The effects of avian leukosis virus subgroup J on broiler chicken performance and response to vaccination.

The effects of avian leukosis virus subgroup J (ALV-J) infection on meat-type chickens reared in a simulated commercial setting were evaluated. Each of three ALV-J isolates was evaluated with both simulated horizontal transmission (SHT) and simulated vertical transmission (SVT). Mortality, morbidity, disease condemnations, and feed conversions were increased and body weights at processing were decreased in ALV-J infected birds as compared to sham inoculated hatch mates. The adverse effects of ALV-J infection were more severe in birds exposed by SVT than in birds exposed by SHT. At 8 weeks of age response to vaccination for infectious bronchitis virus and Newcastle disease virus or prior exposure to a pathogenic reovirus was assessed in the ALV-J and sham inoculated broiler chickens by challenge studies. Although not statistically significant, an overall trend of decreased protection to challenge after vaccination, or prior exposure, was observed in the ALV-J inoculates as compared to sham inoculated hatch mates. Differences in vaccine response were most evident in groups inoculated with ALV-J by the SVT route.

Comparison of a putative second serotype of chicken infectious anemia virus with a prototypical isolate II. Antigenic and physicochemical characteristics.

A putative new serotype of chicken infectious anemia virus (CIAV) isolated from 17-wk-old broiler breeder pullets was compared with a known, previously characterized CIAV isolate, the Del-Ros strain. Physicochemical characteristics evaluated induded thermal stability, size, pH, and chloroform sensitivity. Physicochemically, CIAV-7 was identical to CIAV. The virus isolates were compared antigenically by enzyme-linked immunosorbent assay, virus neutralization, immunofluorescence assay, and western blot. All four serologic assays demonstrated that CIAV-7 is antigenically distinct from the Del-Ros strain of CIAV. Additionally, polymerase chain reaction (PCR) and Southern blot were used to determine if there were similarities in genome sequence between the two viruses. CIAV-7 could not be detected with CIAV-specific PCR primers or a with CIAV-specific probe by Southern hybridization.

Comparison of a putative second serotype of chicken infectious anemia virus with a prototypical isolate I. Pathogenesis.

CIAV-7 is a virus with similar pathogenic and physicochemical characteristics to, but antigenically distinct from, chicken infectious anemia virus (CIAV). The pathogenesis of CIAV-7 was evaluated in a comparative study with a representative isolate of CIAV, the Del-Ros strain. The pathogenesis of CIAV-7 was similar to Del-Ros on the basis of the clinical disease induced and gross and microscopic lesions, although CIAV-7 produced fewer and less severe lesions overall. A second comparative pathogenesis study was performed with Del-Ros and CIAV-7, both alone and in combination with infectious bursal disease virus (IBDV). In this study, the pathogenesis of CIAV-7 was similar to Del-Ros in clinical, gross, and microscopic lesions in the bone marrow. However, thymic lesions were less severe in CIAV-7-inoculated birds. The interaction between Del-Ros and IBDV was synergistic, whereas there was no observed potentiation of CIAV-7-induced disease by IBDV. Progeny from breeder flocks from several geographic locations in the eastern United States were challenged with CIAV-7 or Del-Ros to assess protection by maternal antibodies. Some progeny from all flocks had protection against CIAV-7 challenge, providing evidence for the presence of CIAV-7 in the field. Additionally, the number of birds protected against CIAV-7 or Del-Ros challenge varied within flocks, demonstrating that the agents are serologically distinct.

Genetic variation in susceptibility to Marek's disease in a commercial broiler population.

Two commercial broiler pure lines that were previously identified to differ in their susceptibility to Marek's disease (MD) were line-crossed to generate an F1 population. Eight F1 males were randomly mated to four or five F1 females to produce an F2 test population that would be segregating for genes affecting MD. All F2 progeny (four hatches) were pedigreed at hatch and placed in colony houses as nonvaccinated. At 5 days of age, they were challenged intraabdominally with MD virus RB1B. Clinical signs, mortality, and gross and microscopic lesions were recorded during the MD challenge. At 8 wk postchallenge, all remaining birds were euthanatized and necropsied. During the MD challenge of the first two hatches, we observed that several severely stunted broilers originated from certain families and the differences in body weight among birds appeared as early as 3 wk postchallenge. To confirm this observation, body weight at 6 wk postchallenge was determined for all surviving birds in hatches 3 and 4 (n = 242). Genetic variation in body weight among broiler sire families was apparent; the average body weight for males at this time was 2.07 kg, whereas with females, it was 1.87 kg. At least 12.2% of the broilers, including both sexes, weighed less than 1 kg ("severely stunted") at this time. The incidence of these growth-stunted birds within each broiler sire family ranged from 0 to 26% and for dam families, 0 to 60%. Correlation analyses between stunting and other MD-associated traits revealed that the incidence of stunting had a significant and positive association with paralysis (r = 0.50). Therefore, the data suggest that there may be a genetic component affecting body weight loss during MD infection. The genetic component is speculated to affect susceptibility to MD paralysis with an indirect effect on the body weight of birds. The significance of this finding is best exemplified by the identification of a broiler sire family with over 26% of its progeny affected by this MD-associated trait.

Chicken anemia virus.

Chicken anemia virus is commonly found in commercially produced chickens and has a worldwide distribution. It is difficult to inactivate thermally or with common disinfectants, which limits the utility of normal sanitization practices. The virus is important because of the disease it produces following transovarian transmission and because of its potential for inducing immunosuppression alone or in combination with other infectious agents. Control measures are directed at limiting vertical transmission and preventing coinfections with other lymphocidal agents.

Antigenic and S-1 genomic characterization of the Delaware variant serotype of infectious bronchitis virus.

A previously unrecognized infectious bronchitis virus (IBV) serotype, referred to hereafter as the Delaware variant (DE var), was isolated from commercial broiler chickens during a severe, widespread respiratory disease epornitic in the Delmarva peninsula region of the United States in January-March 1992. The DE var serotype was found to be antigenically unrelated by virus-neutralization (VN) test to nine reference IBV serotypes from North America. Additional VN tests indicated that the DE var isolates (DE/072/92, DE/121/ 92, DE/152/92, and DE/174/92) from broilers were fully or partially neutralized by monospecific antisera prepared against themselves and against two IBV field isolates (DE/492/90 and DE/903/90) recovered from a Delmarva commercial layer flock experiencing egg production losses in 1990. Antigenic relatedness values determined by VN indicated layer isolate DE/492/90 was more closely related to the broiler DE var isolates than was layer isolate DE/903/90. Cross-challenge tests performed in specific-pathogen-free chickens also demonstrated the antigenic similarity of the broiler (DE/072/92 and DE/174/92) and the layer isolates (DE/492/90 and DE/903/90), with heterologous strain protection values ranging from 55% to 100%. Protection values of DE var isolates vs. Massachusetts 41 and Arkansas DPI were considerably lower (0-60%). The S-1 gene of the US/DE/072/92 isolate of the DE var serotype was amplified by reverse transcription polymerase chain reaction, cloned, and sequenced. The DE var S-1 gene sequence was compared with the S-1 gene sequences of IBV serotypes from North America, Europe, and Australia. A dendrogram based on this analysis supported the conclusion that the DE var serotype is highly novel among IBV. A high degree of similarity (> 88%) was observed between the S-1 genes of the DE var broiler isolates (DE/072/92 and DE/174/92) and layer isolates (DE/492/90 and DE/903/90). These data, taken with the VN and cross-challenge results, establish a genetic as well as an antigenic link between the isolates from layers and broilers and indicate the DE var serotype was responsible for both infectious bronchitis outbreaks.

Interferon activity of mitogen-induced chicken splenic lymphocytes which do not express interferon mRNA.

Interferon activity was measured in media from virally infected chicken embryo fibroblasts and Concanavalin A-stimulated splenic lymphocytes using a viral inhibition assay. Both cell types produce interferon activity. A cDNA probe corresponding to a chicken interferon mRNA was used to probe Northern blots of RNA prepared from both cells. A single hybridizing species of 900 bases was detected in virally infected fibroblast RNA, but no hybridizing species was detected in the splenic lymphocytes. These results suggest that the interferon activity produced by lymphocytes is of different molecular origin than the corresponding activity produced by virally infected fibroblasts.

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.

Immune dysfunction following infection with chicken anemia agent and infectious bursal disease virus. II. Alterations of in vitro lymphoproliferation and in vivo immune responses.

To determine the functional impact of alterations in lymphocyte concentrations and ratios following infection with chicken anemia agent (CAA) alone or in combination with infectious bursal disease virus (IBDV) on the immune system of young chickens, in vitro lymphoproliferation assays and in vivo responses to vaccination with several common viral agents were assessed at various time intervals post-inoculation (PI). Concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM) stimulation of splenic lymphocytes (SPL) collected from control birds could not be detected until 10-14 days PI. Infection with CAA was characterized by significantly higher PWM stimulation of SPL at 17 days PI and significantly lower PWM stimulation of peripheral blood lymphocytes (PBL) at 14 days PI, compared with uninfected controls. Concanavalin A and PWM stimulation of SPL was significantly increased in birds inoculated with IBDV alone. Lymphocytes harvested from birds inoculated simultaneously with CAA and IBDV had significantly lower responses. Effects on humoral and cell-mediated immunity following CAA and/or IBDV were determined by evaluating vaccination responses to Newcastle disease virus (NDV), fowl pox virus (FPV) and infectious laryngotracheitis virus (ILTV) during the acute phase of CAA infection (2 weeks PI). Vaccination of birds 2 weeks following CAA infection at 1 day of age resulted in decreased protection against NDV (85.7%) and ILTV (7.1%) challenge compared with protection rates in control birds (100% and 53.3% respectively). Infectious bursal disease virus infection was associated with decreased protection against NDV (60%) only. Concomitant infection at 1 day of age resulted in a greater reduction in NDV challenge protection (33.3%), slightly decreased FPV protection (87.5%), increased numbers of persistent FPV vaccination lesions and increased protection against ILTV challenge (71.4%). Vaccination of birds 2 weeks following CAA infection at 2 weeks of age resulted in slightly decreased NDV humoral antibody, development of persistent FPV vaccination lesions (17%) and increased immunity to ILTV challenge compared with control birds (83.3% vs. 66.7%). Chickens inoculated with IBDV alone displayed a more severe depression in NDV antibody titers and only a slight decrease in ILTV protection. Vaccination following concomitant infection at 2 weeks of age resulted in a higher percentage of FPV persistent vaccination lesions (39%) and greatly enhanced immunity to ILTV challenge (100%).

The isolation and characterization of chicken anemia agent (CAA) from broilers in the United States.

A chicken anemia agent (CAA) isolated from commercial broilers in the United States was characterized in vivo and in vitro. When inoculated into susceptible 1-day-old chickens, the agent induced a severe bone marrow aplasia, thymic atrophy, multiple subcutaneous and intramuscular hemorrhages, and anemia, as evidenced by reduced hematocrits. Chickens derived from different breeder flocks and inoculated in ovo or at 1 day of age varied in their susceptibility to the CAA, with some flocks being highly susceptible, while others were almost totally resistant. This was true for both specific-pathogen-free and commercial chickens. The isolate was able to pass through a 50-nm-pore-size filter and was resistant to inactivation at 56 C for 30 minutes. It failed to agglutinate avian and mammalian erythrocytes and could not be propagated in conventional cell cultures. The physical and biological characteristics of the agent and the disease it induces indicate that it is similar to the CAA found in Japan and Europe.

The effects of age, route of exposure, and coinfection with infectious bursal disease virus on the pathogenicity and transmissibility of chicken anemia agent (CAA).

Specific-pathogen-free (SPF) chickens were inoculated with several different concentrations of chicken anemia agent (CAA) by the intra-abdominal, intratracheal, or oral routes. Based on lowered hematocrit values, the birds were most susceptible to CAA introduced by the intra-abdominal route. When SPF chickens were infected with infectious bursal disease virus (IBDV) at 1 day of age, they remained susceptible to CAA up to at least 21 days, whereas birds inoculated with CAA alone were susceptible only at 1 day of age. Infectious bursal disease virus introduced at 1 day of age also increased the susceptibility of birds to contact infection with CAA and resulted in increased mortality rates in CAA inoculates. The response of SPF birds to CAA infection varied following exposure at 1 day of age to two different strains of IBDV (STC and Variant-E). Chicken anemia agent contacts and inoculates infected with the Variant-E strain were affected 1 week earlier by CAA than by STC inoculates, as evidenced by depressed hematocrits. However, the total number of birds affected was similar for both the Variant-E and STC-inoculated chickens. Commercial broiler chickens inoculated at 1, 7, 10, and 14 days of age by non-parenteral routes with CAA or a combination of CAA and IBDV had mean hematocrits that were lower than controls. Several CAA-inoculated birds were considered anemic, with hematocrit values of 25 or less, while uninoculated birds remained within normal ranges.

Protection afforded infectious bronchitis virus-vaccinated sentinel chickens raised in a commercial environment.

The protective efficacy of three infectious bronchitis virus (IBV) vaccines for sentinel chickens raised with commercial Delmarva broiler chickens was evaluated during winter 1987. Specific-pathogen-free leghorn sentinel chickens were vaccinated with Massachusetts (Mass) alone, Mass and JMK, or Mass and Arkansas (Ark) combination live vaccines, or they remained unvaccinated. Four weeks post-vaccination, sentinels were placed on broiler farms at weekly intervals for 3 weeks corresponding to weeks 4, 5, and 6 of the broiler growing cycle. Vaccine efficacy was evaluated based on IBV reisolation attempts from tracheal swabbings following a 1-week field exposure period. Sentinel chickens vaccinated with Mass and Ark combination vaccine were best protected against IBV field challenge. Only four IBV isolations were made out of a 3-week total of 36 attempts, for an 11% isolation rate. IBV vaccines containing either Mass alone or Mass and JMK offered much lower levels of protection.

In vitro and in vivo characterization of avian Escherichia coli. I. Serotypes, metabolic activity, and antibiotic sensitivity.

Over a 2 1/2-year period (January 1981 to June 1983), 177 Escherichia coli isolates were obtained from 145 field-reared broiler flocks in the Delmarva peninsula, and 20 were obtained from clinically normal day-old hatchery chickens representing an additional 17 flocks in Delmarva. Ninety-one isolates obtained from the field-reared birds between 2 and 8 weeks of age were associated with complicated air-sac disease. Serotyping efforts demonstrated a predominance of O2, O35, and O78 serogroups and a large number of untypable isolates. More than 50% of the isolates in each of the three dominant serogroups were collected from broilers with colibacillosis, but they were never detected in the yolk-sac samples of clinically normal day-old hatchery chickens. In vitro biochemical characterization of the E. coli isolates revealed variable rates of carbohydrate fermentation and amino acid decarboxylation. No common characteristics appeared to be shared by the predominant serogroups isolated from clinically affected birds, although several serogroup-specific reactions were noted. The majority of the isolates were sensitive to chloramphenicol, gentamicin, nalidixic acid, ormethoprim-sulfadimethoxine, spectinomycin, neomycin, and ampicillin. About half of the isolates were sensitive to nitrofurantoin and the sulfa compounds. Less than 25% of the isolates were sensitive to streptomycin, erythromycin, tetracyclines, novobiocin, penicillin, bacitracin, and lincomycin.

In vitro and in vivo characterization of avian Escherichia coli. III. Immunization.

Broiler breeder hens were vaccinated once at 20 weeks or twice at 20 and 25 weeks of age with a formalin-inactivated oil-emulsion Escherichia coli bacterin composed of serogroups O2, O78, and O35. Serological responses as assessed by microagglutination documented an increase in serotype-specific antibody in vaccinated birds. Challenge of progeny from vaccinates and nonvaccinates with homologous E. coli demonstrated that maternally derived antibody could protect against mortality and/or lesions for as long as 2 weeks post-hatching.

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.

Effect of serial embryo passage of an Arkansas-type avian infectious bronchitis virus isolate on clinical response, virus recovery, and immunity.

Infectious bronchitis virus (IBV) Arkansas-type DPI strain (Ark DPI) was attenuated by serial passage in chicken embryos. Virus of passage 50 was less pathogenic for day-old maternally immune broiler-type chickens than virus of passage 10 or 25 as determined by clinical response to vaccination, virus isolation in respiratory (trachea) and nonrespiratory (kidney and cloaca) tissues, and weight-gain studies. Chickens vaccinated with virus of passage 10, 25, or 50 were at least 80% resistant to homologous virus challenge of the upper respiratory tract 4 and 6 weeks postvaccination. Serum antibody production by passage-50-vaccinated chickens was comparatively low at 4 weeks but at 6 weeks was similar to that in chickens vaccinated with virus of passage 10 or 25.

The immunization of broiler chickens against type C botulism.

Broiler chickens were inoculated with different amounts of a Clostridium botulinum type C toxoid at 1 or 14 or both 1 and 14 days of age. Immunity was assessed following challenge with type C botulism toxin at 3, 6, and 8 weeks of age. Protection induced by toxoid injection was affected more by time and number of inoculations than by the amount of toxoid administered. Single toxoid injections at one day of age furnished poor protection, whereas groups injected at 14 days of age were well protected at 6 and 8 weeks of age but not at 3 weeks of age. Variable results were observed in groups inoculated at both 1 and 14 days of age: immunity was evident in some groups following 3-, 6-, and 8-week toxin challenges.

Susceptibility of broiler chickens to Clostridium botulinum type C toxin.

As broiler chickens aged during the first eight weeks of life, they decreased in susceptibility to the lethal effects of Clostridium botulinum type C toxin. Eight-week-old broilers were 2,000 times less susceptible to the lethal effects of the toxin than hatched chicks. Different broiler crosses showed similar susceptibility to the lethal effects of type C toxin at seven weeks of age, but 2.5 times more toxin was required to produce lethality by oral inoculation than was required by subcutaneous inoculation.