Approaches to dog health education programs in Australian rural and remote Indigenous communities: four case studies.

Dog health in rural and remote Australian Indigenous communities is below urban averages in numerous respects. Many Indigenous communities have called for knowledge sharing in this area. However, dog health education programs are in their infancy, and lack data on effective practices. Without this core knowledge, health promotion efforts cannot progress effectively. This paper discusses a strategy that draws from successful approaches in human health and indigenous education, such as dadirri, and culturally respectful community engagement and development. Negotiating an appropriate education program is explored in its practical application through four case studies. Though each case was unique, the comparison of the four illustrated the importance of listening (community consultation), developing and maintaining relationships, community involvement and employment. The most successful case studies were those that could fully implement all four areas. Outcomes included improved local dog health capacity, local employment and engagement with the program and significantly improved dog health.

A single amino acid is critical for the expression of B-cell epitopes on the helicase domain of the pestivirus NS3 protein.

Truncated NS3 proteins, expressed by recombinant baculoviruses, were used to investigate the location of conserved B-cell epitopes on this non-structural bovine viral diarrhoea virus (BVDV) protein. A goat anti-pestivirus antiserum, and a panel of anti-NS3 monoclonal antibodies, including the BVDV-1 specific antibody P1D8, were used to verify the presence or absence of the epitopes. Interestingly, the monoclonal antibodies reacted only with the truncated protein encompassing the helicase domain of NS3. Expression of the B-cell epitopes was dependent on, but not within, a 57 amino acid sequence at the carboxy-terminal end of this protein, supporting observations that these conserved epitopes are conformational in nature. A comparison of deduced amino acid sequences of the helicase domain from BVDV-1, BVDV-2, BDV and CSFV isolates highlighted a single amino acid that appeared to be unique to P1D8-reactive BVDV-1 isolates. Site-directed mutagenesis studies confirmed that this amino acid is critical for the expression of the BVDV-1 specific NS3 epitope recognised by the P1D8 monoclonal antibody. Surprisingly, the amino acid was also important for an epitope recognised by two group-specific monoclonal antibodies, P1H11 and P4A11. Protein modelling studies, based on the structure of the hepatitis C NS3 helicase domain, indicated that this amino acid occupies a prominent position on the surface of the protein.

Lysophosphatidic acid-induced proliferation in opossum kidney proximal tubular cells: role of PI 3-kinase and ERK.

UNLABELLED: Lysophosphatidic acid-induced proliferation in opossum kidney proximal tubular cells: Role of PI 3-kinase and ERK. BACKGROUND: Lysophosphatidic acid (LPA) is a mitogenic lipid bound to albumin in the circulation and implicated in the induction of proximal tubular cell (PTC) injury in proteinuric states. In this study, we investigated the effect of LPA on proliferation of opossum kidney (OK) cells and the roles of the p85/p110 phosphatidylinositol 3-kinase (PI 3-kinase) and extracellular signal-regulated kinases (ERKs) ERK-1 and ERK-2 in LPA-induced proliferation. METHODS: [3H]-thymidine incorporation was used as an index of OK cell proliferation. PI 3-kinase and ERK activities were measured by in vitro kinase assays of immunoprecipitates from both wild-type OK cells and OK cells expressing a dominant negative p85 (Deltap85) subunit of PI 3-kinase in an inducible vector. RESULTS: LPA stimulated a marked increase in [3H]-thymidine uptake in wild-type and Deltap85 OK cells. OK cell PI 3-kinase activity was stimulated by LPA and was inhibited by expression of Deltap85. LPA-induced proliferation was inhibited by wortmannin and the induction of Deltap85 expression. These data suggest that LPA stimulates PI 3-kinase activity, which is essential for signaling the induction of proliferation. LPA also stimulated ERK activity (peak at 5 min, return to baseline by 60 min) maximally at a dose of 100 microM LPA. This increase was approximately 600% above basal and was similar to the effects of 10% fetal calf serum. The proliferative effect of LPA was decreased by the ERK-kinase (MEK) inhibitor PD98059 (5 microM), therefore suggesting that ERK as well as PI 3-kinase activation is important for proliferation. ERK activation by LPA was not affected by pretreatment with wortmannin or by the expression of Deltap85. PI 3-kinase activation by LPA was not affected by pretreatment with PD98059. CONCLUSIONS: We conclude that activation of PI 3-kinase is essential for the LPA-induced proliferation of OK cells and that ERK activation is also important. Therefore, they are both vital elements in separate signaling pathways leading to cell proliferation. LPA filtered into the proximal tubule in proteinuric states is likely to have profound effects on PTC growth.

Lysophosphatidic acid-induced calcium mobilization and proliferation in kidney proximal tubular cells.

Patients with proteinuria tend to develop progressive renal disease with proximal tubular cell atrophy and interstitial scarring. It has been suggested that the nephrotoxicity of albuminuric states may be due to the protein molecule itself or by lipids, such as lysophosphatidic acid (LPA), that albumin carries. LPA was found to cause a transient increase in intracytoplasmic free Ca2+ ([Ca2+]i) in opossum kidney proximal tubule cells (OK) that was maximal at 100 microM LPA and was dose dependent with an EC50 of 2.6 x 10(-6) M. This Ca2+ mobilization was from both internal stores and across the plasma membrane and was pertussis toxin (PTX) insensitive. Treatment of OK cells with 100 microM LPA for 5 min was found to cause a twofold increase in [3H]thymidine incorporation and a three- to fivefold increase over control after 24 h. This was highly PTX sensitive and insensitive to pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A. These findings may be of significance in the progression of renal disease and indicate the potential importance of lipids in modulating proximal tubule cell function and growth.

Rapid screening of embryonated chicken eggs for bluetongue virus infection with an antigen capture enzyme linked immunosorbent assay.

The sensitivity and specificity of an antigen capture ELISA have been compared with virus isolation in cell culture. Bluetongue virus (BLU) (serotype 23) from the blood of a sheep was titrated by inoculating embryonated chicken eggs (ECEs) and detecting viral antigen in chicken embryo livers using an antigen capture enzyme linked immunosorbent assay (ELISA) (Stanislawek et al., 1996. Detection by ELISA of bluetongue antigen directly in the blood of experimentally infected sheep. Vet. Microbiol. 52, 1-12). Five days after inoculation of ECEs with lysed red blood cells from the infected sheep the embryo livers were harvested and homogenised. The supernatant from the homogenate was used in the antigen capture ELISA to determine which livers were infected and the virus titre calculated as CEID50/ml packed red blood cells. These results were compared with a standard cell culture isolation protocol which passaged the liver homogenate supernatant through Aedes albopictus cells and up to three passages in BHK21 cells. The antigen capture ELISA showed 100% sensitivity and specificity with no false negatives or false positives when compared to cell culture isolation of the virus. The major advantage of the combination of ECE inoculation and antigen capture ELISA is the reduction in the time to less than 7 days from a maximum of 35 days for the ECE/cell culture system. The procedure is easy to undertake, cost effective and does not require expensive specialist cell culture facilities.

Tyrosine phosphatase antagonist-induced activation of the neutrophil NADPH oxidase: a possible role for protein kinase C.

To investigate the role of tyrosine phosphorylation in polymorphonuclear leucocyte (PMN) activation we have examined the effect of the potent tyrosine phosphatase (PTPase) inhibitor, vanadyl hydroperoxide, on PMN function. Western blotting of vanadyl hydroperoxide-treated PMN showed that there was a rapid dose-dependent increase in tyrosine-phosphorylated proteins. Vanadyl hydroperoxide also induced superoxide production in PMN over the range 10-100 microM, similar to the concentrations that also induced tyrosine phosphorylation. The tyrosine kinase inhibitor erbstatin totally inhibited the respiratory burst induced by vandyl hydroperoxide, showing that tyrosine kinase activity was necessary for superoxide production. The protein kinase C (PKC) inhibitors chelerythrine and bisidolylmaleimide inhibited the vanadyl hydroperoxide-induced respiratory burst with an inhibitory concentration of 50% (IC50) close to that for PKC inhibition without affecting tyrosine phosphorylation. These results indicate a possible role for PKC in vanadyl hydroperoxide-mediated superoxide production, and that any PKC involvement is downstream of tyrosine phosphorylation. These results further demonstrate that inhibition of phosphotyrosine phosphatases results in the activation of a functional response, indicating a critical role for phosphotyrosine phosphatases in PMN stimulation.

The phosphotyrosine phosphatase inhibitor vanadyl hydroperoxide induces morphological alterations, cytoskeletal rearrangements and increased adhesiveness in rat neutrophil leucocytes.

The functional consequences of treating rat neutrophils with the potent tyrosine phosphatase inhibitor vanadyl hydroperoxide (pervanadate) has been investigated. Pervanadate induced rapid increases in cellular protein phosphotyrosine content in a dose-dependent manner. This treatment also resulted in a change in morphology of the cells from a rounded to a polarised morphology, with many cells exhibiting uropods, pseudopodia and increased membrane activity. Pervanadate induced a transient actin polymerisation and reorganisation similar to that in agonist-stimulated cells. The pervanadate-induced increases in tyrosine phosphorylation, shape change and actin polymerisation were inhibited by the tyrosine kinase inhibitors tyrphostin and erbstatin, indicating that these phenomena were mediated by the constitutive activity of cellular tyrosine kinases. Double fluorescence experiments demonstrated that there was a co-localisation of tyrosine phosphorylated proteins with F-actin in both pervanadate- and agonist-stimulated neutrophils. Pervanadate also induced spreading of neutrophils on tissue culture substrata with concurrent changes in F-actin localisation including unusual F-actin-containing structures. These results demonstrate that morphological changes and cytoskeletal reorganisation in neutrophils are regulated by tyrosine phosphorylation, and that inhibition of tyrosine phosphatase activity in neutrophils is sufficient to activate motile machinery of these cells. These results suggest that an alternative pathway involved in neutrophil stimulation might be via inhibition of endogenous tyrosine phosphatases rather than activation of tyrosine kinases.

Production, hatchability and fertility of eggs from breeding Japanese quail (Coturnix coturnix japonica) fed diets containing furazolidone.

1. Breeding Japanese quail were allocated to 8 groups, each group consisting of 20 females and males. The birds were fed one of 4 diets for up to 33 d: a control diet or a diet containing 200 mg/kg, 400 mg/kg or 1000 mg/kg furazolidone. Subsequently, quails were fed a furazolidone-free diet for up to 21 d. Egg production, quality, hatchability and fertility of the groups were measured. 2. Significant reduction in egg production occurred in birds fed 400 mg/kg and 1000 mg/kg furazolidone, the effect being more pronounced at the higher concentration. 3. Hatchability was reduced significantly for all groups of birds fed furazolidone and this effect was both dose and time dependent. The reduction in hatchability was attributable to an increase in infertile eggs rather than an increase in embryonic mortality. 4. Egg quality was affected, with more small eggs being produced by birds fed 1000 mg/kg furazolidone. 5. After removal of the experimental diets egg production of the affected groups returned to control values. Hatchability and fertility of affected groups also returned toward control values, but had generally not attained these values 21 d after the cessation of the experimental diets. 6. It was concluded that standard recommendations for the therapeutic dosage of poultry with furazolidone may not be appropriate for breeding Japanese quail.

The pathogenesis of the nervous syndrome of Phalaris aquatica toxicity in sheep.

The clinical signs displayed by 96 sheep affected by the nervous syndrome of Phalaris aquatica toxicity and 10 normal sheep injected intravenously with the phalaris alkaloid, 5-methoxy dimethyltryptamine (dose range 0.01 to 5.0 mg/kg), were observed. The distributions of phalaris indole-like cytoplasmic pigments in nuclei of the brains and spinal cords of 9 naturally affected sheep were determined microscopically. Based on the relationship between clinical signs and the central nervous system nuclei involved in their production, the distribution of phalaris indole-like pigments, and the pharmacology of dimethylated tryptamines, it is suggested that the nervous syndrome induced by Phalaris aquatica results from a direct action of phalaris alkaloids upon serotonergic receptors in specific brain and spinal cord nuclei.

Upper motor neurone effects in sheep of some beta-carboline alkaloids identified in zygophyllaceous plants.

The beta-carbolines harmane, norharmane, tetrahydronorharmane, harmine, harmaline and harmol were administered to sheep to assess their effects on upper motor neurone function. Harmane at a dose rate of 54 mg/kg induced hypomotility, head tremors, pelvic limb paresis, hypermetria and a wide based stance. A range of similar effects were observed with norharmane at the same dose rate. Tetrahydronorharmane at a dose rate of 54 mg/kg induced hypermotility followed by hypomotility, asymmetrical pelvic limb paresis, hypermetria, a wide based stance, and stereotyped eating behaviour. Harmine and harmaline at 6 mg/kg induced mild head and body tremors, and at 18 mg/kg induced hypomotility, intense head and body tremors, pelvic limb paresis, crossing over of limbs, neck extension and head swaying. Harmol was not effective at 54 mg/kg by either the subcutaneous or intraperitoneal routes, but at an intravenous dose of 27 mg/kg it induced hypermotility followed by hypomotility, body tremors, limb paresis, muscle asynergy, a wide based stance and jumping behaviour. Harmane, tetrahydronorharmane, harmaline and harmol were convulsive in some sheep at high dose rates.

Reduced duck hepatitis B virus viraemia in ducklings coinfected with the immunodepressive reticuloendotheliosis virus.

Coinfection of avian hosts by duck hepatitis B virus (DHBV) and reticuloendotheliosis virus (REV) was studied to assess the effect of immunodepression by REV on the replication of DHBV. One-day-old ducklings, domestic chickens, and turkey poults were inoculated either with DHBV or DHBV and REV and were bled and weighed at regular intervals. DHBV infection as manifested by viraemia and DHBV DNA in liver was established only in ducklings. All chickens and turkeys were negative for DHBV DNA in serum and liver. However, ducklings coinfected with REV showed a delayed onset and reduced level of viraemia compared to ducklings infected only with DHBV. The narrow host range of DHBV was confirmed even in immunodepressed species. It is suggested that the reduction in DHBV viraemia in ducklings was due to factors not involving the specific immune system.

Natural infection of duck embryos with duck hepatitis B virus: time and tissue sequence of infection.

There have been no studies addressing the detailed sequence of embryonic infection with duck hepatitis B virus (DHBV). Therefore, duck embryos from flocks infected with DHBV were examined to study the sequence of infection by DHBV in various embryonic tissues. Embryos from flocks infected with DHBV were harvested in duplicates from 7 to 25 days of incubation. Whole embryos (to 12 days) or dissected embryonic tissues were fixed, paraffin embedded, and stained for DHBV surface antigen (DHBsAg) using a peroxidase-antiperoxidase technique. Isolated hepatic cells were infected in 7-day-old embryos, and these increased in number until 11 days, when most cells were positive for DHBsAg. Endocrine pancreatic cells were positive from day 10, but only an occasional exocrine pancreatic cell was infected after day 20. Renal tubule cells were positive for DHBV by day 11, increasing in number until about day 18, after which a decline in numbers of infected cells occurred. Renal glomeruli became positive for DHBsAg from day 24. When present in the developing embryo, thymus, bursa of Fabricius, spleen, bone marrow, lung, and duodenum remained negative for DHBsAg. It was concluded that the timing of infection of specific tissues was not necessarily related to cellular maturity but may reflect a need for specific metabolic functions that permit viral replication.

Investigations into the toxicity of Echium plantagineum in sheep. 1. Field grazing experiments.

A field grazing trial was undertaken to monitor the health and production of crossbred sheep grazing pasture where Echium plantagineum constituted a considerable proportion of the available forage. The trial, conducted for 19 months over successive grazing seasons, demonstrated a significant difference in production, with sheep on the E. plantagineum pasture being lighter and growing less wool compared with sheep on Echium-free pasture. No mortalities involving pyrrolizidine alkaloid poisoning were recorded in sheep grazing E. plantagineum, although there was histological evidence of moderately severe liver damage associated with high liver copper concentrations in at least one sheep following the grazing of large quantities of the plant.

Investigations into the toxicity of Echium plantagineum in sheep. 2. Pen feeding experiments.

In a pen feeding trial fresh Echium plantagineum was fed as the sole diet to crossbred sheep with or without a history of previous access to the plant. Control groups received a diet of lucerne chaff and oats. During the trial, sheep on the Echium diet lost weight and deaths occurred with histological evidence of excessive copper accumulation, usually accompanied by pyrrolizidine alkaloid damage, in the liver and biochemical evidence of liver toxicity. It is concluded that E. plantagineum alone is not a suitable fodder for sheep and can be toxic due to its pyrrolizidine alkaloid content and high copper to molybdenum ratio.

Immunity in Pekin ducks experimentally and naturally infected with duck hepatitis B virus.

The immune response to duck hepatitis B virus (DHBV) had not been elucidated. An assay was therefore established to detect the presence of antibody to DHB surface antigen (anti-DHBs) in serum of experimentally inoculated and naturally infected ducks. Anti-DHBs in serum was detected by indirect RIA from the percentage inhibition of binding of rabbit anti-DHBs to purified DHBsAg. Specificity was confirmed by positive and negative controls, infected and noninfected sera, and a mouse monoclonal antibody to DHB core antigen (anti-DHBc). Serum and liver samples were tested for DHBV DNA by dot-blot hybridization assay. Adult ducks repeatedly inoculated with DHBV remained non-viraemic but developed anti-DHBs. This antibody activity neutralized the infectivity of DHBV, which was experimentally inoculated into 1-day-old ducklings. In naturally infected flocks anti-DHBs was detected in a proportion of noninfected adult ducks as well as 1-day-old hatchlings. Anti-DHBs activity in hatchlings neutralized the infectivity of experimentally inoculated DHBV. Pekin ducks can therefore mount a neutralizing antibody response to DHBV, and immunity may be transferred in ovo from dam to off-spring.

Physiological changes associated with the production of defective egg-shells by hens receiving sodium chloride in the drinking water.

1. Supplementing the drinking water of laying hens with 600 or 2000 mg sodium chloride/l induced large increases in egg-shell defects without corresponding changes in egg production, egg weight or food and water intakes. A supplement of 2000 mg NaCl/l resulted in a high incidence of shell-less eggs. 2. The increased incidence of egg-shell damage in hens receiving the NaCl was associated with a decrease in egg-shell quality measured objectively. These responses persisted even after the NaCl was removed from the drinking water. 3. The NaCl treatment had little effect on blood acid-base balance and electrolytes, but significant reductions were observed in the carbon dioxide tension, and bicarbonate and calcium concentrations in the fluid surrounding the egg in the shell gland. 4. The poor shell quality appeared to be associated with a reduced supply of bicarbonate, rather than with an effect on Ca, in the lumen of the shell gland, although a reduced residence time of eggs in the shell gland may also have contributed to the problem.

Experimental evidence that tryptamine alkaloids do not cause Phalaris aquatica sudden death syndrome in sheep.

The acute toxicity for sheep of 3 alkaloids that occur in Phalaris acquatica was examined by intravenous and oral administration. The lowest tested dose rates that produced clinically observed signs were, for 5-methoxy dimethyltryptamine, 0.1 mg/kg body weight intravenously and 40 mg/kg orally; for gramine, 10 mg/kg intravenously and 500 mg/kg orally; and for hordenine, 20 mg/kg intravenously and 800 mg/kg orally. All induced the clinical signs observed in the nervous form of phalaris toxicity, but none induced the cardiac, sudden death, syndrome.

Experimental duck hepatitis B virus infection: pathology and evolution of hepatic and extrahepatic infection.

Seventy, 1-day-old ducklings inoculated intraperitoneally with duck hepatitis B virus and 30 controls have been studied over a 2-year period. Infection with duck hepatitis B virus occurred in all inoculated ducks, although this was not associated with clinical morbidity. Duck hepatitis B virus DNA was first detected in liver on Day 3, in pancreatic acinar cells on Day 4, serum on Day 6, splenic red and white pulp on Day 7 and in the renal glomurulus on Day 14, using a combination of dot, Southern blot and in situ hybridization techniques. Peak levels of circulating virus, as determined by DNA polymerase levels, occurred 1 to 4 weeks postinoculation. Mild degrees of portal inflammation were seen in sections of liver tissue in both infected and control ducks. However, moderately severe inflammatory changes were present in 8 of 22 infected birds compared with 0 of 18 controls (p less than 0.025). Appearance of this inflammatory infiltrate 6 weeks postinoculation coincided with a decrease in levels of duck hepatitis B virus DNA in hepatocytes and within the pancreatic acinar cells. At the same time, duck hepatitis B virus DNA became increasingly localized to the splenic germinal centers, and viral DNA was first detected in pancreatic islet cells. No histological changes accompanied the extra-hepatic tissue infection. The sequence and significance of duck hepatitis B virus infection in liver and extra-hepatic tissues is discussed in relation to the pathogenesis of hepatitis B virus infection in man.

Investigations into the association of retroviruses with ovine lymphoma.

To determine whether retroviruses are associated with sporadic ovine lymphoma, suspension cell cultures of four lymphomas and one control lymph node were labelled with tritiated uridine. Following differential and sucrose density gradient ultracentrifugation of their media a peak of radioactivity was found at a density of 1.15 to 1.18 g ml-1 in preparations from the cell cultures of two lymphomas and the normal lymph node. Sedimentation velocity centrifugation of the sodium dodecyl sulphate-treated radiolabelled material found an approximate sedimentation value of 7S. The assay for RNA directed DNA polymerase in ultracentrifuged pellets of media from cultures of 15 lymphomas and one control lymph node found activity in material from four lymphomas and the control node cultures. However, little variation in incorporation kinetics occurred with changes in assay conditions and activities were not associated with particles of density 1.15 g ml-1. It was concluded that the detected activities were not retroviral in origin.