Phylogeography,genetic stocks,and conservation implications for an Australian endemic marine turtle

1. Identification of the geographic extent of population boundaries, the distribution of genetic lineages, and the amount of genetic exchange among breeding groups is needed for effective conservation of vulnerable marine migratory species. This is particularly true of the flatback turtle (Natator depressus), which only breeds in Australia but has extensive migrations that can include international waters.
2. This study investigated the phylogeography and genetic structure among 17 flatback turtle rookeries across their range by sequencing an 810 bp portion of the mitochondrial DNA in 889 samples and genotyping 10 microsatellite loci in 598 samples.
3. There was low phylogenetic divergence among haplotypes and evidence of recent population expansion, likely in the late Pleistocene. A predominant haplotype was found across all rookeries, but other haplotype groups were regionally specific.
4. In general, there was agreement in patterns of genetic differentiation in the mitochondrial DNA and microsatellite data, and in some pairwise comparisons a higher mutation rate of microsatellites provided stronger evidence of differentiation.
5. These results suggest natal philopatry operates in the choice of breeding locations for males as well as females.
6. Evidence of genetic connectivity among neighbouring rookeries led to the identification of seven genetic stocks. Geographic boundaries of rookeries used by genetic stocks varied widely (160–1,300 km), highlighting a need for field studies to better understand movement patterns.
7. Hierarchical analysis of molecular variance identified significant genetic differentiation based upon genetic stock, nesting phenology (summer vs. winter nesters), and a west–east discontinuity across Torres Strait. A pattern of isolation by distance was identified, which was most strongly observed in the microsatellite data.
8. In combination with tagging and telemetry studies, these results will allow better quantification of stock‐specific threats along migratory routes and in foraging habitats. Implications of climate change will be stock specific and may depend upon the extent of genetic connectivity between neighbouring stocks.

     

Post‐disturbance haulout behaviour of harbour seals

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Comparative study of electron microscopical techniques for the detection of scrapie-associated fibrils

Samples of cervical spinal cord and four anatomical regions of the brains of 12 sheep with natural scrapie and six control sheep were examined by electron microscopy, after the tissues had been stored at 4°C and −20°C. The tissues were tested for the presence of scrapie-associated fibrils by a centrifugal extraction technique and by a touch-grid technique. The touch-grid technique was no better than the centrifugal extraction technique for the detection of fibrils. Structures which could have been classified as tubulofilaments were detected in touch-grid preparations without detergent treatment. With the centrifugal extraction technique there was a significant reduction of the fibril scores in some of the tissue extracts stored at −20°C, but not in any of the extracts stored at 4°C. There was, however, a reduction in the fibril scores when the final extracted pellets were stored at 4°C. The stability of the fibrils on the test grids was unaffected by six months storage at room temperature but the clarity of their ultrastructure did deteriorate. Poor hydrophilic spread of the sample on the test grids did not have a significant effect on the fibril scores.     

Preliminary observations on the experimental transmission of scrapie to elk (Cervus elaphus nelsoni) by intracerebral inoculation

To determine the transmissibility of scrapie to Rocky Mountain elk (Cervus elaphus nelsoni), six elk calves were inoculated intracerebrally with brain suspension from sheep naturally affected with scrapie. One elk developed a brain abscess and was euthanatized at 7 weeks postinoculation (PI), and two others died at 6 and 15 months PI because of physical injuries. At 25 and 35 months PI, two other elk died after brief terminal neurologic episodes. Necropsy of these revealed moderate weight loss but no other gross lesions. Microscopically, characteristic lesions of spongiform encephalopathy were seen throughout the brains and the spinal cords, and in both cases these tissues were positive for PrP(res) by immunohistochemistry. Brains of both animals were positive for PrP(res) by western blot and for scrapie-associated fibrils (SAFs) by negative stain electron microscopy. PrP(res) and SAFs were not detected in the three elk that died or were euthanatized because of coincidental causes. Over 3.5 years after initiation of this experiment, the one remaining inoculated elk and two uninoculated (control) elk are alive and apparently healthy. These preliminary findings demonstrate that 1) sheep scrapie agent can be transmitted to elk by intracerebral inoculation; 2) the infection can result in severe, widely distributed spongiform change and accumulations of PrP(res) in the central nervous system (CNS); and 3) based on the examination of a limited number of CNS sections from two cases, this condition cannot be distinguished from chronic wasting disease with currently available diagnostic techniques.     

Development and validation of an ELISA to detect antibodies to Corynebacterium pseudotuberculosis in ovine sera

Several enzyme-linked immunosorbent assays (ELISAs) have been developed for the detection of antibodies to Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis (CLA). However, none are commercially available in the UK. It was therefore necessary to develop a new, economic ELISA for use in a research project studying the epidemiology of CLA in UK sheep. The ELISA with its diagnostic qualities is presented. The ELISA was developed using sonicated C. pseudotuberculosis and optimised to detect total antibody or IgG class antibody in serum. Receiver operating characteristic (ROC) curves were obtained and the area under the ROC curve was used to compare the sensitivity and specificity of the two ELISAs. Both versions of the ELISA were evaluated on a panel of 150 positive reference sera and 103 negative reference sera. Using the test at 100% specificity, the sensitivity of detection of total antibody was 71% (95% confidence interval 63-78%), and the sensitivity of detection of IgG antibody to C. pseudotuberculosis was 83% (76-89%), which compares favourably with other reported ELISA tests for CLA in sheep. The sensitivity of the IgG antibody assay may be higher because of the greater affinity of IgG class antibodies compared with the IgM antibodies also detected by the total antibody ELISA. The results of ROC analysis indicated that the IgG isotype ELISA was more accurate than the total antibody ELISA. The efficiency of the test was greatest when serum samples were run in a dilution series than when any single serum dilution was used. The ELISA is considered to be suitable for application in field studies of CLA in UK sheep.     

Ontogeny of systemic cellular immunity in the neonatal pig: correlation with the development of post-weaning multisystemic wasting syndrome

The aetiology of porcine post-weaning multisystemic wasting syndrome (PMWS) is poorly understood. Porcine circovirus type 2 (PCV-2) is an essential component of the experimental disease model for PMWS: however, evidence from experimental and field studies indicates that additional factors play a critical role in the aetiopathogenesis of PMWS. Current candidates include (1) immune stimulation (for example, via co-infection or vaccination), and (2) a novel infectious agent. A prospective, longitudinal case-control study was designed to investigate molecular triggers in leucocytes of neonatal piglets that may predispose to the development of PMWS. Blood samples were collected weekly from pigs (n=125) within five farms, from 1 week to 8 weeks of age: that is, before the appearance of clinical signs. Four colour flow cytometry was used to investigate changes in subsets of peripheral blood mononuclear cells, using monoclonal antibodies against the following cell associated markers; sIgG, CD3, MHCII dR, CD14, CD4a, CD8a, CD45RC, CD25, SWC3a, SWC8, CD163 and CD45. Sampling and laboratory analysis was supported by monitoring of clinical signs from 1 week to 20 weeks of age, or until disease supervened. At the conclusion of the study, 68 pigs (54%) were classified in Group 1 (no signs of clinical disease), 34 pigs (27%) in Group 2 (signs of clinical disease but not characteristic of PMWS), 17 pigs (14%) in Group 3 (suspect PMWS case) and 5 pigs (4%) in Group 4 (PMWS case). A single case of Porcine Dermatitis and Nephropathy syndrome (PDNS) was also diagnosed. Significant changes with age were demonstrated in clinically normal, neonatal pigs (Group 1), including an increase in B-cells and T-cells, and an increase in the proportion of total T-cells expressing MHCII. Within the T-cell subset, the proportion of CD8(+high) CD4(-) T-cells increased, in addition to the proportion of CD4(+) T-cells co-expressing CD8. Of the factors recorded, farm was found to have a highly significant effect on immune system development in the neonate. Comparison of Groups 1 and 4 cases identified significant differences between pigs which remained normal and those which subsequently developed PMWS. Pigs which went on to develop PMWS had a greater proportion of T-cells expressing MHCII in early life, higher mean intensity of expression of MHCII on T-cells, higher mean intensity of expression of MHCII on B cells and higher expression of CD25 on CD45RC(-) T-cells. These findings suggest that lymphocyte activation may be a key early event in the aetiology of PMWS.     

Diffuse osteomyelitis of the fourth metacarpal bone in a horse caused by Clostridium perfringens

Osteomyelitis in adult horses, often associated with trauma or iatrogenic infection following surgery, usually presents as a focal area of infected bone. Diffuse osteomyelitis, affecting both the cortex and medulla, along the full length of a bone is encountered less frequently and treatment of such infections is not well reported in horses. The 2-year-old Warmblood mare in this case was diagnosed with diffuse osteomyelitis affecting the 4th metacarpal bone with concurrent unicortical fracture of the third metacarpal bone following traumatic injury. Computed tomography (CT) aided diagnosis in this case, providing critical additional detail to that obtained with radiography and ultrasound. This case highlights the value of CT in the diagnosis of diffuse osteomyelitis. This is the first reported case of diffuse osteomyelitis caused by Clostridium perfringens in horses. Successful treatment in this case consisted of surgical debridement of the associated abscess, followed by systemic and locoregional antimicrobial therapies.