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.

Bovine ocular squamous cell carcinoma: tumor-associated immunity detected by E-rosette augmentation test.

The E-rosette augmentation (ERA) assay was used to study cell-mediated immunity with blood leukocytes from cattle with bovine ocular squamous cell carcinoma (BOSCC), from cattle with benign ocular or cutaneous lesions, and from healthy controls. Potassium chloride extracts (3 M) of BOSCC and skin from the same donor, cutaneous bovine papillomas, allogeneic lymphosarcoma, and xenogeneic sheep squamous cell carcinoma were used as antigens. Of the 21 animals with BOSCC, leukocytes of 19 gave positive ERA reactions to BOSCC extract and only those of 4 reacted nonspecifically to other extracts. Of 21 animals in the control group, only 3 gave positive ERA reactions. In reciprocal tests, cattle with BOSCC showed ERA reactivity only against extracts of BOSCC, and cattle with cutaneous papillomas showed reactivity only against extracts of cutaneous papillomas. Blood leukocytes from tumor-bearing cattle stimulated with related tumor extracts released a soluble factor that enhanced E-rosette formation when tested on normal bovine leukocytes.

Serum blocking factors in bovine ocular squamous cell carcinoma demonstrated by inhibition of erythrocyte rosette augmentation.

Blood leukocytes from cattle with bovine ocular squamous cell carcinoma (BOSCC) react with extracts of BOSCC, and blood leukocytes from cattle with cutaneous papillomas react with extracts of papillomas in the erythrocyte rosette (E-rosette) augmentation test. Sera from cattle with BOSCC, with cutaneous papillomas, and with ocular papillomas and sera from lesion-free cattle were tested for their ability to block antigen-induced E-rosette augmentation and E-rosette augmentation induced by phytohemagglutinin. Autologous serum from 16 BOSCC-affected cattle blocked E-rosette augmentation induced by BOSCC extract, and autologous sera from three cattle with cutaneous papillomas blocked E-rosette augmentation induced by papilloma extract. Sera from cattle with BOSCC did not block E-rosette augmentation induced by papilloma extract, nor did sera from cattle with cutaneous papillomas block BOSCC extract-induced E-rosette augmentation. Sera from cattle with BOSCC blocked BOSCC extract-induced E-rosette augmentation in some but not all allogeneic combinations. E-rosette augmentation was observed when blood leukocytes from all 12 BOSCC-affected and six control cattle were exposed to phytohemagglutinin. This reaction was blocked by autologous sera from 11 of the 12 BOSCC-affected cattle, from one of the three cattle with cutaneous papillomas, but from none of the three other cattle. Sera from BOSCC-affected cattle also blocked phytohemagglutinin-induced E-rosette augmentation in allogeneic combination, regardless of the tumor status of the donor of the leukocytes.

Transmission of virus from serosal fluids and demonstration of antigen in neutrophils and mesothelial cells of cattle infected with bovine ephemeral fever virus.

Following intravenous injection of bovine ephemeral fever (BEF) virus 6 cattle were autopsied after clinical disease became evident. Fluid from serosal cavities with serofibrinous inflammatory changes showed large increases in neutrophil numbers. BEF virus was detected for the first time in pericardial, thoracic and abdominal fluids. Virus was also detected in synovial fluids, confirming an earlier report of transmission with a synovial fluid sample. Using a direct fluorescent antibody technique, BEF virus antigen was identified for the first time in synovial, pericardial, thoracic and abdominal fluids, in synovial membranes and epicardium. In synovial membranes and epicardium, specific fluorescence was observed in two cell types, mesothelial cells and neutrophils. In the fluids, fluorescence was restricted to neutrophils, the predominant cell type. Specific fluorescence was observed in blood smears from only one animal although blood samples collected at autopsy from all animals contained infective virus.

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.

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.

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.

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.

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.

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.

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.

Detection of papillomavirus DNA in precancerous lesions of the ears of sheep.

Sixty-seven benign precancerous cutaneous lesions from the ears of 51 sheep were examined for papillomavirus DNA by hybridisation to radioactively labelled or biotinylated probes of bovine papillomavirus type 1 (BPV 1) DNA under varying conditions of stringency. An additional 16 precancerous lesions from other cutaneous sites on 15 sheep and 15 samples of lesion-free skin from nine sheep were similarly examined. Both total genomic and subgenomic probes were used. DNA from 10 aural lesions and one vulval lesion reacted with the probe in a manner indicative of the presence of episomal papillomavirus DNA. Papillomavirus DNA was detected at low stringency in eight of the 10 aural lesions and the vulval lesions, and at high stringency in two aural lesions. Three out of the 8 aural lesions that were positive at low stringency reacted when re-tested at high stringency. Hybridisation with one of the samples of lesion-free ovine skin produced occasional equivocal signals. One particular positive lesion, an ovine aural cutaneous horn, was studied in more detail. When treated with restriction endonucleases, its restriction enzyme pattern was the same as that for BPV 2 DNA with eight of twelve enzymes and the same as that for BPV 1 DNA with two of the twelve enzymes. It was concluded that this ovine papillomavirus was more closely related to BPV 2 than to BPV 1. The possibility that it could be a subtype of BPV 2 is discussed.

Serological response of chickens to oral vaccination with Newcastle disease virus.

Conventional Newcastle disease vaccines are not suitable for application to village chickens in tropical countries of Asia. Trials with food-based vaccines are being initiated and the following experiments were performed to evaluate oral vaccination with Newcastle disease virus. Experimental chickens were vaccinated orally with the avirulent V4 strain of Newcastle disease virus and haemagglutination-inhibition antibody responses were measured. V4 virus was introduced into the crop by tube and total faecal output was collected daily and assayed for Newcastle disease virus. Virus was recovered on Days 5 and 6 after vaccination from most chickens that had received 10(7.4) and 10(6.4) 50% egg-infectious doses (EID50) of virus. There was no recovery of virus from birds receiving a lower dose of vaccine. Groups of chickens kept in cages with wire floors were given various doses of vaccine into the crop. Higher antibody titres were achieved with higher doses of virus. This dose responsiveness was not observed when various doses of vaccine were presented on food pellets and the groups of chickens were kept on concrete floors. Similar antibody responses were then seen with nominal doses of 10(5.2) and 10(8.2) EID50 per bird, possibly as a result of excretion and re-ingestion of the vaccine virus. Spread of the vaccine virus was demonstrated when control chickens and chickens receiving 10(7.7) EID50 of V4 virus on food pellets were housed together on a concrete floor. Similar antibody titres were achieved in both vaccinated and in-contact chickens.

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.

Tumour-associated antigens in bovine ocular squamous cell carcinoma: studies with sera from tumour-bearing animals.

Sera from 21 cattle (10 bovine ocular squamous cell carcinoma (BOSCC) bearing and 11 controls) were tested for antibody reactivity against various cultured cells (BOSCC, normal skin and tumours other than BOSCC), by an indirect immunofluorescence technique. Most of the BOSCC sera reacted with autologous and allogeneic BOSCC cells and did not show any significant reactivity with normal skin cells (autologous or allogeneic). These sera when further tested on 7 different allogeneic or xenogeneic tumour cell types (other than BOSCC), showed significant reactivity only with cultured equine sarcoid and cutaneous papilloma cells. Three of the BOSCC sera did not react with autologous BOSCC cells but reacted indiscriminately with all other allogeneic BOSCC, normal cells and other tumour cells. Most of the control sera did not show any significant reactivity against BOSCC cells except sera from one cow bearing ocular papilloma and 2 healthy normals that were in contact with BOSCC bearing animals. This study suggests that BOSCC cells possess tumour associated antigens that are common to most (if not all) BOSCC cells.

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.