for chicken lymphocytes may also contribute to the observed individual variation of lymphocyte proliferation responses of these turkeys. In comparison, cellular immunity against avian IBV has been demonstrated by antigen-speci®c proliferation of T lymphocytes, delayed-type hypersensitivity, and increased populations of CD4 or CD8 T cells [10] . Experimental suppression of chicken T cells with CsA enhanced virus titers in the kidney. Spleen mononuclear cells of chicken vaccinated with live IBV showed antigen-speci®c lymphocyte proliferation and produced IL-2 and interferon-g in response to in vitro stimulation with homologous or heterologous strains of IBV [25] . Cytotoxic T lymphocyte (CTL) responses to IBV was demonstrated to be correlated with initial decrease of amount of virus in kidney and lung and clinical respiratory signs of illness [26] . Adoptive transfer of CD8 T lymphocytes from chicks infected with IBV, protected chicks from challenge infection [27] . The functional CTL epitopes were probably present in spike or nucleocapsid structural proteins of IBV [28, 29] .

Treatment of newly hatched turkey poults with CY, an alkylating agent, has been shown to cause de®ciency in antibody responses to SRBC and persistent cellular degeneration in bursa and bursa-dependent areas in all major lymphoid organs [18] . Results from the present study revealed signi®cantly decreased antibody responses to SRBC in turkey Table 4 Effect of CsA and CY on the severity of TCV infection as determined by immuno¯uorescent staining intensity and distribution of TCV in intestines of turkey poults experimentally infected with TCV. (Turkey poults were infected with TCV at 10 days old. Frozen turkey intestine sections were incubated with turkey antiserum speci®c for TCV and subsequently incubated with¯uorescein isothiocyanate (FITC) conjugated goat anti-turkey IgG (H 1 L) antibody. Non-infected control turkey intestines were negative for TCV by IFA throughout the entire experimental period (data not shown). * The difference between groups treated with CY and untreated group is signi®cant p , 0:05) The intensity of IFA staining was scored on a scale of 1±4 with 4 being the most intense staining. The distribution of area with positive staining within section was categorized into diffuse, multifocal, and focal and scored as 3, 2. And 1, respectively. The scores of intensity and area distribution were added. Data were expressed as mean^SD of scores of IFA stainings from ®ve turkey poults in each group. Higher value indicated more intense staining. c ND not done. d Turkey poults were treated with CsA, 100 mg per kg body weight, at 7 days old in trial 1 or 4 days old in trial 2 and every third day thereafter. e Turkey poults were treated with CY, 5 mg per turkey poult per day on the ®rst 3 days post-hatching.

poults treated with CY. These data suggested that B cells were the major targets of CY in turkey poults. The undetectable antibody response of turkey poults treated with CY to SRBC in trial 2 is not caused by any reagent or operation problem because all samples from different groups were treated and examined in the same way simultaneously. This also does not necessarily re¯ect a complete elimination of humoral B cell activity. The antibody response to SRBC may not be detectable by the evaluation method, hemagglutination assay. Nevertheless, treatment of CY in the present study did signi®cantly suppress the humoral B cell activity and, subsequently, signi®cantly enhanced the severity of TCV infection in turkey poults as revealed by markedly decreased body weight gain and higher scores on IFA staining for TCV antigens in the intestine. Humoral antibody responses to avian IBV in chicken are measurable by enzyme-linked immunosorbent assay, hemagglutination-inhibition test, or virus neutralization test. Vaccination studies of IBV generally correlated humoral antibody responses with protection. The protective role of B cells against IBV infections has been shown by selective experimental suppression with the chemical CY, the hormone testosterone propionate, or surgical bursectomy [10] . Chickens treated with CY had longer persistence of IBV and, subsequently, showed increased clinical signs and more severe histopathological lesions in the kidney. High titers of humoral antibodies were correlated with the absence of virus recovery from kidney and genital tract and seemed to protect the tracheal epithelium from challenge infection. Spike protein of IBV was considered the major structural protein that induces protective humoral antibody responses. Antibodies generated in mice using a recombinant spike protein as inoculum could neutralize IBV infectivity in ciliostasis tests with tracheal organ cultures [30] . Immunization of a recombinant fragment (S1) of spike glycoprotein protected chickens from challenge infection of IBV [31] .

Decreased body weight gain is one of the major characteristics of turkey coronaviral enteritis [32] . Infection with TCV causes rapid histopathologic changes in the mucosa of the intestinal tract of turkey poults. Ultrastructural changes included granularity of epithelial cells, loss of microvilli, margination of chromatin in the nucleus, and increased cellularity of the lamina propria [32, 33] . The alteration in the intestinal tract causes failure of absorption and accumulation of¯uids and gases resulting in diarrhea and decreased body weight gain. The body weight gain of turkey poults treated with CsA or CY or without treatment of CsA or CY were all signi®cantly decreased after infection with TCV as compared with that of non-infected controls. Treatment of CY increased the severity of TCV infection in turkey poults because the body weight gain of turkeys treated with CY was signi®cantly lower than that of turkeys not treated with CsA or CY after infection with TCV.

Because considerable lymphocyte proliferation responses were detectable in turkey poults treated with CsA in trial 1, the initial treatment with CsA of turkey poults (group I) beginning at 7 days old in trial 1 was changed to 4 days old in trial 2 in order to enhance the suppression effect on cellular immunity of turkey poults at 10 days old when they were infected with TCV. For detection of TCV in the intestine by IFA, the time point of 10 h PI in trial 1 was not repeated in trial 2 because signi®cant difference in the amount of staining between groups at this interval was not observed in trial 1. The difference in the amount of staining between groups at 10 h PI was not expected in trial 2 as suggested by the similar amount of staining among groups at 3 days PI in trial 1. In contrast, the time point of 9 days PI in trial 1 was changed to 14 days PI in order to examine if there were greater differences between groups treated with CsA or CY and group not treated with CsA or CY.

The level of TCV antigen was high in the intestines of turkey poults within the ®rst week following infection. The level of TCV antigen declined after 7 days PI while the virus-speci®c antibody responses rose markedly (Loa et al., unpublished data), suggesting the protective role of humoral immunity against TCV infection in turkey poults. Consistent with these observations, the results of the present study also revealed the declines of TCV antigens in the intestines of turkey poults not treated with CsA or CY at 9 and 14 days PI. In addition, the present study showed that suppression of humoral immunity by CY enhanced the severity of TCV infection in the intestines of turkey poults infected with TCV. In summary, signi®cantly decreased body weight gain along with increased severity of TCV infection in turkey poults treated with CY and infected with TCV suggest that humoral immunity may provide protective immunity for turkey poults against infection with TCV.

Fringed membranous particles and viruses in faeces from healthy turkey poults and from poults with putative poult enteritis complex/ spiking mortality

Antigenic characterization of a turkey coronavirus identi®ed in poult enteritis and mortality syndrome-affected turkeys

Detection of antibody to turkey coronavirus by antibody-capture enzyme-linked immunosorbent assay utilizing infectious bronchitis virus antigen

Coronaviral enteritis of turkeys (bluecomb disease)

Bluecomb disease of turkeys. IV. Demonstration of a ®lterable agent

Infection and cross-protection studies of winter dysentery and calf diarrhea bovine coronavirus strains in colostrum-deprived and gnotobiotic calves

Cellular immune responses of pigs after primary inoculation with porcine respiratory coronavirus or transmissible gastroenteritis virus and challenge with transmissible gastroenteritis virus

Infectious bronchitis

Antibody responses in spiral colon, ileum, and jejunum of bovine coronavirus-infected neonatal calves

Infectious bronchitis virus: immunopathogenesis of infection in the chicken

Immunity to transmissible (coronaviral) enteritis of turkeys (bluecomb)

Bluecomb disease of turkeys. V. Preliminary studies on parental immunity and serum neutralization

Fluorescent antibody test for rapid diagnosis of coronaviral enteritis of turkeys (bluecomb)

Secretory antibodies against turkey coronaviral enteritis

Immuno¯uorescent studies on localization of secretory immunoglobulins in the intestines of turkeys recovered from turkey coronaviral enteritis

Cell-mediated immunity against turkey coronaviral enteritis (bluecomb)

The effect of cyclosporin A on the chicken immune system

Suppression of immunologic function and degeneration of lymphoid organs in cyclophosphamide-treated turkeys

Coronaviruses associated with outbreaks of transmissible enteritis of turkeys in Quebec: hemagglutination properties and cell cultivation

The mechanism of action of cyclosporin A and FK506

Hemorrhagic enteritis virus induced changes in the lymphocyte subpopulations in turkeys and the effect of experimental immunode®ciency on viral pathogenesis

Genetic control of the response of chicken leukocytes to a T-cell mitogen

Effect of the genetic selection of turkeys for increased body weight and egg production on immune and physiological responses

Effect of selection for increased body weight on mitogenic responses in turkeys

Cross-reactive cellular immune responses in chickens vaccinated with live infectious bronchitis virus vaccine

Speci®c cytotoxic T lymphocytes are involved in in vivo clearance of infectious bronchitis virus

Adoptive transfer of infectious bronchitis virus primed ab T cells bearing CD8 antigen protects chicks from acute infection

The carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic T lymphocytes and protects chickens from acute infection

Cytotoxic T lymphocytes are critical in the control of infectious bronchitis virus in poultry

Expression of the infectious bronchitis virus spike protein by recombinant vaccinia virus and induction of neutralizing antibodies in vaccinated mice

Induction of protective immunity in chickens vaccinated with infectious bronchitis virus S1 glycoprotein expressed by a recombinant baculovirus

Scanning electron, light, and immuno¯uorescent microscopy of coronaviral enteritis of turkeys (bluecomb)

Histopathology of fasting and bluecomb disease in turkey poults and embryos experimentally infected with bluecomb disease coronavirus