Reversion to subgroup J avian leukosis virus viremia in seroconverted adult meat-type chickens exposed to chronic stress by adrenocorticotrophin treatment.

Chickens infected with subgroup J avian leukosis virus (ALV J) early in posthatch life develop viremia followed by a neutralizing antibody (Nab) response that may or may not be able to clear the viremia. Occasionally, chickens that do clear viremia by developing an efficient Nab response revert to viremia, and the factors responsible for this reversion are not clear. In this study, it was hypothesized that stress can cause seroconverted viremia-free chickens to revert to viremia. Adult (52-wk-old) male commercial meat-type chickens that were exposed to ALV J at hatch and had since cleared viremia and remained viremia-free for up to 40 wk, when subjected to chronic stress (for 14 days) induced by porcine adrenocorticotrophin (ACTH), reverted to viremia and cloacal shedding (2/6 [33%]). However, chickens that were contact-exposed to ALV J at 32 wk of age and had seroconverted failed to revert to viremia when subjected to similar chronic stress. Stress did not increase the susceptibility of adult meat-type chickens to ALV J infection by contact exposure. The lack of statistical significance due to the small sample size is a limitation of this study. However, in general, the results suggest that treatment of chickens with ACTH can cause reversion of viremia and cloacal shedding in ALV J-seroconverted adult male chickens that had been exposed to the virus at hatch, but not in chickens that were contact-exposed at 32 wk of age. The results warrant further studies with greater sample size to examine the role of stress in ALV J epidemiology.

[Molecular-genetic analysis of the field isolates of avian leucosis viruses in the Russian Federation].

Results of monitoring of different subtypes of avian leukosis virus (ALV) from commercial poultry farms in 14 regions of Russian Federation were discussed. Only three regions were found to be negative. ALV was detected in other 11 regions in 46-64% cases (for different regions). The phylogenetic analysis of the genomes for the 12 field isolates of ALV was carried out in different regions of Russian Federation. The isolates belong to different subtypes of the virus and form two large groups. The genomic differences between Russian and foreign isolates within each group range from 5% to 10%.

Subgroup J avian leukosis virus neutralizing antibody escape variants contribute to viral persistence in meat-type chickens.

We have previously demonstrated a high incidence of chickens with persistent viremia even in the presence of neutralizing antibodies (V+A+) against the inoculated parental virus in commercial meat-type chickens inoculated at hatch with subgroup J avian leukosis virus (ALV J) field isolates. In this study, we used an ALV J molecular clone, ADOL pR5-4, to determine the role of neutralizing antibody (NAb) escape mutants in maintaining a high incidence of viral persistence, namely, V+A+ infection profile in commercial meat-type chickens. Chickens were housed as a flock in a pen or housed in isolation in solitary Horsfall-Bauer units for testing for NAb escape variants. The emergence of NAb escape variants was evaluated by sequential autologous virus neutralization (VN) (between virus and antibody from the same sampling period) and heterologous VN (between virus and antibody from preceding and succeeding sampling periods). Sequential virus isolates and corresponding antisera from 18 chickens were examined by VN matrix. In all chickens, autologous virus isolates were not neutralized by corresponding antisera. However, some of these resilient autologous virus isolates were neutralized by antibodies from subsequent sampling intervals. Nucleotide sequence analysis of consecutive isolates from three individually housed chickens with V+A+ infection profile revealed distinct changes within the envelope region, suggesting viral evolution to escape the host immune response. These results demonstrate that the emergence of antibody escape variants in commercial meat-type chickens contributes to ALV J persistence.

Tumor latency in avian lymphoid leukosis.

Transfer of bursa cells from older chicken infected with lymphoid leukosis (LL) virus into young recipient chicks depleted of bursal lymphocytes by cyclophosphamide shortened the latency period for lymphoma development in recipient chickens by an amount equivalent to the age of the donor. Chickens were crosses between males of the Regional Poultry Research Laboratory, East Lansing, Michigan, inbred line 151, subline 5, and females of inbred line 7, subline 2. In a further experiment with intact infected chicks, artificial metastasis at various ages by surgical bursectomy and inoculation of bursa cells into the bloodstream of the host did not shorten the latency period. These results suggest that latency in avian LL is a property of target B-cells and is unrelated to maturational events of the host physiology.

Effects of T-2 Toxin on Turkey Herpesvirus-Induced Vaccinal Immunity Against Marek's Disease.

T-2 toxin, a very potent immunotoxic Type A trichothecene, is a secondary metabolite produced primarily by Fusarium spp., which grows on cereal grains and can lead to contaminated livestock feed. Repeated exposure to T-2 toxin has been shown to cause immunosuppression and decrease the resistance of exposed animals to a variety of infectious diseases; however, the effects of T-2 toxin on Marek's disease (MD) vaccinal immunity have not been reported. Four trials were conducted to determine the effects of T-2 toxin on vaccinal immunity against MD. Day-old, white leghorn chicks of Avian Disease and Oncology Laboratory line 15I5 × 71 were treated daily for 7 days via crop gavage with T-2 toxin at a sublethal dose of 1.25 mg/kg body weight. Treated and untreated chicks were also vaccinated with turkey herpesvirus (HVT) at hatch and were challenged with the JM strain of MD virus (MDV) at 8 days of age. Chickens were tested for HVT viremia at 1 wk postvaccination immediately before challenge, and for HVT and MDV viremia at 3 wk postchallenge. Chickens were observed for the development of MD lesions and mortality within 8 wk of age. T-2 toxin significantly reduced body weight and titers of HVT viremia within 7 days after hatch. T-2 toxin shortened the incubation period for the development of MD lesions and mortality, but only in unvaccinated chickens. The percent MD protection in T-2-toxin-treated, HVT-vaccinated chickens ranged from 82% to 96% and was comparable to that in HVT-vaccinated untreated control chickens (89%-100%). The data suggest that exposure of chickens to sublethal doses of T-2 toxin for 7 consecutive days after hatch may influence the development of 1) HVT viremia; and 2) MD lesions and mortality, but only in unvaccinated chickens.

Isolation and identification of avian leukosis viruses: a review.

Avian leukosis virus (ALV) is the most common naturally occurring avian retrovirus that can cause a variety of neoplastic disease conditions in chickens. In addition to causing neoplasia, ALV is known to be associated with reduced productivity and other production problems in affected flocks. Biological and molecular assays for the detection of ALV are very useful in identification and classification of new isolates, safety testing of vaccines and in testing pathogen-free and other breeder flocks for freedom of virus infection. However, such assays are not particularly helpful in the diagnosis of virus-induced neoplastic disease of poultry, as avian oncogenic viruses are widespread and infection in the absence of tumor formation is common. Current technology based on molecular and antigenic characteristics of the virus is being used to develop more sensitive and specific procedures for isolation and identification of ALV. This review is primarily focused on the discussion of current technology most commonly used in isolation and identification of ALV associated with clinical disease in chickens.

Reduction of horizontal transmission of avian leukosis virus subgroup J in broiler breeder chickens hatched and reared in small groups.

Transmission of avian leukosis virus, subgroup J (ALV-J), from donor chickens inoculated as embryos to simulate congenital infection to uninfected hatchmates was studied in two strains of commercial broiler breeder chickens. Chicks of two commercial lines free of ALV-J became infected when hatched (1/2 lots positive) or reared (8/8 lots positive) in direct physical contact with ALV-J-infected donors. Infection also occurred when chicks were exposed in the hatchery to ALV-J-infected donors by cloacal swab transfer (2/2 lots positive), needle transfer during subcutaneous inoculation (2/2 lots positive), or ingestion of infected meconium (2/2 lots positive). However, transmission was delayed or prevented by wire partitions in the hatcher and rearing of small groups in cubicles, and rarely (1/10 lots positive) resulted from short-term direct or indirect contact. In a simulated field test, a flock of 503 broiler breeder chickens with an initial embryo infection rate of 4.6% was hatched and reared as 48 small groups to 4 weeks of age. Groups were tested at hatch and at 3 weeks, and 14 infected groups were eliminated. This flock tested negative for ALV-J infection from 4 to 32 weeks and did not transmit infection to progeny or develop tumours. A control group of 377 chickens with a similar initial infection rate was hatched and reared as a single group. This control flock transmitted virus to 5.7% of its progeny and about 5% of the hens developed tumours. The small-group hatching and rearing practices employed in these studies allowed for the accurate identification and removal of groups containing chickens infected prior to hatching and prevented horizontal transmission of ALV-J between uninfected and infected groups for at least 4 weeks. More importantly, application of these procedures successfully eradicated ALV-J in a single generation under laboratory conditions. This suggests that similar procedures could be a valuable adjunct to virus eradication programmes in the field.

Avian leukosis virus (ALV) infection, shedding, and tumors in maternal ALV antibody-positive and -negative chickens exposed to virus at hatching.

Chickens highly susceptible to avian leukosis virus (ALV) infection and tumors, with and without ALV subgroup A maternal antibody (MAB), were infected with a field strain of ALV subgroup A at hatching. Viremia, antibody development, cloacal and albumen shedding, and tumors in chickens with MAB (MAB+) were compared with those in chickens lacking MAB (MAB-). At 18 weeks of age, the incidence of viremia was significantly lower in MAB+ chickens than in MAB- chickens; further, MAB significantly reduced the proportion of tolerantly infected (viremic antibody-negative) chickens. Cloacal shedding of ALV at 22 weeks of age and shedding of ALV group-specific (gs) antigen in albumen of eggs from all laying hens at 30-32 weeks of age were significantly lower in MAB+ hens than in MAB- hens. The incidence of ALV-induced tumors was lower in MAB+ chickens than in MAB- chickens, significantly so in one of three trials conducted. These results suggest that MAB may influence the development of viremia, antibody, and shedding of ALV following massive exposure to virus at hatching.

Hemorrhagic enteritis of turkeys: influence of maternal antibody and age at exposure.

The effect of maternal antibody (MAB) to hemorrhagic enteritis (HE) on the response of turkeys to infection with virulent and avirulent strains of HE virus (HEV) was examined. The influence of age at exposure and treatment with HEV antibody on development of clinical HE also was studied. MAB protected poults from clinical HE for up to 6 weeks of age. MAB also interfered with vaccination against the disease for at least 5 weeks after hatching, as indicated by absence of HEV antigen in spleens and by poor seroconversion at 6 days and at 3 weeks post-vaccination, respectively. The incidence of clinical HE in MAB-negative poults was significantly higher in poults inoculated with virus at 15 days of age or older than in poults inoculated at 1-13 days of age. Further, MAB-negative poults embryonally inoculated with virulent or avirulent strains of HEV did not develop disease; these poults developed antibody and resisted challenge with virulent virus at 6 weeks of age. Poults treated with HE antibody within 1 hour of challenge or at 1, 3, or 5 weeks before challenge with virulent virus were protected against lesions and mortality induced by HEV. These results suggest that MAB may influence susceptibility of turkeys to infection with HEV for at least 5 to 6 weeks after hatching, unlike the case with most other viral infections of poultry. The results confirm that early age resistance to clinical HE is independent of MAB and suggest that such resistance persists for up to 13 days of age. The data also suggest that turkeys lacking MAB can be immunized against HE by embryo vaccination.

Response of B congenic chickens to infection with infectious bursal disease virus.

Six congenic lines of chickens that differ from the parental inbred line RPRL-15I5 for genes in the major histocompatibility (B) complex were used to study the influence of the B haplotypes on the response of chickens to infection with virulent infectious bursal disease virus (IBDV) at 1 day or 4 weeks of age, and on the antibody response to vaccination with live or inactivated oil-emulsion (OE) IBDV vaccines at 7 weeks of age. IBDV-induced immunodepression and lesions in the bursa, spleen, and thymus in chickens infected with virus at 1 day of age were of the same degree of severity, regardless of line of chickens used. The response of blood cells to the mitogens phytohemagglutinin-M and concanavalin A was elevated in chickens infected with IBDV at 1 day of age. In an experiment conducted to study the effect of the B haplotype on IBDV infection in 4-week-old chickens, B congenic line C-12 (B12B12) showed the highest susceptibility to clinical IBD, with mortality of 79%. No detectable difference in the serological response to vaccination with live or OE IBDV vaccines was noted among chickens of various congenic lines. We conclude that the B haplotypes may influence IBDV-induced mortality, but not immunodepression or severity of lesions in lymphoid organs, or the antibody response to live or OE IBDV vaccines.

Further studies on in vitro and in vivo assays of hemorrhagic enteritis virus (HEV).

Isolation of hemorrhagic enteritis virus (HEV) from spleens of infected turkeys in the MDTC-RP19 lymphoblastoid cell line was compared with detection of HEV antigen in the same spleens using the agar gel precipitation (AGP) test. A concordance of 80% was found between the two assays. Virus isolation had a sensitivity of 84% and a specificity of 88% compared with the AGP test. RP19 cells were also susceptible to infection with several other avian adenoviruses, but such infection was easily differentiated from that of HEV by a fluorescent-antibody (FA) test. Turkeys required 10(2) tissue-culture-infectious doses (TCID) to develop HE-specific lesions and 10(5) TCID to be killed. On the other hand, as little as 10 TCID of apathogenic HEV protected the poults against challenge with virulent HEV. The enzyme-linked immunosorbent assay (ELISA) for detection of HEV antibody was improved by using virus-infected RP19 cells as antigen. The ELISA appears to be more sensitive than the serum-neutralization test.

Pathogenesis of infectious bursal disease in chickens infected with virus at various ages.

Chickens free from infectious bursal disease (IBD) maternal antibody were inoculated with a virulent strain of IBD virus at 1, 5, or 11 weeks of age. Chickens inoculated at 5 weeks developed severe clinical signs and had reduced levels of serum complement within 2-4 days postinoculation, but those inoculated at 1 or 11 weeks did not. However, at 1, 2, 4, and 8 days postinoculation, the rate of virus recovery from different tissues, severity of microscopic lesions, and frequency of detection of viral antigens in lymphoid organs of chickens inoculated at 5 weeks were comparable to those of chickens inoculated at 1 or 11 weeks of age. These findings suggest that age resistance to clinical manifestations of IBD is probably independent of the ability of virus to replicate and induce lesions in the host.

Comparative evaluation of in vitro and in vivo assays for the detection of reticuloendotheliosis virus as a contaminant in a live virus vaccine of poultry.

Indirect immunofluorescence (IFA), polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) were used for detection of reticuloendotheliosis virus (REV) as a contaminant in a live virus fowl pox (FP) vaccine of poultry. A FP vaccine known to be contaminated with REV was tested by in vitro and in vivo assays in chicken embryo fibroblasts (CEFs) and day-old specific-pathogen-free (SPF) chicks, respectively. Using in vitro assays, IFA and PCR were more sensitive than ELISA in detection of REV in CEFs inoculated with REV-contaminated FP vaccine. However, when the vaccine was tested by in vivo assays using SPF chickens, the sensitivity of ELISA was comparable with that of IFA and PCR. Antibody to REV was not detected in SPF chickens within 4 wk postinoculation with REV-contaminated FP vaccine at hatch. Filtration of vaccine to eliminate vaccine virus from the inoculum before testing in CEFs resulted in a significant reduction in the frequency of REV detection by PCR or IFA. The data suggest that the sensitivity of IFA, PCR, and ELISA depends on the concentration of REV in the vaccine and that in vivo assays of vaccines for contamination with REV should include a test for virus because a negative antibody test may be misleading.

Isolation and some characteristics of a subgroup J-like avian leukosis virus associated with myeloid leukosis in meat-type chickens in the United States.

Several subgroup J-like avian leukosis viruses (ALV-Js) were isolated from broiler breeder (BB) and commercial broiler flocks experiencing myeloid leukosis (ML) at 4 wk of age or older. In all cases, diagnosis of ML was based on the presence of typical gross and microscopic lesions in affected tissues. The isolates were classified as ALV-J by 1) their ability to propagate in chicken embryo fibroblasts (CEF) that are resistant to avian leukosis virus (ALV) subgroups A and E (C/AE) and 2) positive reaction in a polymerase chain reaction with primers specific for ALV-J. The prototype strain of these isolates, an isolate termed ADOL-Hc1, was obtained from an adult BB flock that had a history of ML. The ADOL-Hc1 was isolated and propagated on C/AE CEF and was distinct antigenically from ALV of subgroups A, B, C, D, and E, as determined by virus neutralization tests. Antibody to ADOL-Hc1 neutralized strain HPRS-103, the prototype of ALV-J isolated from meat-type chickens in the United Kingdom, but antibody to HPRS-103 did not neutralize strain ADOL-Hc1. On the basis of both viremia and antibody, prevalence of ALV-J infection in affected flocks was as high as 87%. Viremia in day-old chicks of three different hatches from a BB flock naturally infected with ALV-J varied from 4% to 25%; in two of the three hatches, 100% of chicks that tested negative for virus at hatch had evidence of viremia by 8 wk of age. The data document the isolation of ALV-J from meat-type chickens experiencing ML as young as 4 wk of age. The data also suggest that strain ADOL-Hc1 is antigenically related, but not identical, to strain HPRS-103 and that contact transmission of ALV-J is efficient and can lead to tolerant infection.

Efficacy and safety of a cell-culture live virus vaccine for hemorrhagic enteritis of turkeys: laboratory studies.

Poults free from hemorrhagic enteritis (HE) antibody were vaccinated by gavage at 1 day or 2 weeks of age with a live HE vaccine virus that had been propagated in a Marek's disease (MD)-induced B-lymphoblastoid cell line of turkey origin. Vaccinated and unvaccinated poults were challenged with a virulent HE virus at various times postvaccination. One hundred tissue-culture-infectious doses of the vaccine virus per poult were sufficient to induce a serological response as well as to protect poults against HE lesions and mortality. Vaccinated poults were protected against the disease as early as 1 week and as late as 8 weeks PV. The vaccine was efficacious by several routes of application. The vaccine virus spread horizontally from vaccinated to contact-exposed poults, as indicated by seroconversion and resistance of contact-exposed poults to challenge. The vaccine had no detectable harmful effects on the humoral immune response to particulate antigens or on weight gain of vaccinated poults. The vaccine proved to be free from MD virus, as indicated by the absence of MD lesions and antibody in 8-week-old chickens inoculated intra-abdominally with the vaccine at hatching. These findings indicate that the cell-culture-propagated HE vaccine is efficacious and safe.

Studies of reticuloendotheliosis virus-induced lymphomagenesis in chickens.

Seventy-three percent of chickens inoculated with the chick syncytial strain of reticuloendotheliosis virus (REV) at hatching developed lymphomas by 39 weeks of age. Neonatal treatment with cyclophosphamide or surgical bursectomy at 2, 4, 8, or 12 weeks of age significantly (P less than 0.01) reduced lymphoma development. In a further experiment, surgical bursectomy of REV-infected chickens followed by intravenous inoculation of the chickens with a single cell suspension of their own bursa cells at 2, 4, 9, or 13 weeks of age resulted in lymphoid tumors in chickens treated at 9 or 13 weeks but not in chickens treated at 2 or 4 weeks of age. Furthermore, this treatment did not shorten the incubation period for lymphoma development. These findings argue very strongly that transforming target cells are primarily in the bursa of Fabricius. The data also suggest that a minimum residency of 4 weeks in the bursa is required for infected bursa cells to become transformed. Therefore, lymphomagenesis induced by REV in chickens appears similar to that induced by the avian leukosis virus group.

Propagation of virulent and avirulent turkey hemorrhagic enteritis virus in cell culture.

Virulent and apathogenic isolates of turkey hemorrhagic enteritis virus (HEV) were successfully propagated in lymphoblastoid cell lines of turkey origin, whereas spleen and kidney cell cultures from HEV-infected turkeys failed to replicate the virus. The lymphoblastoid cell lines used were MDTC-RP16 and MDTC-RP19, which were previously established from tumors induced by Marek's disease virus in turkeys. Virus replication followed co-cultivation of lymphoblastoid cells with spleen cells from HEV-infected turkeys. Virus replication was demonstrated by immunofluorescence, by agar-gel-precipitin tests, and by electron microscopy. Supernatant fluid of cultures infected with virulent HEV caused death and specific lesions in turkey poults. Poults vaccinated with apathogenic HEV were protected against death and lesions after challenge with pathogenic HEV, which was recovered from infected cultures. The MDTC-RP19 cell line appeared far more susceptible than the MDTC-RP16 cell line to infection with HEV.

Evidence for bursal involvement in the pathogenesis of hemorrhagic enteritis of turkeys.

The pathogenesis of hemorrhagic enteritis in turkey poults infected with hemorrhagic enteritis virus (HEV) at 3 days or at 2 or 5 weeks of age was compared with pathogenesis in poults that had been chemically bursectomized neonatally and exposed to cell-culture-propagated virus at 2 or 5 weeks of age. Conventional poults exposed to HEV at 2 or 5 weeks developed clinical disease, and mortality ranged from 38% to 100%. In addition to the splenic and intestinal lesions usually seen with HEV infection, the pancreas, bursa of Fabricius, and thymus were also affected. In contrast, although they were free from detectable maternal antibody, poults infected with HEV at 3 days of age failed to develop clinical disease or mortality; however, virus was demonstrated by histological and electron microscopic examinations in spleens of these poults. Neonatal chemical bursectomy completely prevented the clinical signs, gross lesions, and mortality induced by HEV in poults at 2 or 5 weeks of age. These findings strongly suggest that an intact bursa is necessary for HEV to induce disease in turkeys.

Influence of maternal antibody on avian leukosis virus infection in White Leghorn chickens harboring endogenous virus-21 (EV21).

Slow-feathering (SF) white leghorn dams harboring the endogenous viral gene ev21, which encodes for complete endogenous virus-21 (EV21), and rapid-feathering (RF) dams lacking EV21 were immunized with a live field strain of avian leukosis virus (ALV) subgroup A. One group of SF dams and one group of RF dams were not immunized and were maintained to produce chicks lacking maternal ALV antibody. When the SF dams were crossed with line 15B1 males, the resulting male progeny were SF, EV21-positive, and the females were RF, lacking EV21 or congenitally infected with EV21. EV21-positive and -negative progeny of immunized and unimmunized SF and RF dams were exposed to ALV at hatching. Viremia, antibody development, cloacal shedding, and tumors in chickens lacking EV21 were compared with those in chickens with EV21. Congenital transmission of EV21 from SF dams to RF female chicks was significantly higher in immunized dams than in unimmunized dams. Maternal ALV antibody delayed infection with ALV and reduced viremia and cloacal shedding of virus in progeny. The effect of maternal antibody on ALV infection was much more pronounced in progeny lacking EV21 than in progeny harboring EV21. The data suggest that the development of ALV infection and tumors may be influenced by status of infection with EV21 and by the immune status of dams.

Some observations on the response of ring-necked pheasants to inoculation with various strains of cell-culture-propagated type II avian adenovirus.

The response of ring-necked pheasants to inoculation with three strains of cell-culture-propagated type II avian adenovirus was examined. Marble spleen disease (MSD) virus of pheasants and both avirulent and virulent strains of hemorrhagic enteritis virus (HEV) of turkeys all induced typical gross and microscopic splenic lesions of MSD; neither MSD-associated lung lesions nor mortality were noted in inoculated pheasants, regardless of strain of virus used. Pheasants inoculated with a cell-culture-propagated avirulent strain of HEV were properly immunized against challenge with virulent HEV, as indicated by seroconversion and by protection against virus-induced splenic lesions. We conclude that these strains of type II avian adenovirus are comparable in pathogenicity for pheasants and cannot be distinguished. Further, absence of MSD-associated lung lesions and mortality in pheasants maintained under controlled laboratory conditions suggest that other environmental factors are probably involved in induction of such lesions and mortality in field cases of MSD.