Effect of avian reoviruses on lymphoid organ weights and antibody response in chickens

Several avian reoviruses were screened to determine their effects on the immune system by inoculating them subcutaneously (SQ) into day-old chicks. For comparison, infectious bursal disease virus (IBDV) was similarly evaluated. The response of the immune system was measured functionally by the hemagglutination-inhibition (HI) response to Newcastle disease virus (NDV) and structurally by changes in the organ-to-body-weight ratios of the bursa of Fabricius, thymus, and spleen. When inoculated SQ, most of the reoviruses caused transient alterations in lymphoid organ weights, decreasing the bursa weight and increasing the spleen weight. Of those reoviruses tested, only one--a commercial vaccine based on the isolate S-1133--demonstrated the ability to interfere significantly with NDV-HI responses, although several had numerically lower titers. Two of the isolates were also evaluated by oral inoculation. Giving the viruses orally did not cause any alterations in organ weights; however, both isolates depressed the HI response of chicks to NDV. Compared with reoviruses, IBDV significantly depressed NDV-HI titers. The structural responses to IBDV differed, however: IBDV significantly depressed bursa weights for all 3 weeks of the test period without affecting spleen weights. Some of the reovirus isolates inoculated SQ were lethal for day-old chicks. This, and their ability to alter the lymphoid-to-body-weight ratios of the spleen and bursa, could be considered valid criteria by which to study the pathogenesis of these agents.     

Response of embryonic chicken lymphocytes to in ovo exposure to lymphotropic viruses.

OBJECTIVE: To examine effects of virus exposure on embryonic lymphoid organ structure, apoptosis, and lymphoid cell subpopulations. ANIMALS: Eggs of specific pathogen free (SPF) White Leghorn chickens at embryonation day (ED) 17. PROCEDURES: Eggs were inoculated with 2,000 plaque-forming units (PFU) of serotype 1 herpesvirus (Marek's disease virus [MDV 1]), 2,000 PFU of herpesvirus of turkeys (MDV 3), or 1,000 embryo infectious doses (EID50) of infectious bursal disease virus (IBDV). On post-inoculation days (PID) 3 and 5, lymphoid organ to body weight ratios were determined, and bursa of Fabricius, thymus, and spleen were evaluated for lesions and apoptosis. Proportions of lymphoid cell subpopulations of PID-3 chicken embryos and 7- to 10-day-old chicks were quantitated by flow cytometry. RESULTS: Lymphoid organ weights were similar in virus-free, MDV1, and IBDV groups. Embryos inoculated with 2,000 PFU MDV 3/egg had lower bursal weights than virus-free controls. In a repeated trial, MDV 3 (1,000 PFU to 4,000 PFU) did not reduce bursal weights among groups. Histologic changes were seen in bursae after MDV 1 and IBDV inoculation. Apoptosis was greater in bursae of MDV 1-infected embryos than controls. Lymphoid cell subpopulations were similar among all groups with the exception of CD8+ and IgM+ cells in spleens of IBDV-infected 10-day-old chicks. CONCLUSIONS AND CLINICAL RELEVANCE: Infection with pathogenic strains of MDV 1 and IBDV did not alter lymphocyte subpopulations in embryos or cause complete destruction of lymphoid organs. Changes in lymphoid cell subpopulations exposed as embryos to IBDV were seen only after hatching.     

Responses of specific-pathogen-free chicks to concomitant infections of reovirus (WVU-2937) and infectious bursal disease virus

Specific-pathogen-free white leghorn chicks concomitantly infected with both infectious bursal disease virus (IBDV) and reovirus (WVU-2937) on day 1 had significantly lower (P less than 0.05) virus-neutralizing- and precipitating-antibody geometric mean titers (GMT) to reovirus than chicks infected with only reovirus on day 1 but had a similar incidence of inflammation of the metatarsal digital flexor tendons. Chicks infected with IBDV on day 7 and reovirus on day 14 had a greater incidence of inflammation of the tarsometatarsal digital flexor tendons and lower neutralizing- and precipitating-antibody GMT to reovirus than chicks infected with only reovirus on day 14. Chicks infected with both viruses on day 1 had a significantly lower (P less than 0.05) neutralizing-antibody GMT to IBDV than chicks infected with only IBDV.     

Role of route of exposure, age, sex, and type of chicken on the pathogenicity of avian reovirus strain 81-176

Chickens were evaluated by age, sex, and type for susceptibility to reovirus strain 81-176 inoculated subcutaneously. Chicks were most susceptible to the lethal effects of reovirus infection at hatching, after which resistance increased rapidly. By 1 week of age, mortality was negligible, but chicks were still susceptible to the less lethal effects of the virus. Mortality rates of males and females were equal. Leghorn and broiler-type chicks did not differ appreciably in their response to viral inoculation. An effort was made to find a more "natural" means of exposure to reovirus than parenteral inoculation. Neither oral nor aerosol exposure was as effective as subcutaneous inoculation. Attempts to transmit reovirus to susceptible hatching chicks, starting when they were in ovo (19 days of incubation), also failed. Decreased weight gain proved to be a valid criterion for judging reovirus infection. Reovirus infection lowered the mean values of body weights, and the standard deviations were substantially greater, indicating an unevenness in size of the affected chickens.     

Protective immunity against infectious bursal disease virus in chickens in the absence of virus-specific antibodies

The role of cell-mediated immunity (CMI) in pathogenesis of infectious bursal disease virus (IBDV) was investigated. One-day-old specific pathogen-free chickens were treated with 3mg of cyclophosphamide (Cy) per chicken for 4 consecutive days and, 3 weeks later, infected with the IBDV-IM strain. Chickens were examined for: (a) mitogenic response of splenocytes to ConA, as an indicator of T-cell functions in vitro, (b) antibody against IBDV by ELISA, (c) IBDV genome in various tissues by RT-PCR and (d) immunological memory. At the time of IBDV infection, Cy-treated chickens had depleted bursal tissue (an avian primary B-cell lymphoid organ), severely compromised antibody-producing ability, but normal T-cell response to ConA. In primary infection, no detectable antibody against IBDV antigen in Cy-treated, IBDV-infected chickens was observed up to 28 days post-infection (PI), while IBDV genome was detected by RT-PCR in spleen, thymus, liver and blood until 10 days PI. Like intact control chickens infected with IBDV, Cy-treated, IBDV-infected chickens suppressed splenocytes responses to ConA from 5 to 10 days PI, suggesting that intact control as well as Cy-treated chickens responded similarly to IBDV infection in the early phase. Following re-infection with IBDV, no detectable secondary antibody response to IBDV as well as IBDV genome in tissues were observed in Cy-treated chickens, while intact control chickens developed vigorous secondary antibody response. Similar to intact control chickens infected with IBDV, Cy-treated chickens after second infection with IBDV did not suppress splenocyte response to ConA. These results suggested that in the absence of detectable anti-IBDV antibodies, protection of Cy-treated chickens from IBDV infection may occur via immunological memory mediated by CMI. We concluded that under normal conditions, IBDV induces a protective antibody response, however, in the absence of antibody, CMI alone is adequate in protecting birds against virulent IBDV.     

Failure of two serotype II infectious bursal disease viruses to affect the humoral immune response of turkeys

The effect of serotype II infectious bursal disease virus (IBDV) isolates from turkeys on the homoral immune response of turkey poults was determined. Following exposure to the OH IBDV isolate, poults in two experiments were inoculated with sheep red blood cells, which is a T-dependent antigen, and poults in two other experiments were inoculated with Salmonella heidelberg O antigen, which is a T-independent antigen. Prior exposure to serotype II IBDV did not affect serum antibody titers to these antigens. IBDV infection also did not affect the concentrations of serum immunoglobulin M (IgM), IgG, or IgA in these poults. Bursa:body weight ratios of OH IBDV-infected poults were not significantly different from those of uninfected controls. In one experiment, the humoral immune response of poults to the LaSota Newcastle disease vaccine was not affected by infection with the MO IBDV isolate. Although no clinical infectious bursal disease was observed in any poult in these experiments, the serotype II IBDV isolates were infectious and transmissible in poults.     

Persistence of commercial modified live reovirus vaccines in chicks

Four modified live reovirus vaccines were compared with a field isolate (81-176) of reovirus for safety in 1-day-old and 1-wk-old specific-pathogen-free chicks. At 1, 3, 5, and 7 wks after vaccination, blood, cloacal swabs, and hock tendon samples were collected and assayed for residual virus. Tendon samples were also examined microscopically and scored for histological changes. Vaccine efficacy was monitored by challenging chicks 7 wks postvaccination via the footpad with the 81-176 isolate. Serum samples were collected throughout the trials and assayed for reovirus antibodies using an enzyme-linked immunosorbent assay. In chicks vaccinated at 1 day of age, three of the four vaccine viruses were consistently recovered from the hock tendon tissues. Post-challenge virus recoveries were similarly positive for the same vaccine viruses. Histological lesion scores of the tendons paralleled the degree of virus recovery. In the chicks vaccinated at 1 wk old, none of the vaccine viruses were recovered, nor were tendon lesion scores substantially elevated. Following challenge, however, virus was recovered from all vaccinated groups.     

Effect of infectious bursal disease virus vaccines on persistence and pathogenicity of modified live reovirus vaccines in chickens.

Two commercially available live reovirus vaccines, alone or in combination with two infectious bursal disease virus (IBDV) vaccines, were evaluated for safety and efficacy in specific-pathogen-free leghorn chicks. Four trials were conducted to evaluate the vaccine combinations. At periodic intervals during the trials, tissues were collected and assayed for residual reovirus and examined for histological changes. Six weeks following reovirus vaccination, all treatment groups were challenged with a virulent field isolate of reovirus and sampled 1 week later for the final time. The two reovirus vaccines were safe and effective if given at 1 week of age, regardless of whether the vaccinates had been exposed to IBDV at 1 day. However, both reovirus vaccines persisted in the tendons of 1-day-old vaccinates. The effects of IBDV vaccines were generally minor and reflected by increases in the number of pre-challenge or post-challenge virus recoveries from some of the treatment groups receiving both type vaccines.     

鸡新城疫－传染性支气管炎－传染性法氏囊病三联灭活疫苗对不同日龄雏鸡的免疫效力研究

为评估鸡新城疫（ND）-传染性支气管炎（IB）-传染性法氏囊病（IBD）三联灭活疫苗对不同日龄和不同水平母源抗体雏鸡的免疫效力和持续期,本试验用该疫苗免疫7、14、21日龄SPF雏鸡和有母源抗体的普通雏鸡,免疫后采血测定ND血凝抑制抗体（HI Ab）、IB血凝抑制抗体（HI Ab）及IBD中和抗体（NA）,并用传染性法氏囊病病毒（IBDV）强毒攻击。结果显示,7日龄SPF雏鸡免疫后21 d ND HI抗体、IB HI抗体及IBD中和抗体效价分别为7.9log2、6.9log2和14.1log2,SPF鸡日龄越大,抗体水平越高;28日龄SPF鸡免疫后3个月,0.3 mL免疫剂量组试验鸡ND HI、IB HI及IBD中和抗体效价分别达6.5log2、6.1log2和13.6log2,IBDV攻毒保护率均为100%（10/10）;不同日龄普通雏鸡免疫效果与SPF鸡试验一致,抗体水平随鸡日龄增大而升高,IBD攻毒保护率也都达到100%（10/10）。试验结果证实,鸡新城疫-传染性支气管炎-传染性法氏囊病三联灭活疫苗可使7、14及21日龄SPF雏鸡和普通雏鸡产生良好的免疫力,对雏鸡的免疫期至少为3个月。     

Maternal antibody and its effect on infectious bursal disease immunization

Chickens vaccinated with infectious bursal disease virus (IBDV) early in life and revaccinated with an inactivated, oil-adjuvant IBDV vaccine at 18 weeks of age produced and maintained high levels of virus-neutralizing (VN) antibody through 10 months of lay. VN-antibody titers of chicks hatched from eggs laid during the same period closely matched the average VN-antibody titers of the dams. A sequential study of the decline rates of IBDV maternal antibody (MAB) in unvaccinated and IBDV-vaccinated chicks showed that the vaccine virus did not accelerate the antibody depletion rate in vaccinated chicks. Chicks carrying high IBDV MAB showed no active immune response to vaccination with commercial IBDV vaccines. They were also refractory to a pathogenic field isolate of IBDV (FV). However, chicks with low levels of MAB responded to both vaccine virus and the FV, although their response to vaccine virus was milder and delayed.     

Immunostimulant effect of a mixed herbal extract on infectious bursal disease virus (IBDV) vaccinated chickens in the context of a co-infection model of avian influenza virus H9N2 and IBDV

This study was conducted to assess the comparative effects of a mixed herbal extract (MHE) containing Ocimum sanctum, Withania somnifera, Emblica officinalis, Tinospora cordifolia, Mangifera indica, and Asphaltum (shilajit) on infectious bursal disease virus (IBDV)-vaccinated (VAC) chickens infected with IBDV and avian influenza virus (AIV) H9N2. The experiment included three groups (G1-G3): G1, the negative control group; G2, the VAC + challenged (Ch) group; and G3, the VAC + Ch + MHE group. MHE was orally administered continuously for 5 weeks post-vaccination (PV) with IBDV at 12 days of age, and the chicks were simultaneously challenged with virulent IBDV (intraocularly) and AIV H9N2 (intranasally) at 21 days PV. Blood and tissue samples as well as tracheal and cloacal swabs were gathered at different times PV and post-challenge. Immunological and haematological parameters, histopathological lesions, relative organ weights and final live weights revealed significant differences (P ≤ 0.05) between G2 and G3 groups. Furthermore, in the G3 group, the protection rates, ELISA and HI titers and CD4+/CD8+ ratio were significantly increased, whereas viral shedding titers and the heterophil/lymphocyte ratio were decreased. In conclusion, the oral administration of the mixed herbal extract for 5 weeks can stimulate the immune response to IBDV vaccination and relieves the pathogenicity of an AIV H9N2 and IBDV co-infection in chickens.     

Specific suppression of the bursa-dependent immune system of chicks with infectious bursal disease virus.

Chicks which had been inoculated with infectious bursal disease virus (IBDV) at 1 day of age had a severe depression of bursa-dependent humoral immune functions by day 42. Antibody responses against rabbit red blood cells or to immunization with bovine serum albumin were significantly suppressed. In contrast, chicks inoculated with IBDV at 21 days of age produced near normal antibody responses as compared with the responses in noninfected control chicks. The IBDV had no significant effect on the thymus-dependent cellular responses as measured by skin graft rejection or delayed type hypersensitivity reactions to tuberculin.     

传染性法氏囊病疫苗免疫母鸡后子代雏鸡外周血液的B细胞和免疫球蛋白含量变化

应用免疫SPA菌体花环法和间接ELISE法对传染性法氏囊病（IBD）疫苗免疫母鸡后，其子代雏鸡外周血液B细胞数量和IgG、IgM、IgA含量进行了动态研究。结果发现：IBD疫苗免疫后，其子代雏鸡外周血液B细胞数量和IgG、IgM、IgA含量均不同程度地高于未免疫的相应对照雏鸡，表明IBD疫苗免疫母鸡后，其子代雏鸡外周血液的体液免疫功能明显增强。而传染性法氏囊病超强毒株攻击子代雏鸡后，未免疫子代雏鸡外周血液的上述各项指标均明显低于疫苗免疫的子代雏鸡，这与IBDV感染雏鸡后，其体液免疫中枢器官法氏囊严重损害，淋巴细胞变性坏死等有关，也是导致感染雏鸡免疫抑制的基础。     

Comparative study of the pathogenicity of avian reoviruses

Reovirus strains CO8 and 81-5, isolated from chickens with malabsorption syndrome, and reovirus strain 176, isolated from chickens with tenosynovitis, were each individually inoculated into 1-day-old specific-pathogen-free chicks. Strain CO8 caused tenosynovitis and pericarditis following footpad inoculation, but it was of low pathogenicity when given by either oral or subcutaneous route. In contrast, strains 81-5 and 176 were highly pathogenic and caused severe mortality following subcutaneous inoculation. Lesions included hepatic necrosis, bursal atrophy, thymitis, and splenic hyperplasia; strain 81-5 also caused pericarditis and myocarditis. Although strain 176 caused higher mortality than strain 81-5, these two strains differed little in the severity and distribution of the lesions they caused. No signs or lesions of typical malabsorption syndrome were observed.     

Relationship of common avian pathogen antibody titers in so-called chicken anemia agent (CAA)-antibody-positive chicks to titers in CAA-antibody-negative chicks.

Antibody titers for infectious bursal disease virus (IBDV), infectious bronchitis virus, Newcastle disease virus, and reovirus from chicks with chicken anemia agent (CAA) antibodies were compared with antibody titers from their CAA-antibody-negative counterparts. These comparisons were made in 396 chickens that were 1 day, 2 weeks, 8-9 weeks, 10 weeks, 17 weeks, or 29-32 weeks old. Only one serum sample was collected from any given chick or chicken. There were no significant differences between the antibody titers at any age for any antigen, with one exception: at 29-32 weeks, the IBDV titers were higher (t = 2.62, df = 142, P less than 0.01) in chickens with CAA antibody. Although not at all likely, we believe that the observation of high IBDV antibody titers in CAA-antibody-positive chicks could have been a spurious one.     

Single and combined infections of specific-pathogen-free chickens with infectious bursal disease virus and an intestinal isolate of reovirus

The susceptibility of 1-day-old and 7-day-old specific-pathogen-free chickens to infection with a virulent strain of infectious bursal disease virus (IBDV) or an intestinal isolate of avian reovirus, or a combination of the two, was investigated. Chickens infected with IBDV and reovirus had more severe pathological lesions than chickens infected with either virus alone, and prior infection with IBDV enhanced the pathogenicity of enteric reovirus. Virus recovery was attempted from bursa, spleen, thymus, liver, intestine, pancreas, cecal tonsils, heart, and tarso-metatarsal tendons. Viruses were recovered from all tissues sampled for either IBDV or reovirus isolation, and indications were that infection with IBDV before infection with reovirus led to longer persistence of reovirus in some tissues. Antibodies to IBDV or reovirus were measured by the virus neutralization test and enzyme-linked immunosorbent assay. Chickens infected with IBDV had lower (P less than 0.05) antibody responses to reovirus than chickens infected with reovirus alone.     

Immune profile of infectious bursal disease. III. Effect of infectious bursal disease virus on the lymphocyte responses to phytomitogens and on mixed lymphocyte reaction of chickens

A whole-blood-culture technique was used to sequentially evaluated peripheral blood lymphocyte responses to phytohemagglutinin (PHA) and concanavalin A (Con A) of normal chickens and chickens infected at 1 day or 3 weeks of age with infectious bursal disease virus (IBDV). This method had numerous advantages over the more conventional techniques. A comparative study was made on the percentage of inhibition of responses of peripheral blood lymphocytes to PHA and Con A of 1-day- and 3-week-old IBDV-infected chickens. In both groups, there was a minimum inhibition between 3 and 4 weeks postinfection (PI) and a maximum inhibition at 6 weeks PI. A one-way mixed lymphocyte reaction (MLR) was performed using mitomycin-C-treated cells as stimulator cells obtained from chickens of genetically different strains. Lymphocytes from the experimental birds (control, 1-day-infected, and 3-week-infected groups) were used as the responder cells. The results showed that MLR response of the IBDV-infected chickens was significantly reduced compared with those of the uninfected controls. The degree of lowered response was much more severe in chickens infected at 1 day of age than in those infected at 3 weeks of age.     

IBDV-induced bursal T lymphocytes inhibit mitogenic response of normal splenocytes

We examined the suppressive activity of bursal T cells induced by infectious bursal disease virus (IBDV) in inbred (15x7) and outbred commercial specific-pathogen-free (SPF) chickens. The suppressive activity was measured by the ability of bursal and splenic T cells from IBDV-infected chickens to inhibit mitogenic responses of normal splenocytes. The bursacytes but not the splenocytes of IBDV-infected chickens inhibited the mitogenic responses of normal splenocytes. The mitogenic inhibition by the bursacytes of IBDV-infected chickens was dose-dependent. The suppression was observed both in inbred and non-inbred chickens, and thus, was non MHC-restricted. Cell-sorting experiments revealed that both CD4(+) and CD8(+) cells from the bursa of IBDV-infected chickens, as well as cell-culture supernatants conditioned by these cells, mediated suppression. Suppressor T (Ts) cells may therefore be involved in the immunosuppression induced by IBDV.     

The effect of infectious bursal disease virus infection on local and systemic antibody responses following infection of 3-week-old broiler chickens

Broiler chickens infected at 3 weeks of age with infectious bursal disease virus (IBDV) were given Brucella abortus (BA) or sheep red blood cell (SRBC) antigens before, during, and after the acute phase of the infection. Gland of Harder (GH) extracts and serum samples were used to assay local and systemic antibody titer to each antigen 7 days after antigen was administered. Antibody titers to both BA and SRBC antigens were lower (P less than 0.05) in GH extracts and serum of IBDV-infected broilers than uninfected controls. The responses to BA, a thymus-independent antigen, took longer to become depressed than the responses to SRBC, a thymus-dependent antigen. The depression of antibody titers following IBDV inoculation suggests compromise of both local and systemic immune function, a finding of importance to the broiler industry.     

鸡贫血病—法氏囊病联合免疫母鸡后子代雏鸡免疫器官T细胞和抗体生成细胞数量的动态变化

对鸡传染性贫血病（CIA）－传染性法氏囊病（IBD）联合免疫母鸡后的子代雏鸡免疫器官的免疫学化变化进行了研究。结果发现,混合感染CIAV、IBDV雏鸡免疫器官T细胞和IgG,IgM,IgA抗体生成细胞数量在27日龄内明显未免疫对照组、联合免疫组和联合免疫攻毒组,表明感染CIAV、IBDV的雏鸡全身免疫功能显著下降,CAI－IBD联合免疫母鸡后,子代雏鸡T细胞胞和IgG,IgM,IgA抗体生成细胞数量在27日龄内,较未免疫对照组明显增加,表明CIA－IBD联合免疫母鸡可使子代雏鸡免疫器官的免疫功能增强,能抵御强毒攻击。