Influence of vaccine deposition site on post-vaccinal viraemia and vaccine efficacy in broiler chickens following in ovo vaccination against Marek's disease.

In ovo vaccination against Marek's disease is a widely used technology in the broiler industry.A series of experiments was carried out to determine the site of vaccine deposition in the egg during automated in ovo vaccination, and the effect of vaccine deposition site and dose on vaccine responses following vaccination with cell-associated herpesvirus of turkeys in commercial broiler chickens. Vaccine deposition site following automated in ovo vaccination was principally influenced by the age of embryo, with egg size having a smaller effect. The frequency of vaccine deposition inside the embryo body increased as incubation progressed from day 17.5 to 19.5. In experiments using manual vaccine deposition intra-embryonically (IE) or extra-embryonically (EE) at day 18.5, EE vaccine deposition resulted in a significantly delayed development of post-vaccinal viraemia relative to both IE vaccination and subcutaneous vaccination at hatch. There were no effects of vaccine dose (2000, 4000 or 8000 plaque forming units) on the timing of post-vaccinal viraemia. The timing of post-vaccinal viraemia was found to be a good indicator of the level of protection provided by the vaccine against challenge with earlier viraemia associated with better protection. IE vaccine deposition induced significantly greater protection than EE deposition against challenge with a virulent strain of Marek's disease virus. IE deposition consistently produced a high level of protection (68 to 84%) irrespective of vaccine dose or challenge day, while EE vaccine deposition produced no or low levels of protection (0 to 27%) depending on the vaccine dose and day of challenge. The growth of challenged chickens was also affected by site of vaccine deposition, with significantly higher live weights at day 56 of age in IE compared with EE vaccinated groups. These data suggest that the site of vaccine deposition within the embryo is an important determinant of the success of in ovo vaccination.

Isolation of Hendra virus from pteropid bats: a natural reservoir of Hendra virus.

Since it was first described in Australia in 1994, Hendra virus (HeV) has caused two outbreaks of fatal disease in horses and humans, and an isolated fatal horse case. Our preliminary studies revealed a high prevalence of neutralizing antibodies to HeV in bats of the genus PTEROPUS:, but it was unclear whether this was due to infection with HeV or a related virus. We developed the hypothesis that HeV excretion from bats might be related to the birthing process and we targeted the reproductive tract for virus isolation. Three virus isolates were obtained from the uterine fluid and a pool of foetal lung and liver from one grey-headed flying-fox (Pteropus poliocephalus), and from the foetal lung of one black flying-fox (P. alecto). Antigenically, these isolates appeared to be closely related to HeV, returning positive results on immunofluorescent antibody staining and constant-serum varying-virus neutralization tests. Using an HeV-specific oligonucleotide primer pair, genomic sequences of the isolates were amplified. Sequencing of 200 nucleotides in the matrix gene identified that these three isolates were identical to HeV. Isolations were confirmed after RNA extracted from original material was positive for HeV RNA when screened on an HeV Taqman assay. The isolation of HeV from pteropid bats corroborates our earlier serological and epidemiological evidence that they are a natural reservoir host of the virus.

Reactivation of a macropodid herpesvirus from the eastern grey kangaroo (Macropus giganteus) following corticosteroid treatment.

The family Herpesviridae is a large group of viruses which contain double stranded DNA genomes. Biological characteristics, such as host signs, site of replication and site of latency have been used to describe three major subfamilies, Alphaherpesvirinae, Betaherpesvirinae and Gammaherpesvirinae within the family Herpesviridae. Macropodid herpesviruses (MaHV) have been implicated in fatal outbreaks amongst the captive marsupial populations of Australia. These outbreaks have resulted in the isolation of nine MaHV strains which have been classified into two species called macropodid herpesvirus 1 and 2 (MaHV-1 and MaHV-2). Biological characteristics have been used to place MaHV-1 and -2 within the subfamily Alphaherpesvirinae. Molecular phylogenetic reconstructions indicate an unusual position for MaHV-1 and -2 within the alphaherpesviruses. Current isolates of MaHVs have all been obtained from marsupials exhibiting clinical disease. A common biological characteristic of herpesviruses is the establishment of latent infections in nervous tissue. We have determined that MaHV are able to latently infect eastern grey kangaroos through reactivating and isolating a herpesvirus by inducing immunosuppression. We have investigated the possible sites of latency for MaHV-1 using molecular techniques. Detection of herpesvirus DNA in the trigeminal ganglia taken from two naturally infected eastern grey kangaroos indicates dissemination via a respiratory route.

Newly discovered viruses of flying foxes.

Flying foxes have been the focus of research into three newly described viruses from the order Mononegavirales, namely Hendra virus (HeV), Menangle virus and Australian Bat Lyssavirus (ABL). Early investigations indicate that flying foxes are the reservoir host for these viruses. In 1994, two outbreaks of a new zoonotic disease affecting horses and humans occurred in Queensland. The virus which was found to be responsible was called equine morbillivirus (EMV) and has since been renamed HeV. Investigation into the reservoir of HeV has produced evidence that antibodies capable of neutralising HeV have only been detected in flying foxes. Over 20% of flying foxes in eastern Australia have been identified as being seropositive. Additionally six species of flying foxes in Papua New Guinea have tested positive for antibodies to HeV. In 1996 a virus from the family Paramyxoviridae was isolated from the uterine fluid of a female flying fox. Sequencing of 10000 of the 18000 base pairs (bp) has shown that the sequence is identical to the HeV sequence. As part of investigations into HeV, a virus was isolated from a juvenile flying fox which presented with neurological signs in 1996. This virus was characterised as belonging to the family Rhabdoviridae, and was named ABL. Since then four flying fox species and one insectivorous species have tested positive for ABL. The third virus to be detected in flying foxes is Menangle virus, belonging to the family Paramyxoviridae. This virus was responsible for a zoonotic disease affecting pigs and humans in New South Wales in 1997. Antibodies capable of neutralising Menangle virus, were detected in flying foxes.

Recombinant fowlpox virus vaccines against Australian virulent Marek's disease virus: gene sequence analysis and comparison of vaccine efficacy in specific pathogen free and production chickens.

We have cloned and sequenced the glycoprotein genes gB, gC and gD of the Australian virulent Marek's disease virus (MDV) isolate Woodlands No. 1. The glycoprotein gB and gC sequences were identical to the homologs of other virulent MDV type 1 strains, and the glycoprotein gD sequence contained only one unique amino acid substitution. Recombinant fowlpox viruses (rFPVs) expressing the MDV glycoprotein genes were constructed and their efficacy as vaccines was evaluated in specific pathogen free (SPF) and production chickens. Vaccination with the FPV-gB recombinant protected SPF chickens from Marek's disease mortality and tumour formation following challenge with virulent MDV Woodlands No. 1. The degree of protection from Marek's disease was dependent on the vaccine dose and route of inoculation. The rFPVs expressing gC or gD did not provide protection from Marek's disease. A rFPV expressing both gB and gC did not provide enhanced protection in comparison with the rFPV-gB alone. The rFPV-gB vaccine failed to protect commercial chickens from MD mortality and provided little protection from tumour formation in comparison with the commercial herpesvirus of turkey (HVT) vaccine. The failure to provide protection against MD may be related to the impact of maternally derived immunity to MDV and FPV and possibly the genotype of the chickens.

Nursing futures: a process to promote change in the delivery of care.

In this era of healthcare reform, performing care using resources in an efficient manner is essential. "Nursing Futures," a process used by a 24-bed general rehabilitation unit in a 530-bed facility, helped the unit to identify key components of care, determine opportunities for improvement, and create a new system for the delivery of care that maximized resources and improved customer satisfaction. A Nursing Futures Committee, composed of nursing staff from all levels and from all shifts, used a continuous quality improvement process to focus on the problems in care delivery and developed ways to solve these problems using the time and talents of registered nurses in the most effective way. The committee also identified expectations of staff by various customer groups; analyzed the delivery system and defined its shortcomings; developed the ideal patient care unit within financial and institutional constraints; and executed the plan, considering cost and evaluation of patient and staff satisfaction before and after the system was implemented. The new system provided consistency in patient care assignments by reorganizing the unit into two nursing teams and by creating a new nursing position, the patient care manager.

Genetically altered herpesviruses as vaccines.

Herpesviruses are a common and important cause of disease in most domestic animals. While many virus diseases have been successfully controlled by conventional vaccines, genetically modified vaccines offer distinct advantages. They are less virulent, less likely to result in latency and they include genotypic and phenotypic markers which allow differentiation of vaccine virus from wild-type virus and serological differentiation of vaccinated animals from infected animals. These benefits are particularly useful in eradication campaigns for herpesvirus diseases such as Aujeszky's disease and infectious bovine rhinotracheitis. Neither conventional nor genetically modified vaccines prevent super-infection. This is a major problem for diseases such as Marek's disease where virulent virus continues to be excreted from vaccinated animals, thus contaminating the environment and making control more difficult. To prevent infection, new strategies will need to be developed such as transgenic animals which are innately resistant.

Development and trial of a bovine herpesvirus 1-thymidine kinase deletion virus as a vaccine.

An Australian bovine herpesvirus 1 (BHV1) isolate with a defined (427 base pair) deletion in the protein coding region of the thymidine kinase gene was obtained by standard marker rescue procedures. After selection in the presence of the nucleotide analogue 5'-iodo-deoxy-uridine the virus was analysed by hybridisation with three differential oligonucleotide probes, restriction endonuclease profile studies and DNA sequence analysis. The virus elicited an immune response in recipient animals after either intramuscular or intravenous administration and produced no significant deleterious side-effects when administered at a dose sufficient to stimulate the host immune response. The safety and immunogenicity of the recombinant BHV1 virus 39B1 were similar to those reported for other registered BHV1 vaccines and the virus would appear to be suitable for the production of a vaccine seed lot and more exhaustive field trials as a prelude to commercial vaccine production and registration.

National trends in the management of tubal pregnancy, 1970-1987.

Tubal pregnancy leads to reduced childbearing potential and is a major cause of maternal morbidity and mortality in the United States. Several hospital-based studies have shown a trend toward more conservative management of tubal pregnancies, which reflects attempts to reduce morbidity and preserve fertility; however, the impact on future fertility remains unclear. To study national trends in the management of tubal pregnancy from 1970-1987, we analyzed data from the National Hospital Discharge Survey. Tubal pregnancies managed conservatively, using operative procedures that attempt to preserve the function of the involved fallopian tube, increased from approximately 2% in 1970-1978 to 12% in 1984-1987. During 1979-1987, conservative procedures were more than twice as common for women with private insurance as for those without it. The use of diagnostic laparoscopy increased from 10% of tubal pregnancies in 1970-1978 to 33% in 1979-1987, whereas the use of diagnostic laparotomy decreased from 24 to 2%.

Antibodies against certain bluetongue and epizootic haemorrhagic disease viral serotypes in Indonesian ruminants.

The orbiviruses contain several important viruses of livestock including bluetongue (BT) and epizootic haemorrhagic disease of deer (EHD) which share some group antigens. Preliminary screening of sera for antibodies to orbiviruses by the agar gel immunodiffusion (AGID) test has previously revealed widespread infections with the BT group in Indonesia. However serum neutralization (SN) tests give a more accurate estimate of exposure to each serotype in the BT and EHD groups, and in this study were applied to sera that had reacted previously in the AGID test. Five different serotypes of BT and one serotype of EHD virus were studied. Reactors to BT serotype 20 were the most prevalent, followed by EHD type 5 and BT types 21, 12, 1 and 17. Antibodies against BT serotype 20 were present in cattle, buffaloes, goats and sheep, but were most common in buffaloes. Buffaloes showed the highest exposure to the BT serotypes tested. Antibody to EHD type 5 occurred most frequently in cattle. Antibodies against all BT and EHD serotypes tested were found in buffaloes and cattle while goats had antibodies against BT types 20, 21 and EHD type 5 and sheep had antibodies only against BT type 20.

Nucleotide and amino acid sequence analysis of the thymidine kinase gene of a bovine encephalitis herpesvirus.

The nucleotide and predicted amino acid sequence of the thymidine kinase (TK) gene of N 569, a bovine encephalitis herpesvirus (BEHV), has been determined and compared with those of avian, bovine and other mammalian herpesvirus TK genes. Striking differences were observed between the nucleotide sequence of this BEHV TK gene and those reported for bovine herpesvirus 1 (BHV-1). A total of 118 base changes, 39 base deletions and 14 base insertions were identified relative to the TK sequence of a BHV-1.2a strain, resulting in a net loss of seven residues. Comparison of the TK sequences of BEHV and the BHV-1 Q 3932 strain with that reported for the BHV-1 6660 strain suggested that the latter may have contained sequencing errors. The most significant difference between the BEHV TK sequence and those of BHV-1 strains was the absence of a contiguous stretch of ten codons.

The pathogenesis of velogenic Newcastle disease virus infection of chickens of different ages and different levels of immunity.

Chickens of 7 weeks or 20 weeks of age were divided into three groups according to their antibody status (high, low, absent) and were infected with a velogenic viscerotropic Newcastle disease virus. To follow patterns of viral replication, birds were necropsied at regular intervals up to 22 days and organs were sampled from each bird. In non-immune birds, virus could be isolated from all organs examined. In birds with antibody, virus was most frequently isolated from the proventriculus, cecal tonsil, bursa, and brain. However, because no one organ could be recommended for all situations, all four should be sampled for field diagnosis. In immune birds, although clinical signs were either mild or absent, widespread virus replication occurred up to 19 days post-challenge.

The location and nucleotide sequence of the thymidine kinase gene of bovine herpesvirus type 1.2.

On the basis of their restriction endonuclease digestion patterns, four Australian bovine herpesvirus type 1 (BHV-1) isolates were classified as belonging to the BHV-1.2a subtypes. The thymidine kinase (TK) genes of all four BHV-1.2a isolates were located on a 3.5 kb SalI restriction fragment. This is in contrast to North American and European BHV-1.1 isolates whose TK genes are contained on a 2.6 to 2.8 kb SalI fragment. The restriction fragments containing the TK genes were cloned into phagemid vectors and their sequences determined using the dideoxynucleotide chain termination method. The BHV-1.2a isolates possessed identical TK gene sequences, which differed from previously published TK sequences for the LA and 6660 BHV-1.1 strains. In addition to five single base alterations, there were six separate base insertions which resulted in two major frameshifts which spanned an area of 72 amino acids or 20% of the expressed TK gene product. The predicted amino acid sequence exhibited a higher degree of similarity to other herpesvirus TKs, suggesting that previously published TK gene sequences may have been incorrect. The present nucleotide sequence and corresponding amino acid composition reinforces previous observations concerning regions of herpesvirus TK amino acid conservation and should assist in future studies into the evolution and functional domains of herpesvirus TKs.

Demonstration of vascular permeability changes in cattle infected with bovine ephemeral fever virus.

Five cattle infected with bovine ephemeral fever virus were necropsied on the day after onset of clinical disease, when clinical signs of lameness were most severe. Gross lesions observed included a serofibrinous polyserositis involving the synovial, pericardial, thoracic and abdominal cavities. The associated histological changes consisted primarily of oedema and an influx of neutrophils into affected tissues and fluids. In a further eight infected cattle, increases in permeability of vessels associated with serosal surfaces were demonstrated by labelling with either colloidal carbon or Evans blue. Intravenous injections of carbon provided both macroscopic and histological labelling of affected vessels. Evans blue appeared to be more sensitive than carbon but did not provide a histological marker of vascular permeability and provided labelling of tissues rather than individual vessels. The main sites of increased permeability were synovial, pericardial, thoracic and abdominal serosae.