Lythrum hyssopifolia (lesser loosestrife) poisoning of sheep in Victoria.

OBJECTIVE: To document an ovine disease attributed to the consumption of Lythrum hyssopifolia (lesser loosestrife). PROCEDURES: Historical and histological review of field and experimental cases. RESULTS: 1-20% mortality occurred in sheep flocks grazing paddocks where L. hyssopifolia was the predominant green vegetation. Well-documented disease outbreaks occurred in summer on nine farms across Victoria between 1974 and 2002. Liver damage occurred in all nine outbreaks, with kidney damage in at least eight. Hepatocyte necrosis was usually zonal to midzonal (zone 2) in the liver samples from four farms and periacinar (zone 3) in those from three farms, but some livers showed only single-cell necrosis. Multinucleate hepatocytes near necrotic areas were a feature in six cases. Proximal tubular epithelium appeared to be the primary renal target and brown granules were often present in renal tubules. Biochemical and histological evidence of liver and kidney damage was obtained from two sheep experimentally pen-fed harvested L. hyssopifolia. CONCLUSION: Chemicals in L. hyssopifolia are toxic to ovine hepatocytes and renal tubular epithelial cells.

Development of a Johne's disease infection model in laboratory rabbits following oral administration of Mycobacterium avium subspecies paratuberculosis.

To assess the rabbit as a model for the study of Johne's disease pathogenesis, a breeding group of adult and juvenile New Zealand white rabbits were orally challenged with three doses of the Mycobacterium avium subspecies paratuberculosis wildtype bovine strain, CLIJ623, on three occasions. Faecal culture, post-mortem tissue bacteriological culture and histopathology were used to monitor the disease progression in the rabbits for more than 2 years. Of 4 adult and 16 juvenile orally dosed rabbits M. paratuberculosis organisms were recovered bacteriologically from two and three animals, respectively, using the BACTECtrade mark radiometric culture system. Tissue sites from which the bacteria were recovered included the mesenteric lymph nodes, ileocaecal valve, vermiform appendix, caecum, proximal colon and jejunum. Body weight loss, reduced abdominal fat and mild lesions were observed at necropsy in four infected rabbits. Diarrhoea and persistent faecal shedding of bacteria were not observed. Faecal culture did not yield any cultivable mycobacterial organisms on solid media.

Improved vectors for expression library immunization--application to Mycoplasma hyopneumoniae infection in pigs.

Expression library immunization (ELI) has previously been used in a number of disease models in mice. Here, we describe the first example of the application of ELI to a large animal model with the immunization of pigs against enzootic pneumonia, a disease caused by Mycoplasma hyopneumoniae. The development of new plasmid vectors and library screening methods facilitated the application of ELI to this disease by allowing random libraries to be screened for clones expressing recombinant proteins. In this way the vast majority of clones in random libraries that are unproductive can be eliminated, meaning that libraries are more likely to give protection and are subsequently easier to further screen and analyze. By using this approach we have used one library screen and two animal trials to progress from an original library of 20,000 clones to a group of just 96 clones.

A prime-boost vaccination strategy using naked DNA followed by recombinant porcine adenovirus protects pigs from classical swine fever.

Weaned pigs (6-week-old) and 7-day-old pre-weaned piglets were vaccinated with naked plasmid DNA expressing the gp55/E2 gene from classical swine fever virus (CSFV). Both groups of pigs were then given a booster dose of recombinant porcine adenovirus expressing the gp55 gene (rPAV-gp55). Following challenge with CSFV, 100% of weaned pigs and 75% pre-weaned piglets were protected from disease. Weaned pigs given a single dose of rPAV-gp55 were also protected, but showed a slight increase in temperature immediately post-challenge. However, weaned animals given a DNA prime before rPAV-gp55 showed no fluctuation in body temperature following challenge and no pathology in spleen or lymph nodes upon post-mortem. In addition, no CSFV could be re-isolated from the rPAV vaccinated group and from only one pig in the prime-boost group following challenge, suggesting that both vaccination regimes have the potential to reduce or prevent virus shedding following experimental challenge.

Protection of pigs against classical swine fever with DNA-delivered gp55.

Classical swine fever virus causes significant mortality and morbidity in commercial piggeries in many countries in Europe and Asia. The protective antigen, gp55, is highly conformation-dependent and thus killed virus or bacterially produced proteins are not protective. This report demonstrates that DNA vaccination with the gene encoding gp55 can provide protective immunity with inoculation of two doses of 25 microg DNA or a single shot of 200 microg. Furthermore, the DNA can be delivered intramuscularly or by a simple spring-loaded needleless inoculator. In addition it is shown that inoculation of the DNA at a single site conveys the same level of immunity as division of the dose between two sites.

Vaccination and protection of pigs against pleuropneumonia with a vaccine strain of Actinobacillus pleuropneumoniae produced by site-specific mutagenesis of the ApxII operon.

The production of toxin (Apx)-neutralizing antibodies during infection plays a major role in the induction of protective immunity to Actinobacillus pleuropneumoniae reinfection. In the present study, the gene encoding the ApxII-activating protein, apxIIC, was insertionally inactivated on the chromosome of a serovar 7 strain, HS93. Expression of the structural toxin, ApxIIA, and of the two genes required for its secretion, apxIB and apxID, still occurs in this strain. The resulting mutant strain, HS93C- Ampr, was found to secrete the unactivated toxin. Pigs vaccinated with live HS93C- Ampr via the intranasal route were protected against a cross-serovar challenge with a virulent serovar 1 strain of A. pleuropneumoniae. This is the first reported vaccine strain of A. pleuropneumoniae which can be delivered live to pigs and offers cross-serovar protection against porcine pleuropneumonia.

The complete nucleotide sequence of rabbit haemorrhagic disease virus (Czech strain V351): use of the polymerase chain reaction to detect replication in Australian vertebrates and analysis of viral population sequence variation.

The complete nucleotide sequence of the Czech strain of rabbit haemorrhagic disease virus (RHDV) was determined to be 7437 nucleotides in length with a 5-terminal non-coding region of 9 nucleotides and a 3'-terminal non-coding region of 59 nucleotides. Two open reading frames (ORFs) were found within this sequence coding for polypeptides of 2344 (nucleotides 10-7044) and 117 amino acids (nucleotides 7025-7378). The sequence of this isolate was approximately 1% different from that reported by Meyers et al., having 78 nucleotide changes which resulted in 30 amino acid differences, the majority of these clustering in the N-terminus of the large ORF and the middle of the viral coat protein. Only a single conservative amino acid change was seen in the smaller 3'-terminal ORF. Since the virus cannot at present be propagated in tissue culture, but isolated only after replication in rabbits, the reported sequence must be considered as a consensus sequence from the viral population. To gain some understanding of the possible sequence diversity within this virus population, 97 clones were sequenced from a polymerase chain reaction (PCR) fragment to determine the sequence diversity of the virus population. Four major classes of variant were described with mutations generally in the third base position of codons. A nested reverse transcriptase (RT) PCR (using sequence derived for the coat protein of RHDV) was used to determine the presence or absence of RHDV inoculated into non-host animal species. No replication of the virus was detected in 28 different vertebrate species other than rabbits. PCR tests on both mosquitoes and fleas feeding on RHDV infected rabbits were positive. The RT-PCR test was more sensitive when compared with an antigen capture ELISA to detect the presence of genomic RNA/or virus in infected rabbits.

Presence of rabbit haemorrhagic disease virus antigen in rabbit tissues as revealed by a monoclonal antibody dependent capture ELISA.

The production of monoclonal antibodies (mAbs) to a common antigenic region on rabbit haemorrhagic disease virus (RHDV) has enabled the development of a capture ELISA for virus detection. The assay was shown to detect reliably the presence of viral antigen in crude homogenates of a range of rabbit tissues and has provided the first evidence for the presence of RHDV in blood, serum and heart tissue. A limited time course study of the progression of virus infection revealed viral antigen is first detected in the liver at between 18 and 24 h post-infection (p.i.) and in the spleen between 24 and 30 h p.i. The assay displayed a high level of specificity, clearly differentiating between groups of infected (lowest observed optical density (O.D.) 0.45) and uninfected (highest O.D. 0.06) rabbits. No viral antigen was detected in the urine or faeces collected from infected rabbits at post-mortem. Experiments employing the 'spiking' of pools of urine and faecal homogenates collected from uninfected rabbits, with a known amount of virus, indicated that RHDV may not be present in the faeces of infected animals and if present in urine, it appears to undergo substantial degradation.

A competition ELISA for the detection of antibodies to rabbit haemorrhagic disease virus.

Monoclonal antibodies (mAbs) were raised to a preparation of rabbit haemorrhagic disease virus (RHDV) purified from the livers of experimentally infected rabbits. Rabbit antisera to RHDV significantly blocked the binding of two mAbs (2D3(3) and 2D4(5)) to RHDV-coated microplate wells in a competition ELISA. The virus-specific nature of these mAbs was confirmed by immunoperoxidase and immunofluorescence assays on formalin-fixed and fresh infected liver tissue. Utilization of one of these mAbs (2D3(3)) in a competition ELISA, resulted in an RHDV antibody assay which proved more specific than an indirect ELISA and more rapid and reliable than a haemagglutination inhibition assay for screening serum samples from wild and experimental rabbits.

Self-assembly, antigenicity, and immunogenicity of the rabbit haemorrhagic disease virus (Czechoslovakian strain V-351) capsid protein expressed in baculovirus.

Rabbit haemorrhagic disease virus (RHDV) capsid protein was expressed in a baculovirus system. Analysis of the expressed product showed that the recombinant protein, which is 60 kDa in size, was antigenic as revealed by its reactions in ELISA and Western blot with the antibodies raised against RHDV. Direct electron microscopy of the cell culture supernatant and the purified protein demonstrated that the capsid protein expressed in insect cells self-assembled to form empty virus-like particles (VLP) which are similar in size and morphology to that of native virus. These particles were immunoreactive with polyclonal anti-RHDV antibodies and with four monoclonal antibodies which recognise conformational epitopes of the virus. The results indicated that the VLPs were morphologically and antigenically indistinguishable from native virus. The recombinant VLPs induced high levels of RHDV-specific antibodies in rabbits and mice following immunisation. The immune response to the VLPs protected the rabbits following challenge with the virulent RHDV. In haemagglutination assays, the VLPs bound to human red blood cells similar to the native virus particles. The recombinant protein and or VLPs is suitable for the development of a rapid, sensitive and reliable test for detection of antibodies to RHDV and for use as a vaccine for domestic rabbits.

A review of heavy metal and organochlorine levels in marine mammals in Australia.

Study of toxic contaminants in marine mammal specimens collected around Australia is currently uncoordinated and piecemeal. Most states collect samples but there is little or no financial support for their analysis. This study combines data, published or unpublished, from 13 sources. Heavy metals have been analysed in about 676 specimens; over 400 were for mercury levels in P. macrocephalus taken at a whaling station. The remaining samples were mostly from toothed whales, a few baleen whales (< 20), pinnipeds (41) and dugongs (49). The most consistently analysed metals were lead, mercury and cadmium. Liver and kidney lead levels ranged from < 1-3 ppm; levels in bone were 0-418 ppm, with most less than 10 ppm. Mercury levels in a large sample of P. macrocephalus muscle were < 12.2 ppm. Mercury levels in the small number of samples from other species were 0.51-143 ppm (kidney), 1.52-479 ppm (liver) and < 0.1-36 ppm (muscle). Cadmium levels in liver (0-52 ppm) and kidney (0-106 ppm) were extremely variable. Levels greater than 10 ppm were recorded in many species and were especially high in Hydrurga leptonyx, Dugong dugon, Mesoplodon layardii and Pseudorca crassidens. Adult Tursiops truncatus inhabiting the inshore gulfs of South Australia had considerably higher levels of cadmium compared with other regions. Information on organochlorine levels is sparse (approximately 39 specimens) and suggest low levels when compared to other parts of the world. Total DDT was highest (28.4 ppm) in a neonatal Orcinus orca. Some high levels of DDT were recorded in Tursiops truncatus, Delphinus delphis and Arctocephalus pusillus doriferus. PCBs ranged from < 0.05 to 3.87 ppm. A comprehensive pathological assessment of marine mammals is needed in order to evaluate the effects of toxic contaminants.

Granulocytopaenia and thrombocytopaenia in dairy cattle--a suspected mycotoxicosis.

During a 6-week period, 22 Dairy Shorthorn cows and heifers died with granulocytopaenia and thrombocytopaenia. Clinical signs observed in the affected animals included increased salivation, pyrexia, depression, rumenal stasis, bilateral epistaxis, melaena, increased bleeding after removal of retained foetal membranes and rapid weight loss. Despite intensive antibiotic and vitamin K therapy and blood transfusions, all affected animals died. The aetiological agent, thought to be a fungal toxin, could not be isolated from post mortem specimens or pasture samples.

Feline panleukopenia virus replicates in cells in which cellular DNA synthesis is blocked.

The components of the cell cycle for a feline embryo cell line were defined. Thymidine (6mM)-supplemented medium reversibly arrested cells 1 h into the S phase of the cell cycle and was used in a double blocking procedure to synchronize cells to the early S phase. The kinetics of feline panleukopenia virus replication in synchronized cells was studied by using (i) inclusion body formation, (ii) a plaque assay for cell-associated and cell-free virus under one-step growth conditions, (iii) an enzyme immunoassay for viral protein, (iv) electron microscopy of infected cells, and (v) the detection and identification of viral replicative form DNA by restriction endonuclease analysis. Parallel studies by each of these procedures of the replication of feline panleukopenia virus in cells in which a 6 mM thymidine block was maintained indicated that parvovirus replicated with essentially similar kinetics in both unblocked, synchronized cells and in cells in which the block was maintained. Accordingly, a 6 mM thymidine-supplemented medium, although it effectively blocks cellular DNA synthesis, does not block the replication of parvovirus.

Generalized parvovirus disease in neonatal pups.

Generalized canine parvovirus disease was diagnosed in a litter of pups that died when 3 to 9 days old. Parvovirus was isolated from the heart, lungs, liver, kidneys, and small intestine. Histologically, lesions were characterized by hemorrhage and necrosis in the brain, liver, lungs, kidneys, lymphoid tissues, and gastrointestinal mucosa. Intranuclear inclusions were found in vascular endothelium, heart, lungs, liver, kidneys, adrenal cortex, and gastrointestinal tract.

Acute and chronic canine parvovirus myocarditis following intrauterine inoculation.

Canine myocarditis virus was inoculated into puppies in utero 8 days before parturition. Puppies were clinically normal at birth. One puppy died 23 days after inoculation and a second puppy was comatose before euthanasia on day 27 post inoculation. Both had acute, non-suppurative myocarditis, and parvovirus was isolated from the heart of each pup. Two other puppies remained clinically normal until euthanasia at 87 and 131 days after inoculation. These latter puppies had extensive, focal fibrosis within the myocardium. Despite weekly monitoring, ECG changes were not recorded. The apparent predilection of canine parvovirus for cardiac myocytes is discussed.