Sensitivity and specificity of serological tests for infectious bronchitis virus antibodies in broilers.

Broilers with maternally-derived immunity (MDI) to infectious bronchitis (IB) were either spray-vaccinated with H120 at 1 day old, or not vaccinated, then challenged at 28 days with one of four different IBV serotypes. Birds were bled frequently and the sera tested by agar gel precipitation (AGP), haemagglutination inhibition (HI), 2 commercial ELISAs, and virus neutralization (VN) to compare the sensitivity and specificity of the assays. The AGP detected a transient response to challenge with a specificity of 100% and a sensitivity of approximately 40%. The ELISAs showed moderate sensitivity and high specificity with sera from non-vaccinated broilers, and high sensitivity and variable specificity with vaccinated birds. Depending on the cut-off value used, the specificity of HI tests was 55 to 100%, while the sensitivity varied widely, making identification of the serotype of an IB challenge unreliable. In vaccinated broilers the sensitivity of the VN tests (used at 21 days post-challenge only) varied from 20 to 100%, while the specificity was dependant on the cut-off value selected. Increases in HI, VN and ELISA titres in vaccinates were generally about half those in non-vaccinates. It is concluded that AGP and ELISA are adequate to detect antibody responses to IBV challenge in both vaccinated and non-vaccinated broilers. In the ELISA, a cut-off value higher than that suggested by the manufacturers is preferred in vaccinated broilers. Similarly, a cut-off value of at least log(2)7 is desirable when attempting to use HI for IB serotyping.

Molecular and biological characteristics of avian polyomaviruses: isolates from different species of birds indicate that avian polyomaviruses form a distinct subgenus within the polyomavirus genus.

The isolation and characterization of two avian polyomaviruses, from chicken (BFDV-2) and a parrot (BFDV-3), is reported. Both isolates are closely related to the non-mammalian polyomavirus budgerigar fledgling disease virus (BFDV) isolated from budgerigars (now called BFDV-1), and all three viral genomes are shown to have the same basic size of 4981 bp. A 151 bp insertion was, however, observed in the non-coding region of BFDV-2 which represented an exact duplication of the left half of the non-coding region, including the putative early promoter and amino terminus of the large T antigen. With a further 15 base pairs exchanged elsewhere throughout the three genomes, these viruses have distinct degrees of tropism for various avian species. The production of antibodies directed against a beta-galactosidase-large T antigen fusion protein of BFDV-1 is described. These antibodies detected the large T antigen, with an M(r) of approximately 80K, and the small t antigen, with an M(r) of approximately 24K, in cells infected with BFDV isolates. Whereas these antibodies bind with low affinity to the large T antigen of simian virus 40 (SV40), SV40- or mouse polyomavirus-specific antibodies will not bind to the BFDV large T antigen. Antibodies directed against BFDV structural polypeptides exhibit broad, reciprocal cross-reactivities with all three structural proteins of mammalian polyomaviruses. The significance of polyomavirus infections in various avian species is discussed. Based on unique structural and biological properties we propose that these viruses should be placed in a distinct subgenus (Avipolyomavirus) within the polyomaviruses.

Epithelia-damaging virus infections affect vitamin A status in chickens.

The effect of infection with infectious bronchitis virus (IBV) and reovirus (RV) on vitamin A status was investigated in chickens with a normal or marginal intake of vitamin A. At the age of 4 wk, chickens were infected with either IBV or RV, primarily affecting the respiratory or intestinal tract, respectively. Both viruses lowered plasma retinol levels significantly. The effect was more pronounced in chickens fed a diet marginally deficient in vitamin A than in those fed a diet adequate in vitamin A. Concentrations of retinol-binding protein, transthyretin and albumin in RV-infected chickens were also significantly lower than in noninfected chickens fed the same diets; in chickens infected with IBV, there was no effect. These results suggest that the reduced vitamin A status of IBV-infected chickens could be attributed to increased rate of utilization by tissues. In RV infection, this mechanism could be involved but impaired absorption of nutrients (including vitamin A) and direct loss of nutrients via the intestinal tract could also be important.

Serological classification of recent infectious bronchitis virus isolates by the neutralization of immunofluorescent FOCI.

The neutralisation of immunofluorescent foci test was adapted to the grouping of 14 recent Dutch infectious bronchitis virus isolates. This test provides a distinct grouping of the isolates and the corresponding sera. Evaluation of the tests was carried out by means of the computer program called 'Taxonomic', designed for the calculation of taxonomic order from serological data. The taxonomic order, depicted in the form of a tree, facilitates the judgement of the degree of resemblance between viruses or groups of viruses as well as sera. Using this test the isolates of infectious bronchitis virus can be classified into distinct groups.

Investigations into the role of reovirus in the malabsorption syndrome.

Malabsorption syndrome, defined by five criteria, could not be reproduced by oral inoculation of newly hatched chicks with six reoviruses isolated from six different cases. Passage in birds of four reoviruses with intestinal homogenates did not result in increased pathogenicity. In contrast, inoculation of complete infectious intestinal homogenate caused great weight loss, long lasting excretion of yellow-orange mucoid and wet faeces, increased plasma alkaline phosphatase activity, decreased carotene concentration and bone abnormalities. Malabsorption syndrome could not be reproduced with infectious intestinal homogenate comprising only reovirus and possibly other non-enveloped viruses after treatment with methanol or chloroform. Infectious homogenate made reovirus-free by incubation with anti-serum was as pathogenic as homogenate that had been treated the same way with broth and that still contained viable reovirus. While infectious homogenate was almost apathogenic for 3-day-old chicks, its pathogenicity for birds of this age was greatly enhanced by a pre-infection with reovirus immediately after hatching. Reovirus therefore may act as a trigger in the malabsorption syndrome. This enhancing effect, however, was not specific for reovirus; it was also achieved with an adenovirus. Vaccination of two groups of breeders with two different inactivated reovirus vaccines, resulted in effective transfer of antibody to the offspring, but did not protect the offspring against malabsorption syndrome.

Comparison of the effect of live Newcastle disease vaccine Clone 30 in broilers administered at day 1 or at day 7 and the effect of H120 vaccination at 17 days of age: a field experiment.

To analyse the results of a vaccination on the first day of age against Newcastle disease (ND) and on the 17th day of age against Infectious Bronchitis (IB) resp. with spray vaccines with Clone 30 and H120 vaccine. These vaccinations are compared in field circumstances with other vaccination methods. A serological examination and challenge test were used to be informed about the response and protection. From the present study the following conclusions can be drawn: Clear indications are obtained that following a spray vaccination against ND with Clone 30 vaccine of one-day-old broilers which possessed maternal antibodies, birds received a moderately good protection against ND, in spite of very low levels of HI antibodies. A spray vaccination against IB with H120 vaccine of broilers at 17 days of age gave some protection from two weeks after vaccination, however making a good conclusion about the protection is impossible and further investigation is required.

Application of monoclonal antibodies in the Dutch avian leukosis control programme.

Comparative testing with series of albumen samples obtained from seven Dutch poultry flocks was performed for avian leukosis virus group-specific antigen (ALV-gsa) by complement fixation test (CFT) and by a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) employing selected monoclonal antibodies directed to different ALV-p27 epitopes. The higher sensitivity for ALV-gsa detection of the DAS-ELISA resulted in increased scores of ALV-shedding hens in all seven flocks. Relatively low absorbance values seem to be associated with low rates of congenital ALV transmission.

Influence of feed on the course of tenosynovitis caused by REO virus infection in broilers.

The first outbreak of tenosynovitis caused by infection with REO virus in the Netherlands, involving 15 broiler flocks, is described. The disease could be reproduced easily by subcutaneous, oral and contact infection of susceptible broilers with the isolated virus. The 15 affected flocks all came from one broiler parent flock and were fed with feed from one mill (A). Twenty other flocks, also coming from this parent flock and reared in the same period on feed from another feed mill (B) did not suffer from tenosynovitis. The parent flock showed no disease symptoms but seroconversion to REO virus occurred after the onset of the problems in the offspring. The influences of the different feeds on the course of the synovitis could be reproduced experimentally, but with less extreme differences than observed in the field. The possible ways in which feeds may influence tenosynovitis causing REO virus infection are discussed.

Occurrence and significance of infectious bronchitis virus variant strains in egg and broiler production in the Netherlands.

Despite vaccination against Infectious bronchitis virus (IBV) with the Massachusetts type vaccine viruses H120 and H52 in the Netherlands, an increasing number of properly vaccinated flocks have suffered from the disease since 1978. In the years 1978-1982, the virus was isolated from 162 IBV suspected flocks. Cross-virus-neutralization tests showed that the majority (67 per cent) of these isolates belonged to serotypes other than the Massachusetts type, the Connecticut-, Florida-, Iowa 97-, Iowa 609- and JMK serotype. The majority of these Dutch isolates could be divided into 4 serogroups, called D207, D212, D3128 and D3896. Only a few isolates were not related to these serotypes. A survey of 328 flocks for antibody against these serotypes demonstrated that antibody against one or more of these novel serotypes were present in most of the flocks. Experiments demonstrated that vaccination with the H120 vaccine virus was not able to protect chickens against the adverse effects of a challenge with the novel serotypes.

A case of Aujeszky's disease virus infection in young chicks.

On a rearing farm with 96,000 birds, 10,000 three and four days old chicks died with nervous symptoms. A virus was isolated from the brains and identified as an Aujeszky's disease virus. The isolate was very pathogenic for chickens up to about 7 days of age, causing mortality after parenteral injection (intracerebral, intraperitoneal, intramuscular) but not after oral, eye drop or spray application. An Aujeszky vaccine virus, made apathogenic by passages in chicken cells for use in swine, had the same pathogenic properties for chicks. The isolated Aujeszky's disease virus is regarded as the agent responsible for the death of the 10,000 chicks on the farm. This virus most likely had been injected in just hatched chicks instead of or together with the Marek vaccine virus. In addition to meningitis, edema, neuronophagia and cuffing of blood vessels with mononuclear cells, haemorrhages were observed in thin sections of brain and spinal cord. After injection of isolate and vaccine virus in the leg muscle intranuclear inclusion bodies were observed in the ganglion cells in the spinal cord. Inclusion bodies have not been described before in pathological conditions of the nervous tissue of chickens.

The immune response of the chick following viral vaccinations and immunization with sheep red blood cells.

The effects of viral vaccinations and immunization with sheep red blood cells (SRBC) on the humoral response of pullets were investigated. Pullets were vaccinated with Marek's disease virus, Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and infectious bursal disease virus at appropriate ages used in commercial practice. At seven weeks, the pullets were intramuscularly immunized with SRBC. NDV and IBV antibodies were detected by hemagglutination-inhibition tests. Hemagglutination (HA) titers were established against SRBC. IBV antibody titers were not affected by vaccination or by immunization with SRBC. NDV antibody titers were significantly increased by vaccination and by immunization with SRBC. The SRBC agglutinin response was also positively affected by vaccination. The HA titer increase consisted of a rise in 2-mercaptoethanol (2-ME)-sensitive antibodies and a fall in 2-ME-resistant antibodies.

Study on the local effect of eye-drop vaccination against infectious bronchitis in 1-day-old chicks with maternal antibodies.

One-day-old chicks with maternal antibodies to infectious bronchitis virus (IBV) were vaccinated by eye-drop with H120 vaccine strain of IBV. Four weeks later the chicks were challenged by eye-drop or intratracheally with virulent IBV (Massachusetts-type field strain). The chicks were resistant to ocular challenge, but highly susceptible to an intratracheal challenge. After intratracheal challenge the birds showed clinical signs of infectious bronchitis (IB). The immunofluorescence test on IBV was positive. Macroscopical and microscopical lesions were present in the trachea. From these observations it was concluded that the protection against virulent IBV after eye-drop vaccination is localised mainly in the conjunctival and nasal tissues. Thus in vaccination studies with IBV the result of challenge depended highly on the route of application of the challenge virus. Ten days after challenge the neutralisation index of serum for IBV was significantly higher in the intratracheally-challenged chicks as compared with their eye-drop challenged or/and unchallenged mates.

Effect of the removal of the Harderian gland in 1-day-old chicks on immunity following IB vaccination.

After removal of the Harderian gland in 1-day-old chicks (Hx birds), protective immunity to infectious bronchitis virus (IBV) was decreased 3 weeks after vaccination with the H120 strain of IBV. Protective immunity was measured by challenge. The decreased protection was not reflected in the neutralisation indices of the Hx birds. In contrast, the mean neutralisation index was higher in the Hx birds than in the vaccinated controls. The role of the lachrymal gland in the development of immunity to IBV is discussed.

Vaccination of 1-day-old broilers against infectious bronchitis by eye drop application or coarse droplet spray and the effect of revaccination by spray.

Broilers hatched with maternal antibodies against infectious bronchitis virus (IBV) had developed protective immunity by 3 weeks after vaccination by the conjunctival route with H120 vaccine virus at 1-day-old. Immunity was still present when birds were 7 weeks old. Following vaccination of similar 1-day-old chicks by the same route by a coarse droplet spray, immunity developed more slowly; 50% of the 3-week-old birds developed clinical and/or pathological changes typical of infectious bronchitis following challenge, and at 5 weeks of age some birds were still susceptible to the virulent IBV. While this retarded development of protection coincided with delayed lymphocytic infiltration and follicle formation in the Harderian gland, protection was not associated with circulating antibody levels against IBV, nor was it after eye drop application of the vaccine. Birds first vaccinated at 3 weeks by spray had developed protective immunity by 6 weeks, which was complete by 8 weeks of age. Revaccination by spray at 3 weeks of age of birds first vaccinated by spray at 1-day-old caused a depression of the protective immunity for approximately 1-2 weeks. This coincided with the presence of a few plasma cells and their destruction in the Harderian gland. Immunity in revaccinated birds first vaccinated at 1-day-old by eye drop was neither depressed nor increased. Formation of circulating antibody could not be demonstrated in the birds 2 weeks after they were vaccinated for the first time at 3 weeks of age. Revaccination at 3 weeks of age of their mates first vaccinated at 1-day-old resulted in a great decrease of circulating antibody. In contrast, application of virulent virus by eye drop at 3 weeks of age caused increasing titres. These different reactions are discussed.

Infectious stunting and leg weakness in broilers: I. Pathology and biochemical changes in blood plasma.

A syndrome of stunting and leg weakness could be reproduced experimentally by inoculation of 1-day-old broilers with homogenised intestines from affected birds. Inoculated birds kept in isolators showed highly impaired growth until 3 weeks p.i. Birds produced mucoid yellowish coloured droppings and at post mortem thin liquid intestinal contents were found. Biochemical examination of blood plasma showed low plasma carotenoid concentrations and an increased alkaline phosphatase (ALP) activity, mainly caused by one isoenzyme, which was most likely of intestinal origin. These findings implicate infectious causal agents with the intestines as the site of primary involvement. Bone abnormalities consisted of rickets-like changes at the age of 3 weeks, whereas a distinct dyschondroplasia was seen at 4 weeks. The syndrome could also be transmitted to uninoculated birds kept in contact with birds inoculated at 1 day of age. Birds inoculated at 7 days of age also showed greatly impaired growth but developed no macroscopical bone disorders. Inoculation at 14 days of age did not result in impaired growth or bone abnormalities. Following inoculation with REO virus, isolated from a field case, no bone abnormalities occurred. However, a shortlived impaired growth, diarrhoea, increased plasma ALP activity and decreased carotenoid concentration were observed. The rapid spread of the disease and the role of REO virus are discussed.

Egg production in relation to the results of a long term serological survey of 73 flocks of fowl.

Seventy-three flocks of fowl were tested at regular intervals for the presence of precipitins to fowl adenovirus (AV) and infectious bronchitis virus (IBV), haemaggluinating inhibiting antibodies to BC in 14 virus, and of agglutinins to Mycoplasma gallisepticum (M.g.) and Mycoplasma synoviae (M.s.). In all the eight flocks affected with Egg Drop Syndrome (EDS '76), egg production problems were associated with increasing numbers of BC14 virus reactors and AV reactors. In flocks showing production problems other than EDS'73 without any apparent cause, the average percentage of AV reactors increased significantly after the rearing period; this was not true of IBV reactors. BC14 reactors were either absent or present only once, in small numbers and with low titres, during the test period. The average percentage of AV reactors did not increase after the rearing period either in normally producing flocks or in flocks with production problems for which other diseases or dietary errors plausibly accounted for these problems. All this suggests a pathogenic role of AV in production problems. One can conclude from the high percentage of reactors in all groups of flocks that subclinical IBV infections are common. The percentage of IBV reactors during the laying period of flocks with EDS'76 was significantly higher than that of normally producing flocks. It is therefore suggested that subclinical IBV infection could be among the factors causing stress, acting as a trigger for EDS'76. All M.g.-infected flocks showed production problems; M.s. infections could not be related to egg production disturbances.

Egg production in relation to the results of a long term serological survey of 73 flocks of fowl.

Summary Seventy-three flocks of fowl were tested at regular intervals for the presence of precipitins to fowl adenovirus (AV) and infectious bronchitis virus (IBV), haem-agglutinating inhibiting antibodies to BC14 virus, and of agglutinins to Mycoplasma gallisepticum (M.g.) and Mycoplasma synoviae (M.s.). In all the eight flocks affected with Egg Drop Syndrome (EDS '76), egg production problems were associated with increasing numbers of BCI4 virus reactors and AV reactors. In flocks showing production problems other than EDS '76 without any apparent cause, the average percentage of AV reactors increased significantly after the rearing period; this was not true of IBV reactors. BC14 reactors were either absent or present only once, in small numbers and with low titres, during the test period. The average percentage of AV reactors did not increase after the rearing period either in normally producing flocks or in flocks with production problems for which other diseases or dietary errors plausibly accounted for these problems. All this suggests a pathogenic role of AV in production problems. One can conclude from the high percentage of reactors in all groups of flocks that sub-clinical IBV infections are common. The percentage of IBV reactors during the laying period of flocks with EDS '76 was significantly higher than that of normally producing flocks. It is therefore suggested that subclinical IBV infection could be among the factors causing stress, acting as a trigger for EDS '76. All M.g.-infected flocks showed production problems; M.s. infections could not be related to egg production disturbances.

Immunosuppressive effect of infectious bursal disease virus on vaccination against infectious bronchitis.

Groups of broiler chicks hatched with parental antibodies to infectious bursal disease virus (IBDV) and infectious bronchitis virus (IBV) were vaccinated against IBV at 1 day of age via the oculonasal routes and inoculated with virulent IBDV at 1, 5, 10, 15 or 20 days of age. While the non-IBDV inoculated birds were solidly immune against IBV challenge at an age of 29 days, immunity in the IBDV infected birds was depressed. This depression, which was most serious in the birds IBDV inoculated at 1 or 5 days of age, coincided with a delayed infiltration of the Harderian gland by lymphocytes and immunoglobulin-bearing cells. In the groups inoculated at older ages infiltration did not seem to be delayed but moderate destruction of plasma cells was observed 7 to 14 days later. The neutralisation index to IBV, which was significantly higher in the non-IBDV infected than in the infected birds at the day of challenge, increased sharply after challenge in the IBDV infected but not in the non-infected birds. All IBDV-inoculated birds developed typical lesions when 17 to 26 days old whereas precipitins reappeared when birds were 29 to 33 days old.