Deduced amino acid sequences surrounding the fusion glycoprotein cleavage site and of the carboxyl-terminus of haemagglutinin-neuraminidase protein of the avirulent thermostable vaccine strain I-2 of Newcastle disease virus.

A single-tube RT-PCR technique generated a 387 bp or 300 bp cDNA amplicon covering the F(0) cleavage site or the carboxyl (C)-terminus of the HN gene, respectively, of Newcastle disease virus (NDV) strain I-2. Sequence analysis was used to deduce the amino acid sequences of the cleavage site of F protein and the C-terminus of HN protein, which were then compared with sequences for other NDV strains. The cleavage site of NDV strain I-2 had a sequence motif of (112) RKQGRLIG(119), consistent with an avirulent phenotype. Nucleotide sequencing and deduction of amino acids at the C-terminus of HN revealed that strain I-2 had a 7-amino-acid extension (VEILKDGVREARSSR. This differs from the virulent viruses that caused outbreaks of Newcastle disease in Australia in the 1930s and 1990s, which have HN extensions of 0 and 9 amino acids, respectively. Amino acid sequence analyses of the F and HN genes of strain I-2 confirmed its avirulent nature and its Australian origin.

Thermostability profile of Newcastle disease virus (strain I-2) following serial passages without heat selection.

I-2 is an avirulent strain of Newcastle disease virus. During establishment of the I-2 strain master vaccine seed, a series of selection procedures was carried out at 56 degrees C in order to enhance heat resistance. This master seed is used to produce a working seed, which is then employed to produce the vaccine. These two passages are done without further heat selection; however, it is not known how rapidly and to what extent thermostable variants would be lost during further passage. The study was therefore conducted to determine the effect of passage on thermostability of strain I-2. The virus was serially passaged and at various passage levels samples were subjected to heat treatment at 56 degrees C for 120 min. The inactivation rates for infectivity and haemagglutinin (HA) titres were assayed by use of chicken embryonated eggs and HA test, respectively. Thermostability of HA and infectivity of I-2 virus were reduced after 10 and 5 passages, respectively, without heat selection at 56 degrees C. These results suggest that 5 more passages could be carried out between the working seed and vaccine levels without excessive loss of thermostability. This would result in increased vaccine production from a single batch of a working seed.

Vietnamese trials with a thermostable Newcastle disease vaccine (strain I2) in experimental and village chickens.

The Australian I2 strain of Newcastle disease virus was tested as a vaccine in the laboratory and in Vietnamese villages. The infectivity litre of lyophilised vaccine fell less than 1 log10 unit on storage for 6 days at 26-32 degrees C. Vaccine stored at similar temperatures induced high levels of immunity in laboratory chickens after storage for 17 days and in village chickens after storage for 21 days. I2 vaccine protected for at least 24 weeks after vaccination, and for 16 weeks after application in drinking water. Under laboratory conditions, I2 vaccine given by eye drop spread by contact to unvaccinated chickens, inducing in them both an antibody response and protective immunity. In villages, chickens vaccinated by eye drop, chickens receiving vaccine on food and chickens in contact with vaccinated chickens all resisted artificial challenge 6 weeks after vaccination. There were no adverse reactions to vaccination. Strain I2 was judged to be thermostable, avirulent and immunogenic, and suitable for use as a vaccine under village conditions.

Field isolates of fowlpox virus contaminated with reticuloendotheliosis virus.

The polymerase chain reaction (PCR) method was used to examine samples from field cases of fowlpox for the presence of reticuloendotheliosis virus (REV). The S-strain fowlpox vaccine, known to be contaminated with REV, served as a positive control. Fowlpox virus was grown from field samples and vaccines by inoculation of embryonated hen eggs by the chorioallantoic membrane (CAM) route. DNA was extracted from the CAM lesions and examined for REV proviral sequences using primers specific for the long terminal repeats of REV. Amplicons of the expected length were detected in all the 45 field samples from poultry and in the S strain vaccine. Two other vaccines and two isolates from wild birds contained no detectable REV sequences. The PCR products from the vaccine and one field isolate were sequenced and were identical. These products showed 81 to 87.5% homology with the published sequences for the long terminal repeats of REV. It was not determined whether the REV proviral DNA was integrated with cellular DNA, fowlpox DNA or both. Inoculation of day-old chickens with the S-strain vaccine resulted not only in the production of fowlpox lesions but also feathering defects and proventriculitis. This suggests that the REV present in the vaccine is replication competent. Problems being encountered with protection from fowlpox following vaccination in Australia might be attributed to simultaneous challenge with fowlpox virus and REV.

Receptors for the V4 strain of Newcastle disease virus in the digestive tract of chickens.

Enterocytes were detached from various parts of the digestive tract of chickens by treatment with DTT or with hyaluronidase. Isolated enterocytes were exposed to suspensions of the V4 strain of Newcastle disease virus (NDV). Removal of virus from the supernatant fluid was taken as evidence of binding of virus to enterocytes and residual virus was measured both by infectivity assay and by ELISA. Enterocytes from duodenum, jejunum, ileum, caecum, and rectum bound the virus; enterocytes from oesophagus, crop and proventriculus did not.

Bovine papillomavirus type 4 DNA isolated from a skin lesion in a steer.

A lesion on the head of a steer, defined histologically as an epithelial papilloma, yielded DNA which did not hybridise with any of the bovine papillomavirus DNAs usually associated with the formation of skin lesions. DNA from the lesion did hybridise with DNA from bovine papillomavirus 4, even under stringent conditions, and contained a sequence that could be amplified by polymerase chain reaction with primers specific for that virus. Bovine papillomavirus 4 had previously been isolated only from lesions of the upper alimentary canal.

Australian studies on Newcastle disease virus. The French heritage.

Eric French contributed greatly to the early Australian studies on Newcastle disease virus, producing the foundations on which subsequent Australian studies were based. In 1964 he conducted the first major serological survey for Newcastle disease in the Australian poultry flock, and showed that the pathotypes of the virus recognised at that time were not present. After the isolation of strain V4 in 1966, he initiated some of the first studies on the nature of this stain. In particular, he demonstrated the avirulence of this virus, its ability to infect chickens when delivered orally with food and its potential utility as a vaccine. Subsequent studies by other workers included the development of strain V4 as a conventional vaccine and as a vaccine suitable for use in village chickens.

Recent isolates of Newcastle disease virus in Australia.

Forty-five recently isolated strains of Newcastle disease virus and the V4 vaccine strain of Newcastle disease virus were used to infect experimental chickens. Neither V4 nor any of the new strains produced detectable clinical disease. All the viruses produced an antibody response and spread by contact. Some of the newly isolated viruses produced a more rapid serological response than V4 virus did. Dual or multiple infections with one of the new strains of Newcastle disease virus, infectious bronchitis virus and Escherichia coli did not enhance the pathogenicity of any of the agents.

Approaches to food-based vaccines for domestic chickens.

Domestic chickens were fed viral vaccines that were applied to the surface of food pellets. Responses were judged by the production of specific antibodies, and compared with the responses obtained when the same vaccines were given by conventional routes. Chickens responded similarly to commercial avian infectious encephalomyelitis vaccine given on food or by eyedrop when antibodies were measured by ELISA, and the vaccine virus spread by contact. Increasing the dose of oral vaccine tenfold gave a more rapid serological response but the levels of antibody were not increased. There was no serological response to commercial infectious laryngotracheitis virus vaccine given on food. An experimental avian adenovirus vaccine produced a serological response when given on food, but higher levels of antibody were produced in response to vaccination by eyedrop. The vaccine virus spread by contact. It was concluded that current avian infectious encephalomyelitis vaccines, and prospective recombinant vaccines based on avian adenovirus vectors, could be delivered on food.

Molecular studies on avian strains of Pasteurella multocida in Australia.

A collection of 45 strains of Pasteurella multocida was assembled. The strains had been isolated from cases of fowl cholera in eastern Australia over 8 years, and included mainly type A strains. All the strains were examined for plasmids and resistance to 10 antimicrobial agents and most of the strains were examined for restriction fragment length polymorphism. Nine strains were assayed for pathogenicity for mice. Twenty strains yielded no plasmid. Seven contained a single plasmid of 1.3 kbp and 18 contained 2 plasmids, of 2.4 and 7.5 kbp. All the strains were resistant to streptomycin, trimethoprim and lincomycin while one strain was resistant to tetracycline. There was no correlation between plasmid content and resistance to antimicrobial agents. Three strains that lacked plasmids were highly virulent for mice, 6 strains containing plasmids were not. Restriction fragment length polymorphism generated by HpaII allowed the 39 strains that were tested to be divided into 10 groups.

Cell-mediated immunity in chickens vaccinated with the V4 strain of Newcastle disease virus.

A leukocyte migration inhibition assay was used to demonstrate antigen-specific cell-mediated immunity (CMI) in chickens vaccinated with the V4 strain of Newcastle disease virus. Chickens were vaccinated when 5 weeks old, and again 3 and 7 weeks later. CMI was detected 9 days after initial vaccination by eyedrop, but only after the second dose of vaccine delivered into the crop. In some chickens there was a temporary suppression of CMI to Newcastle disease virus, especially after the initial application of vaccine to the crop. Haemagglutination inhibition antibodies developed to similar levels in chickens after vaccination by either route. There was no obvious correlation between antibody and CMI responsiveness.

Receptors for the V4 strain of Newcastle disease virus in the digestive tract of chickens.

Enterocytes were detached from various parts of the digestive tract of chickens by treatment with DTT or with hyaluronidase. Isolated enterocytes were exposed to suspensions of the V4 strain of Newcastle disease virus (NDV). Removal of virus from the supernatant fluid was taken as evidence of binding of virus to enterocytes and residual virus was measured both by infectivity assay and by ELISA. Enterocytes from duodenum, jejunum, ileum, caecum, and rectum bound the virus; enterocytes from oesophagus, crop and proventriculus did not.

Lactose pellets--a new approach to oral vaccination of village chickens against Newcastle disease.

Newcastle disease in village chickens in developing countries can now be controlled with vaccines containing thermostable, avirulent V4 virus delivered on food. Logistical problems arise because 7 to 10 g of food vaccine must be allowed for each chicken. Lactose-based pellets have been prepared that contain an immunizing dose of V4 virus in a single pellet, even after long periods of storage. Protective levels of antibody were generated in chickens fed individual pellets, or in groups of chickens fed vaccine pellets mixed with normal food. Chickens receiving vaccine pellets developed a level of protection against challenge with virulent Newcastle disease virus similar to that achieved with vaccine added to food. This process when refined will allow the preparation of vaccine in regional laboratories and delivery without refrigeration to villages.

The contribution of lectins to the interaction between oral Newcastle disease vaccine and grains.

Successful oral vaccination of chickens with Newcastle disease (ND) depends on the survival of vaccine virus on the grains that are used as carriers. Some interactions between grains and the V4 strain of ND virus (NDV) were studied. Crude saline washings were prepared from several grains - rice (unhusked, brown, white and boiled white), sorghum, millet, wheat, maize and barley - and tested for lectin activity, as indicated by agglutination of chicken erythrocytes. Only washings from unhusked rice, sorghum and millet failed to haemagglutinate. None of the crude washings antagonised the haemagglutinating activity of NDV, and the washing from white rice produced an 8-fold enhancement. The presence of lectins in the washings from rice, wheat and barley was confirmed by purifying a substance with N-acetyl-D-glucosamine specificity. Only the crude extract from white rice had any profound effect on infectivity, reducing the infectivity titre by 99.99%. It is not known if the viricidal substance is identical with the lectin. Of 9 commercial lectins tested, only ConA bound the V4 virus.

The influence of adjuvants on oral vaccination of chickens against Newcastle disease.

Living V4 strain Newcastle disease vaccine was given to chickens orally. The inclusion of DEAE-dextran, Quil-A or TiterMax in the vaccine, or delivering the vaccine as Iscoms, did not enhance the serological response. The immediate serological response to living V4 vaccine was enhanced in the presence of Avridine. Chickens produced a low serological response to oral administration of inactivated V4 vaccine. This response was not enhanced in the presence of Avridine.

Mucosal immunity in chickens vaccinated with the V4 strain of Newcastle disease virus.

Chickens vaccinated orally with the V4 strain of Newcastle disease virus (NDV) and possessing low levels or undetectable levels of serum haemagglutination-inhibition (HI) antibodies against NDV may resist challenge with virulent virus. Evidence for vaccine-induced mucosal immunity was sought. HI antibodies were detected in serum, lachrymal fluid and tracheal washing after vaccination with V4 virus by intranasal, eyedrop or intracrop routes. IgA was detected by immunodiffusion in lachrymal fluid of both vaccinated and control birds. Lymphoid accumulations were detected in tracheas of chickens after vaccination and there were significant increases in the numbers of plasma cells in sections of Harderian glands from chickens after vaccination. In a second experiment, ELISA was used to demonstrate the production of NDV-specific IgA which was detected in serum, lachrymal fluid, tracheal washing and intestinal washing after intracrop or eyedrop vaccination with V4 virus. It was concluded that oral vaccination of chickens with V4 virus induces a mucosal immune response.

Genomic sequences of bovine papillomaviruses in formalin-fixed sarcoids from Australian horses revealed by polymerase chain reaction.

Seventy six formalin-fixed paraffin-embedded sarcoids from 62 Australian horses, collected over a ten year period, were examined for the presence of genomic sequences from bovine papillomavirus 1 and 2 (BPV1, BPV2) with the polymerase chain reaction (PCR). Sequences that could be amplified by primers specific for BPV1 and BPV2 were present in 56 of the 76 sarcoids (73%). A restriction site present in BPV1 and absent from BPV2 was detected in 28 of 34 amplified products that were treated with endonuclease.

Bovine cutaneous papillomas associated with bovine papillomavirus type 5.

Squamous papillomas obtained from bovine facial skin yielded viral DNA indistinguishable from that of bovine papillomavirus type 5. It was separated from recognised bovine papillomaviruses by its restriction endonuclease pattern and hybridization tests with DNA.