Pathogenicity of various adenovirus serotypes in the presence of Escherichia coli in chickens

Ten strains of adenovirus representing 10 serotypes were administered intratracheally to 3-week-old specific-pathogen-free chickens, which also received 2.9 X 10(5) colony-forming units of a pathogenic Escherichia coli intranasally. One group was given only E. coli, and one was retained as an uninoculated control. Gross pathologic alterations post-mortem were minimal and limited to multiple scattered, pale areas in the lungs of an occasional chicken in various groups. Histopathologic changes in the lungs were those of multifocal, interstitial, and occasionally diffuse pneumonia. Moderate to marked interstitial pneumonia was incited by adenovirus strains 75-1A, B-3 A-2, C-2B, and X-11; Ind-C, Stein, Tipton, J-2, and T-8 caused similar but milder lesions. Strains 75-1A, A-2, C-2B, T-8, and X-11 incited moderate to marked multifocal pneumonia; Ind-C, Stein, Tipton, J-2, and B-3 caused mild multifocal pneumonia. In all groups, the pneumonic lesions were more severe 5 days postinoculation than 12 days postinoculation. Bronchiolitis and tracheitis lesions also varied in severity with serotype. A mild hepatitis was seen with serotypes T-8 and 75-1A. Neither the uninoculated control group nor the group inoculated with only E. coli exhibited gross or histopathologic alterations.     

Adenovirus infection associated with respiratory disease in commercial chickens

The lesions and etiologic agents associated with 13 outbreaks of respiratory disease in commercial chickens were investigated. Adenoviruses were isolated from tracheal and lung tissues of affected chickens in all 13 outbreaks. Escherichia coli was isolated from the lung of an occasional bird. The tracheal specimens were consistently negative for Bordetella avium, but E. coli and occasionally Staphylococcus aureus were isolated. There was also serological evidence in one outbreak, and pathological evidence in another, of a concurrent infectious bursal disease virus (IBDV) infection of chickens affected with the disease. Gross and microscopic alterations in the tracheas and lungs of affected chickens were similar in all outbreaks and consisted of catarrhal tracheitis and occasionally multifocal pneumonia with mononuclear cell infiltrates. Hepatitis and splenitis with heterophil infiltrates occasionally were seen in birds with coliform septicemia. The tracheal and lung lesions in the present investigation were considered primarily of adenovirus etiology, complicated by secondary bacterial infection.     

Experimental cryptosporidiosis and infectious bursal disease virus infection of specific-pathogen-free chickens

Specific-pathogen-free chickens orally inoculated at 4 days of age with a moderately pathogenic vaccine strain of infectious bursal disease virus (IBDV) and/or at 5 days of age with Cryptosporidium baileyi oocysts remained free of overt clinical signs throughout a 16-day period postinoculation (PI). The prepatency period for C. baileyi oocyst shedding was shorter in chickens receiving higher numbers of oocysts, but once shedding was detected, there were no obvious differences in shedding patterns among groups receiving 10(3) through 10(6) oocysts. On days 8 and 16 PI, cryptosporidia were located primarily in the bursae of Fabricius. IBDV exposure was associated with bursal follicle atrophy, whereas C. baileyi infection resulted in bursal epithelial hypertrophy and hyperplasia, mild follicle atrophy, and heterophil infiltration of the bursal mucosa. Examination of experimental groups of 30 birds each indicated that concurrent infection with both agents resulted in more severe bursal lesions, more infected birds, and greater numbers of cryptosporidia in infected tissues. At the termination of the trial, 16 days PI, Cryptosporidium infection was associated with a 6% decrease in mean body weight compared with controls.     

Recovery of antibody-producing ability and lymphocyte repopulation of bursal follicles in chickens exposed to infectious bursal disease virus.

We studied the long-term effect of infectious bursal disease virus (IBDV) in chickens. Specifically, the restoration of virus-induced bursal lesions and the duration of humoral immunodeficiency were examined. One-week-old specific-pathogen-free chickens were intraocularly inoculated with an intermediate vaccine strain (IBDV-Vac) or a virulent strain (IM-IBDV). At intervals postinoculation (PI), chickens were examined for histopathologic lesions. At 1, 3, 5, 10, or 15 wk PI, the chickens were injected with a mixture of antigens, and primary antibody responses were examined at 10 days postimmunization. Initially, the virus caused extensive necrosis of bursal B lymphocytes. This lesion was accompanied by an infiltration of T lymphocytes. With time, the necrotic lesion in the bursa was resolved. The follicles became partly repopulated with B lymphocytes. The repopulation occurred faster in the chickens exposed to IBDV-Vac than in the chickens exposed to IM-IBDV. By 7 wk PI, 40% and 80% of bursal follicles in IM-IBDV- and IBDV-Vac-inoculated chickens, respectively, were repopulated with immunoglobulin M+ B lymphocytes. Both IBDV-Vac and IM-caused suppression of the primary antibody response to antigens. However, the antibody responses of the chickens exposed to either of the two IBDV strains used were compromised only during the first 6 wk of virus exposure. Subsequently, the antibody response returned to near normal levels.     

Rocket Immunoelectrophoresis in the Diagnosis of Infectious Bursal Disease

The rocket immunoelectrophoresis (RIE) test was used for the qualitative detection and quantitative estimation of infectious bursal disease virus (IBDV) specific antigen in experimentally infected chickens and samples collected from suspected outbreaks. The IBDV specific antigen was detected in the bursae of experimentally inoculated chickens up to 5 days post infection (PI) by the agar gel precipitation (AGP) test and 7 days PI by the RIE test. The RIE detected IBDV specific antigen in a significantly greater number of samples collected from the field outbreaks than the conventional AGP test. Exudative bursae were found to have a higher antigen content than haemorrhagic bursae and are recommended as the material of choice for diagnosis of IBD. This test could also be used to quantify IBDV specific antigen in commercial killed vaccines.     

In vitro and in vivo characterization of avian Escherichia coli. II. Factors associated with pathogenicity

The pathogenicity of 197 Escherichia coli isolates obtained from clinically affected commercially grown broiler chickens and normal hatchery chicks was assessed by inoculating day-old broilers intratracheally. The degree of pathogenicity (high, intermediate, low) was judged according to mortality and lesions occurring within 7 days following inoculation. Serotype, metabolic activity, motility, and in vitro antibiotic sensitivity of each isolate were evaluated and related to pathogenicity. Seventy-five of the isolates of high to intermediate pathogenicity belonged to serogroup O2, O78, or O35. In addition, 51 pathogenic E. coli isolates could not be serotyped, and several had multiple serotypes. Most isolates had similar metabolic activity, as determined by amino acid decarboxylation and carbohydrate fermentation, regardless of pathogenicity. An exception was the fermentation of adonitol, which occurred more frequently with the highly pathogenic strains. Motility and in vitro antibiotic sensitivity were not related to pathogenicity. An age-associated resistance to intratracheal E. coli administration occurred by 15 days of age in uncompromised birds. Relative susceptibility of birds older than 2 weeks to intratracheal and/or intravenous E. coli inoculation could be increased by prior exposure to pathogenic reovirus 1733, adenovirus 3167, or infectious bursal disease virus (IBDV). Birds infected with IBDV at 3 weeks failed to clear apathogenic and pathogenic E. coli from circulating blood.     

传染性法氏囊病免疫保护试验中3种评定指标的比较

100只SPF鸡均分为5组，A、B、c3组免疫后攻毒，接种3批次自制的IBD基因工程重组亚单位油乳剂疫苗；D组不免疫不攻毒，E组不免疫而攻毒。免疫后第22天，感染IBDV强毒株BC-6／85。攻毒后第4天，将所有存活的鸡只以颈脱臼致死，收集法氏囊，以3种方法和指标（法氏囊眼观病变；法氏囊显微病变；法氏囊中IBDV抗原检测）进行分析，以评定免疫保护率。结果显示，以这3种方法和指标评定，A组免疫保护率为90％，B、C组免疫保护率均为95％；试验鸡A3、A14、B7、C16、E1～E20，法氏囊眼观病变明显，法氏囊显微病理损伤评分为3～5分，琼脂免疫扩散试验检测法氏囊中IBDV抗原均为阳性；其他试验鸡，法氏囊眼观无明显异常，法氏囊显微病理损伤评分在3分以下，琼脂免疫扩散试验检测法氏囊中IBDV抗原均为阴性。结果表明，这3种方法和指标在IBD疫苗免疫保护试验评定中具有很好的一致性。     

Pathogenicity of fowl adenovirus isolated from gizzard erosions to immuno-suppressed chickens

Pathogenicity of a fowl adenovirus (FAV), JM1/1 strain of serotype 1 derived from gizzard erosions of a broiler chicken, was examined to specific pathogen-free (SPF) chickens pre-treated with infectious bursal disease viruses (IBDVs) or cyclophosphamide (CY). Virulent IBDVs, classical type, were inoculated orally at 3 days of age of SPF chickens. CY was treated subcutaneously for 3 days after hatch. FAV was given orally at 30 days of age. At 40 days of age, all chickens were bled and autopsied for serology and gross observation. Gizzard lesions were ranked by the scores depending on their severities. IBDV- or CY-treated chickens showed significantly higher gizzard lesion scores than non treated birds. There were no gross lesions in any other organs except for bursal atrophy. Serologically, antibody production against FAV was highly suppressed by IBDV infection or CY treatment.     

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.     

Susceptibility of Local Nigerian and Exotic Chickens to Infectious Bursal Disease by Contact Exposure

One hundred 6-week-old susceptible cockerels were inoculated with a pathogenic strain of infectious bursal disease virus (IBDV) and kept in the same pen as 100 each of 6-week-old pullets, local chickens and broilers. The cockerels developed depression and diarrhoea on day 3 post inoculation (PI) and most of the pullets and some of the local chickens and broilers showed similar signs on day 4 PI. Loss in weight was severe and similar in the pullets and local chickens, being significantly greater than that in the broilers from days 3–11 PI. The total mortality was 85%, 66.7%, 30% and 20% for the pullets, cockerels, local chickens and broilers, respectively. The lesions were more severe in the pullets and local chickens than in the broilers. IBDV antigen and antibody were detected, respectively, in all the bursal and serum samples from the infected chickens tested. The contact exposure method used in this study simulates better what happens in nature than inoculation with IBDV. The reduced mortality observed among the local chickens, compared with that (61.5%) seen in earlier studies using intraocular inoculation of IBDV, may have been due to behavioural differences that tend to result in their ingesting a relatively low dose of the virus.     

Differential modulation of cytokine, chemokine and Toll like receptor expression in chickens infected with classical and variant infectious bursal disease virus

ABSTRACT: Infectious bursal disease (IBD) is an important immunosuppressive disease of chickens. The causative agent, infectious bursal disease virus (IBDV), consists of two serotypes, 1 and 2. Serotype 1 consists of classic IBDV (cIBDV) and variant IBDV (vIBDV). Both of these strains vary in antigenicity and pathogenesis. The goal of this study was to compare the immunopathogenesis of cIBDV and vIBDV. Three-week-old specific pathogen free chickens were inoculated intraocularly with standard challenge strain (STC) (cIBDV) and a variant strain Indiana (IN) (vIBDV). The cIBDV produced more pronounced bursal damage, inflammatory response and infiltration of T cells as compared to vIBDV. There were significant differences in the expression of innate (IFN-α and IFN-β), proinflammatory cytokine and mediator (IL-6 and iNOS) in cIBDV- and vIBDV-infected bursas. The expression of chemokines genes, IL-8 and MIP-α was also higher in cIBDV-infected chickens during the early phase of infection. The expression of Toll like receptor 3 (TLR3) was downregulated at post inoculation days (PIDs) 3, 5, and 7 in the bursas of vIBDV-infected chickens whereas TLR3 was upregulated at PIDs 3 and 5 in cIBDV-infected bursas. In vIBDV-infected bursa, TLR7 expression was downregulated at PIDs 3 and 5 and upregulated at PID 7. However, TLR7 was upregulated at PIDs 3 and 7 in cIBDV-infected bursas. The expression of MyD88 was downregulated whereas TRIF gene expression was upregulated in cIBDV- and vIBDV-infected bursa. These findings demonstrate the critical differences in bursal lesions, infiltration of T cells, expression of cytokines, chemokines and TLRs in the bursa of cIBDV-and vIBDV-infected chickens.     

Differentiation between antibodies to serotypes 1 and 2 infectious bursal disease viruses in chicken sera

The usefulness of the virus neutralization (VN) test, the enzyme-linked immunosorbent assay (ELISA), and the agar gel precipitin (AGP) test in differentiating antibodies to infectious bursal disease virus serotypes 1 and 2 was investigated. Sera examined were from chickens that were challenged with live virus or inoculated with inactivated oil-emulsion IBDV vaccines or were both challenged and inoculated. Antibodies to serotypes 1 and 2 were differentiated by the VN test but not by the ELISA, and the AGP test was less than satisfactory.     

Comparison of the dot blot hybridization assay with antigen detection assays for the diagnosis of infectious bursal disease virus infections

The use of cDNA probes as diagnostic tools for detecting infectious bursal disease virus (IBDV) antigens was compared with the agar-gel precipitin (AGP) and immunofluorescence (IF) assays. Specific-pathogen-free chickens were inoculated with the STC or IN strains of IBDV in three separate experiments. Tissue samples were collected at various intervals postinoculation (PI) and examined for viral antigens using the IF assay, the AGP assay, and the hybridization assay. Viral antigen was detected by the AGP assay for a period of approximately 3 days beginning 2 days PI and by the IF assay for approximately 4 days beginning 2 days PI. Virus was detected by the hybridization assay through the length of all the experiments (longest experiment was 24 days) beginning 1 day PI. These results suggested that the hybridization assay is more sensitive than the IF assay and the AGP assay.     

An age-related coagulation disorder associated with experimental infection with infectious bursal disease virus

Specific-pathogen-free White Leghorn chickens were inoculated with a field strain of infectious bursal disease virus. One group (A) was inoculated at 17 days after the chicks were hatched, and the other groups (C and E) were inoculated at posthatch day 42. Blood samples were obtained for determination of clotting times (whole blood recalcification, prothrombin, and activated partial thromboplastin times), virus-neutralizing antibody, and total hemolytic complement. There were significant increases in clotting times for groups C and E at 3 and 5 days after they were inoculated. There were no significant increases in clotting times at 3 days after inoculation in the group A chickens (inoculated at 17 days after hatching). There were no significant decreases in total complement activity in any of these chickens (groups A, C, and E). This study indicates that the mortality and clinical symptoms observed in chickens experimentally infected with infectious bursal disease virus may be associated with a clotting abnormality because it was noted only in chickens that developed severe clinical disease (inoculated at 42 days after hatching) and was not noted in chickens that remained clinically normal (inoculated at 17 days).     

Induction of protective immunity in chickens immunised with plasmid DNA encoding infectious bursal disease virus antigens

Direct DNA inoculations were used to determine the efficacy of gene immunisation of chickens to elicit protective immune responses against infectious bursal disease virus (IBDV). The vp2 gene of IBDV strains GP40 and D78, and the vp2-vp4-vp3 encoding segment of strain D78 were cloned in an expression vector which consisted of human cytomegalovirus (HCMV) immediate early enhancer and promoter, adenovirus tripartite leader sequences and SV40 polyadenylation signal. For purification of vaccine-quality plasmid DNA from E. coli, an effective method was developed. Chickens were vaccinated by inoculation of DNA by two routes (intramuscular and intraperitoneal). Two weeks later, chickens were boosted with DNA, and at 2 weeks post-boost, they were challenged with virulent IBDV strain. Low to undetectable levels of IBDV-specific antibodies and no protection were observed with DNA encoding VP2. However, plasmids encoding VP2-VP4-VP3 induced IBDV-specific antibodies and protection in the chickens. DNA immunisation opens a new approach to the development of gene vaccines for chickens against infectious diseases.     

Immune dysfunction following infection with chicken anemia agent and infectious bursal disease virus. I. Kinetic alterations of avian lymphocyte subpopulations.

The potential effect of chicken anemia agent (CAA) alone or in combination with infectious bursal disease virus (IBDV) on the immune system of young chickens was determined by measuring alterations in hematocrit values, lymphoid organ-to-body weight ratios and lymphoid cell concentrations at 4, 7, 10, 14, 17, 21, 28 and 42 days post-inoculation (PI). Lymphocyte subpopulations were identified and counted by flow cytometry using cell suspensions stained with monoclonal antibodies (Mabs) for panlymphocytes (K55), cytotoxic T-cells (CTLA3), T-helper cells (CT3), Ia-expressing cells (P2M11) and macrophages (P7). Chicken anemia agent induced a substantial but transient decrease in hematocrit value, thymus-to-body weight ratio and bursa-to-body weight ratio between 7 and 21 days PI corresponding to a generalized lymphocytopenia in the thymus, bursa and spleen. However, cytotoxic T-cell, T-helper cell and Ia-expressing cell concentrations increased in the bone marrow of birds inoculated with CAA alone or in combination with IBDV during the same time period. T-helper-to-cytotoxic T-cell ratios increased in the thymus and spleen during severe lymphocytopenia, indicating a selective decrease in cytotoxic T-cells. T-helper-to-cytotoxic T-cells ratios increased in the bone marrow, indicating a selective increase in T-helper cell concentrations. The increase in Ia-expressing cells in the bone marrow may be a reflection of increased number of activated T-cells which express Ia antigen. Infectious bursal disease virus alone induced a persistent depression of Ia-expressing cells in the bursa and the spleen and no measurable change in the bone marrow lymphocyte subpopulations. Chickens inoculated simultaneously with CAA and IBDV experienced clinical signs observed in chickens inoculated with each virus separately with a prolonged acute phase prior to recovery or mortality.     

Effect of infectious bursal disease on the response of chickens to Mycoplasma synoviae, Newcastle disease virus, and infectious bronchitis virus.

At 35 days of age, chickens which as 1-day-old chicks were inoculated with the infectious bursal disease virus (IBDV) had significantly lower antibody titers against Mycoplasma synoviae, Newcastle disease virus, and infectious bronchitis virus than did those never inoculated with IBDV. The IBDV also had a marked effect on the development of air-sac lesions. Birds infected with IBDV that were later inoculated with M synoviae (day 14), Newcastle disease virus (days 14 and 28) experienced an increased incidence and greater seversity of airsacculitis than did chicks which were not exposed to IBDV.     

The exacerbating effect of infectious bronchitis virus infection on the infectious bursal disease virus-induced suppression of opsonization by Escherichia coil antibody in chickens

Chickens infected with infectious bronchitis virus (IBV) and infectious bursal disease virus (IBDV) commonly develop secondary infection of the respiratory tract with Escherichia coli, resulting in significant economic losses. To understand the host factors that may contribute to the E. coli infection, we investigated macrophage-mediated E. coli phagocytosis, intracellular bacterial killing, and development of opsonizing antibody in previously uninfected chickens and in those infected with IBV, IBDV, and IBDV plus IBV. Macrophages from the peripheral blood and the respiratory tracts of chickens infected with IBV or IBDV plus IBV efficiently performed in vitro phagocytosis of E. coli in the presence of positive-control serum (i.e., E. coli antiserum produced in normal chickens). Those macrophages also had adequate bactericidal activity, indicating that IBV and IBDV infections had not affected their phagocytic activity or bactericidal function. The phagocytic activity of macrophages remained unaffected (P < 0.05) when the positive-control serum was replaced with E. coli antiserum produced in chickens infected with IBV alone. However, when E. coli antisera raised in IBDV-infected and, especially, that produced in IBDV plus IBV-infected chickens were supplemented, the percentage of phagocytosis and number of bacteria ingested per phagocyte were significantly (P < 0.05) less. These results indicate that although IBDV alone has the potential to markedly reduce opsonizing ability of antibody, this effect is significantly (P < 0.05) exacerbated by IBV infection.