Big liver and spleen disease of broiler breeders

Five adult broiler breeders were inoculated with faeces and tissue extracts from birds with big liver and spleen (BLS) disease. Five birds were placed in contact with the inoculated birds. After 8 weeks all inoculated and four of the five in-contact birds were shown to be infected as evidenced by the detection of an immune response and/or the demonstration of BLS antigen in spleen and tissue smears, by an immunofluorescence test and by agar gel immunodiffusion. Using appropriate antisera BLS antigen was found mainly in phagocytic liver cells and in splenic macrophages and dendritic cells. Fewer cells containing antigen were found in the kidney. Double staining of liver and spleen tissue for BLS antigen and chicken immunoglobulin indicated that many cells were positive for both. It is possible that BLS antigen in most cells may represent phagocytosed material rather than a replicating agent. If so, while this could explain the failure to detect virus-like particles, it implies that the primary site of replication of the BLS agent and its nature remain to be established.     

An outbreak of lymphomas in commercial broiler breeder chickens vaccinated with a fowlpox vaccine contaminated with reticuloendotheliosis virus

Gross and microscopic examinations of affected tissues from chickens of two commercial broiler breeder flocks aged 27 and 31 weeks revealed lesions of visceral lymphomas with bursal involvement in some chickens. Reticuloendotheliosis virus (REV), but not avian leukosis virus (ALV), was isolated from blood of affected chickens. Furthermore, DNA extracted from tumours tested positive for REV, but not for ALV or Marek's disease virus by polymerase chain reaction (PCR) test. Attempts to determine the source of REV infection included testing a commercial fowlpox (FP) vaccine used to immunize flocks at 7 days of age. Chicken-embryo fibroblasts inoculated with the FP vaccine tested positive for REV by PCR and immunofluorescent tests. REV was also isolated from plasma of pathogen-free chickens experimentally inoculated with FP vaccine at hatch; two of eight (25%) inoculated chickens developed lymphomas by 34 weeks of age. Antigenic characterization of REV isolated from commercial broiler breeder chickens and from FP vaccine, using monoclonal antibodies, revealed that both isolates belong to subtype 3 of REV. The data represent the first report of an outbreak of REV-induced lymphomas in commercial chickens. The data also indicate that the source of REV infection is an REV-contaminated commercial FP vaccine.     

Susceptibility of thymocytes for infection by chicken anemia virus is related to pre- and posthatching development.

To investigate the age-dependent mechanism of susceptibility for chicken anemia virus (CAV) infection, we inoculated embryos and chickens of ages between day 9 of embryonic development and day 28 after hatching with CAV. Chicken embryos inoculated at days 9 and 11 of development showed no CAV-infected cells in the thymus, nor in other lymphoid organs. Many CAV-infected cells were detected in the thymic cortex of all chicken embryos inoculated at days 13 and 16 of development and of all chickens inoculated 1, 3, and 7 days after hatching. All embryos and chickens that contained CAV-infected cells in the thymus also contained CAV-infected cells in the bone marrow, but not in the bursa of Fabricius or the spleen. In chickens inoculated at days 14 and 21, only few CAV-infected cells were detected in the thymus, whereas these cells were not detected in thymi of 28-day-old inoculated chickens. Depletion of the thymic cortex was only detected in chickens inoculated from day 16 of embryonic development till day 21 after hatching. Only hematocrit values of the chickens inoculated 1 and 3 days after hatching were below normal. The rationale for the simultaneous susceptibility of cells of the T-cell lineage and cells of the erythrocyte lineage is discussed. As far as the thymus is concerned, the absence of clinical and microscopical signs of CAV infection in older chickens and the inability of CAV to infect embryos at days 9 and 11 of embryonic development may be caused by a lack of susceptible thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Significance of paroviruses, entero-like viruses and reoviruses in the aetiology of the chicken malabsorption syndrome

Specific-pathogen-free White Leghorn chickens and commercial broilers were inoculated orally at 1 day of age with different intestinal preparations containing a chicken parvovirus, an entero-like virus associated with a reovirus from field materials, or the entero-like viruses and reovirus alone. Despite viral multiplication in inoculated birds, no clinical signs or growth retardation were observed in SPF and broiler chickens infected with the reo or parvo viruses. Abnormal faeces and reduction in weight gains were observed after infection with the field materials and the entero-like viruses. Some easily sedimentable particles could be involved with the entero-like virus in the aetiology of ranting syndrome. Proventriculitis was present in chickens inoculated with one of the field materials and with the entero-like virus isolated from that material. Specific-pathogen-free White Leghorn chickens were as susceptible as commercial broiler chickens to weight gain depression after oral inoculation with crude homogenates at 1 day of age.     

Kinetics of serum antibody responses in broiler chicks against Cryptosporidium baileyi

The kinetics of serum antibody responses in broiler chickens against Cryptosporidium baileyi were studied. Broilers were inoculated intratracheally with 250,000 C. baileyi oocysts at 1, 7, or 14 days of age. Antibody was quantified by an enzyme linked immunosorbent assay. Anti-cryptosporidial serum immunoglobulins (IgM and IgG) were detected 9 days post-inoculation (DPI) in birds inoculated at 1 or 7 days of age with oocysts and 4 DPI when 14-day-old birds were inoculated. Results also reaffirmed age related susceptibility, with day-old birds being more susceptible than 7-day, and 14-day-old birds were not susceptible to clinical disease. The susceptibility to infection correlated with the amount and duration of the IgM response. Day-old inoculated birds developed a higher, longer-lasting response than 7 or 14-day-old infected birds.     

Experimental infections with rifampicin-resistant Clostridium perfringens strains in broiler chickens using isolator facilities.

Experimental infection studies were carried out on the ability of three Clostridium perfringens type A rifampicin-resistant strains to colonize the intestinal tract of broiler chickens kept in isolators from 1-day-old. Various doses of C. perfringens were given orally at 22 days, 9 days or at 1 day old. At 22 days none of the strains, given in doses of approximately 10(10) colony-forming units, caused mortality or clinical necrotic enteritis. None was able to colonize the intestine permanently and all were eliminated within 9 days. One strain given to groups of 9-day-old birds was recovered only from those receiving high doses, but for no longer than 13 days. In chicks infected at 1-day-old there was transient colonization up to 15 days, and the most persistent colonization was in a group given a fresh broth culture of unwashed cells, including extracellular products. Test strains were rapidly replaced by naturally occurring strains of C. perfringens in all groups but they persisted for considerably longer in chickens inoculated at 1-day-old or at 9 days than those at 22 days, indicating a possible resistance to colonization with increasing age. The findings emphasize the difficulties of establishing a reproducible model for infection with C. perfringens in broiler chickens.     

Experimental infection of chickens with an Australian strain of reticuloendotheliosis virus. 2. Serological responses and pathogenesis

Fifteen SPF chickens were inoculated with an Australian strain of reticuloendotheliosis virus (REV) at 1 day of age and five uninoculated chickens were readily infected by horizontal spread from this group. Antibody detectable by the immunofluorescent antibody (IFA) test developed 3 to 6 weeks after infection, and usually persisted for 20-35 weeks, with maximum titres (40-1280) at 8 to 13 weeks. Agar gel precipitin (AGP) reactions developed more slowly and were variable in duration, the highest proportion of positive reactions being detectable 8 to 13 weeks after infection and persisting for 8 to 30 weeks. Infectious REV was readily detected in the plasma and serum of inoculated chickens 6 weeks after infection and a non-infectious REV antigenaemia usually persisted for at least a further 7 weeks, in the presence or absence of antibody. Development of a detectable REV viraemia was strongly associated with poor body development and premature mortality among the inoculated chickens. In two inoculated chickens which failed to develop detectable serological reactions, a REV viraemia occurred which persisted throughout life. At autopsy, REV was re-isolated from the kidneys of most of the inoculated chickens and from the reproductive and intestinal systems of two birds 22 and 56 weeks after infection.     

Study on experimental infections of Spiroplasma from the Chinese mitten crab in crayfish, mice and embryonated chickens

Tremor disease (TD) of the Chinese mitten crab Eriocheir sinensi has become a serious disease in the Chinese freshwater culture industry in recent years. The agent, belonging to Spiroplasma, was purified from yolk and allantoic fluids of chicken embryos and was then inoculated into the body of crayfish, the abdominal cavity of ICR mice and the allantoid of chicken embryos, respectively. Thirty-eight days after the inoculation, the mice and crayfish were dissected and their tissues sampled and observed with both the light and electron microscope. No infection was detected in mouse or crawfish tissue. But when the different tissues from the inoculated embryonated chickens were tested, the agent was detected only in the brain of embryonated chickens. This indicated that the agent represented a neurotropic characteristic in embryonated chickens, just as it had in the crab. This infective characteristic may be due to the development and maturation of host immunity.     

Response of meat-type chickens to infection with RAV-1 avian leukosis virus

Meat-type and White Leghorn chickens were inoculated with the RAV-1 strain of avian leukosis virus at 1 day of age and the severity of infection was assessed by clinical illness, haematology and post-mortem findings. The following were examined from selected birds: histological section for chronic mononuclear myocarditis, immunohistochemically-stained sections of myocardium, spleen, bursa of Fabricius and kidney for group-specific viral antigen, and ultrathin sections of these tissues for virus particles by electron microscopy. The experiment was terminated at 115-122 days. Approximately one-third of the 52 inoculated White Leghorns appeared anaemic at 3-4 weeks of age whereas there was no evidence of anaemia in 177 inoculated meat-type birds or in uninoculated birds of either type. Haemograms confirmed these observations. The first tumour found was a myelocytoma and it was in a meat-type bird at 65 days. Of the 151 meat-type birds of the inoculated group alive at 65 days, 8.5% developed nephroblastomas, but there were no cases of lymphoid leukosis. Myocarditis and virus replication in myocardium were usually more extensive in chickens that developed nephroblastomas than in those without such lesions. In 17 uninoculated control chickens examined between 26 and 122 days of age there were no virus particles or lesions in myocardium.     

Variation in tolerance induction and oncogenicity due to strain of avian leukosis virus

Chickens were inoculated as embryos or orally at hatching with various doses of four strains of avian leukosis virus (ALV) subgroup A (RAV-1, RPL40, RPL41 and RPL42). Viraemia, antibody and tumours in chickens of various groups were compared; ALV shedding was determined, but only in chickens inoculated with virus at hatching. Results indicate that 95% to 100% of chickens embryonally inoculated with 105 infectious units of virus were viraemic at hatching, regardless of the strain of virus used. However, the incidence of viraemia in groups of chickens embryonally inoculated with 100 infectious units of virus varied, depending on the strain of virus, from 5% to 72%. All embryonally inoculated chickens, that had detectable virus at hatching and survived to 16 weeks of age were immunologically tolerant to the virus. ALV-induced tumours ranged from 4% to 47% depending on the strain of virus. Chickens inoculated orally at hatching did not develop immuno-logical tolerance to the virus; 13% to 76% of these chickens, depending on the strain of virus, had antibody by 18 weeks of age. ALV shedding in albumen of eggs or cloacal swabs at 42 weeks of age varied from 7% to 9%. Data from this study indicate that the strain of ALV may influence induction of immunological tolerance and tumours in embryonally inoculated chickens and induction of antibody in nontolerantly infected chickens. The data also suggest that chickens that are viraemic at hatching are probably incapable of breaking tolerance and developing antibody.     

Experimental transmission of hydropericardium syndrome and protection against it in commercial broiler chickens

Liver collected aseptically from broiler chickens suffering from natural outbreaks of hydropericardium syndrome was homogenised in phosphate buffered saline (1:5) or normal saline (1:10). A dose of 0.25 ml of 800 g supernatant (inoculum S) in the first case or homogenate without centrifugation (inoculum T) in the second case was injected subcutaneously into 12- to 15-day-old broiler chickens. Lesions similar to natural outbreaks of the syndrome appeared in 2 to 5 days. Inoculum S inactivated with 0.1% formalin for 24 h (Vac S) or inoculum T inactivated with 0.5% formalin for 72 h (Vac T) was injected into commercial broiler chickens at 10 or 15 days of age. The birds were challenged with inoculum S or inoculum T at 20 or 25 days of age. Experimentally both vaccines gave satisfactory protection, but under field conditions, Vac T was superior.     

Maternally derived antibody--effect on susceptibility of chicks to infectious bursal disease

Levels of infectious bursal disease (IBD) antibody in young chicks from infected parents were measured using the quantitative gel diffusion precipitation test (QGPT). The decay of maternally derived antibody was approximately linear with a half life of about 4 days. Groups of chickens from this flock were inoculated with infectious bursal agent. All chicks which were inoculated at 8 days of age were resistant to infection and all inoculated at 17 days of age were susceptible. About half of the chicks inoculated at 14 days of age were susceptible to infection. One hundred chicks hatched from 10 hens showed considerable variation in their QGPT titres. Precipitin levels in one-day-old chicks from the same parent showed little variation, and were usually one or 2 log(2) lower than their dams.     

Age related resistance to avian leukosis virus. II. Influence of age at inoculation on mortality and congenital transmission

Six age groups of specified pathogen free White Leghorn chickens housed in the same filtered air, positive pressure building, were inoculated at 1 day, 2, 4, 6, 8 and 10 weeks of age respectively, with a mixture of leukosis viruses of subgroups A and B. Birds which died during the experiment were examined for gross and microscopical lesions. The incidence of lymphoid leukosis (LL) in the various groups was inversely proportional to the age of first virus exposure, Le., mortality was 62.5% in the group inoculated at 1-day-old and decreased to zero in the group inoculated at 10 weeks of age. In the age group inoculated at 8 weeks, only one chicken succumbed to the disease. Congenital transmission of leukosis virus (LV) was detected only in chickens inoculated immediately after hatching or at 2 weeks of age. In the latter group, all leukosis virus positive embryos were derived from one hen only. A seventh age group, housed in a separate filtered air, positive pressure building, served as uninoculated controls. Challenge infection was performed on the same day with chickens of all groups at ages varying from 59 to 71 weeks. Testing of pooled embryo extracts collected between 1 and 15 weeks post challenge did not change the virus shedding pattern. Even the chickens of the control group which were challenged (for the first time) at 71 weeks of age remained negative for congenital transmission. The results of this experiment show that the chickens inoculated with LV at 4 weeks of age or older had a low incidence of LL (decreasing to zero when the chickens were inoculated at 10 weeks of age) but did not congenitally shed virus.     

Effects of reticuloendotheliosis virus on the response of chickens to infectious laryngotracheitis virus

Effects of reticuloendotheliosis virus (REV) on the response to infectious laryngotracheitis virus (ILTV) were investigated in young chickens with and without maternally derived antibody (MAb) to REV. In the first experiment a group of 1-day-old chickens without REV MAb were inoculated at 1 day of age with REV whilst another group of similar chickens were left uninoculated. All chickens were vaccinated with ILTV at 7 days of age. There was a significantly higher proportion of infectious laryngotracheitis (ILT) post-vaccinal ophthalmia (p.v.o.) in the group inoculated with REV. In the second experiment chickens with and without MAb to REV were inoculated at 1 day old with REV. These chickens, together with others not inoculated with REV, were vaccinated with ILTV isolate SA-2 8 days later. A virulent ILTV isolate, G, was used to challenge all the chickens 20 days after vaccination. Again the chickens without MAb to REV inoculated with REV showed a higher proportion of ILT p.v.o. and a significantly higher mortality rate due to ILT following vaccination. In the chickens inoculated with REV at 1 day of age and not vaccinated but challenged with ILTV there was a significantly higher mortality and rate of clinical signs due to ILT in those birds without than in those with REV MAb. In both experiments chickens from REV negative parents were found to be free of REV neutralising MAb. However, only 30% of chickens originating from a flock known to be infected with REV had a titre of 1/40 or higher. In spite of this, this group was significantly more resistant than the group without REV MAb to the immunosuppressive effect of inoculation at 1 day old with REV. This was demonstrated by their lower susceptibility (i.e. less p.v.o. and mortality) to the vaccination and challenge with ILTV. Chickens without REV MAb developed neutralising antibodies within 2 weeks of inoculation with REV. Irrespective of the REV MAb status 1-day-old chickens inoculated with REV were viraemic within a week.     

Organ culture studies on adenoviruses isolated from tenosynovitis in chickens

Five strains of adenovirus isolated from tenosynovitis in chickens were examined for their ability to grow in vitro in pieces of explanted tendon from 20-day-old chicken embryos from light or heavy breeds. Replicate tendon organ cultures (TOCs) were infected with three concentrations of each strain of virus and supernatant fluids were assayed for virus at intervals after infection up to 11 days. Two strains grew well in TOC, one moderately well, and the other two failed to grow. A strain of Newcastle disease virus (NDV), an agent not associated with tenosynovitis, also failed to grow. TOC from broiler embryos seemed slightly more susceptible to the two actively growing viruses than explants from a light breed. Growth of the adenovirus was demonstrated by immuno-fluorescence staining of sections of TOC but the appearance of stained antigen was dependent on virus titre. One of the strains which showed no activity in TOC grew in tracheal organ cultures (TrOC), as did one which multiplied in tendon, and NDV. In a more prolonged experiment, one of the viruses was found to persist in TOC at high titre for at least 37 days. The likelihood of the in vitro behaviour of these adenoviruses reflecting that occurring in vivo in tendon tissue of infected chickens is discussed.     

The anti-nuclease humoral immune response of broiler chickens exposed to Staphylococcus aureus, infectious bursal disease virus and chicken anaemia virus in an experimental model for bacterial chondronecrosis and osteomyelitis.

This study aimed to develop an enzyme-linked immunosorbent assay to detect antibody associated with Staphylococcus aureus that is produced during the chicken immune response to this organism. The protein nuclease was tested for suitability as an antigen to detect antibody in sera from broiler chickens that had been exposed to aerosolized S. aureus on day 1 post hatch during experiments to reproduce bacterial chondronecrosis and osteomyelitis. An enzyme-linked immunosorbent assay was developed to measure the levels of nuclease antibody in 509 chicken sera from various experiments, which also enabled the examination of the influence of factors such as the S. aureus infection status and co-infection with chicken anaemia virus (CAV) and infectious bursal disease virus (IBDV) on nuclease antibody levels. Positive levels of nuclease antibody were detected in 71% of serum samples from chickens inoculated with S. aureus, CAV and IBDV, while positive levels were detected in 35% of chickens inoculated with S. aureus only. The influence of CAV and IBDV on the number of chickens with positive levels was most prominent in chickens aged up to 42 days. The study showed that nuclease-specific antibodies form part of the humoral immune response in broiler chickens that have been exposed to S. aureus. Co-infection with CAV and IBDV appeared to promote development of antibody in birds younger than 42 days; however, the presence of antibody did not necessarily prevent systemic infection.     

Serological and pathogenic characterization of avian reoviruses isolated in Quebec

Reoviruses were isolated from intestinal contents of broiler chickens from nine flocks in Quebec with malabsorption syndrome. Serum neutralization test demonstrated the existence of antigenic differences between the isolates and the reference vaccine strain. The isolated reoviruses were inoculated orally and into the foot pad in one-day-old chicks, resulting in a transient, but significant depression in body weight gains. Chickens infected with isolate 615, showed in addition to growth problems, clinical signs and tissue lesions similar to those observed in field cases. When isolate 615 was inoculated into SPF chicks at one day of age, intestinal absorption of D-xylose in infected chicks at 7 days post-infection was significantly lower (P <0.05) than for corresponding controls. This study suggests the implication of some reovirus isolates, such as 615 which was serologically distinct from the vaccine strain S1133, as infectious agents associated with pathological conditions other than viral arthritis.     

Observations on viral tenosynovitis (viral arthritis) in Scotland

The isolation of reoviruses from broiler breeder and layer replacement stock affected with tenosynovitis is recorded. Experimental inoculation of the reovirus isolates in Brown Leghorn chickens produced mild clinical and pathological changes. Virus isolations from the affected joints ceased 15 days after inoculation but continued until day 35 from the caecal tonsil. All inoculated birds developed precipitating antibodies which persisted for at least 46 days.