Rinderpest and peste de petits ruminants viruses exhibit neurovirulence in mice

Members of the morbillivirus genus, canine distemper (CDV), phocine distemper virus (PDV), and the cetacean viruses of dolphins and porpoises exhibit high levels of CNS infection in their natural hosts. CNS complications are rare for measles virus (MV) and are not associated with rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) infection. However, it is possible that all morbilliviruses infect the CNS but in some hosts are rapidly cleared by the immune response. In this study, we assessed whether RPV and PPRV have the potential to be neurovirulent. We describe the outcome of infection, of selected mouse strains, with isolates of RPV, PPRV, PDV, porpoise morbillivirus (PMV), dolphin morbillivirus (DMV), and a wild-type strain of MV. In the case of RPV virus, strains with different passage histories have been examined. The results of experiments with these viruses were compared with those using neuroadapted and vaccine strains of MV, which acted as positive and negative controls respectively. Intracerebral inoculation with RPV (Saudi/81) and PPRV (Nigeria75/1) strains produced infection in Balb/C and Cd1, but not C57 suckling mice, whereas the CAM/RB rodent-adapted strain of MV infected all three strains of mice. Weanling mice were only infected by CAM/RB. Intranasal and intraperitoneal inoculation failed to produce infection with any virus strains. We have shown that, both RPV and PPRV, in common with other morbilliviruses are neurovirulent in a permissive system. Transient infection of the CNS of cattle and goats with RPV and PPRV, respectively, remains a possibility, which could provide relevant models for the initial stages of MV infection in humans.

Specialised pacemaking cells in the rabbit urethra.

1. Collagenase dispersal of strips of rabbit urethra yielded, in addition to normal spindle-shaped smooth muscle cells, a small proportion of branched cells which resembled the interstitial cells of Cajal dispersed from canine colon. These were clearly distinguishable from smooth muscle in their appearance under the phase-contrast microscope, their immunohistochemistry and their ultrastructure. They had abundant vimentin filaments but no myosin, a discontinuous basal lamina, sparse rough endoplasmic reticulum, many mitochondria and a well-developed smooth endoplasmic reticulum. 2. Interstitial cells were non-contractile but exhibited regular spontaneous depolarisations in current clamp. These could be increased in frequency by noradrenaline and blocked by perfusion with calcium-free solution. In voltage clamp they showed abundant calcium-activated chloride current and spontaneous transient inward currents which could be blocked by chloride channel blockers. 3. The majority of smooth muscle cells were vigorously contractile when stimulated but did not show spontaneous electrical activity in current clamp. In voltage clamp, smooth muscle cells showed very little calcium-activated chloride current. 4. We conclude that there are specialised pacemaking cells in the rabbit urethra that may be responsible for initiating the slow waves recorded from smooth muscle cells in the intact syncitium.

Evaluation of a multi-functional nanocarrier for targeted breast cancer iNOS gene therapy.

The present study determines whether the novel designer biomimetic vector (DBV) can condense anddeliver the cytotoxic iNOS gene to breast cancer cells to achieve a therapeutic effect. We have previouslyshown the benefits of iNOS for cancer gene therapy but the stumbling block to future development hasbeen the delivery system.The DBV was expressed, purified and complexed with the iNOS gene. The particle size and chargewere determined via dynamic light scattering techniques. The toxicity of the DBV/iNOS nanoparticleswas quantified using the cell toxicity and clonogenic assays. Over expression of iNOS was confirmed viaWestern blotting and Griess test.The DBV delivery system fully condensed the iNOS gene with nanoparticles less than 100 nm. Transfectionwith the DBV/iNOS nanoparticles resulted in a maximum of 62% cell killing and less than 20%clonogenicity. INOS overexpression was confirmed and total nitrite levels were in the range of 18M.We report for the first time that the DBV can successfully deliver iNOS and achieve a therapeuticeffect. There is significant cytotoxicity coupled with evidence of a bystander effect. We concludethat the success of the DBV fusion protein in the delivery of iNOS in vitro is worthy of future in vivo experiments.

Getting priorities straight: Risk assessment and decision-making in the improvement of inherited disorders in pedigree dogs

The issue of inherited disorders in pedigree dogs is not a recent phenomenon and reports of suspected genetic defects associated with breeding practices date back to Charles Darwin's time. In recent years, much information on the array of inherited defects has been assimilated and the true extent of the problem has come to light. Historically, the direction of research funding in the field of canine genetic disease has been largely influenced by the potential transferability of findings to human medicine, economic benefit and importance of dogs for working purposes. More recently, the argument for a more canine welfare-orientated approach has been made, targeting research efforts at the alleviation of the most suffering in the greatest number of animals.

Rescue of glandular dysmorphogenesis in PTEN-deficient colorectal cancer epithelium by PPARγ-targeted therapy

Disruption of glandular architecture associates with poor clinical outcome in high-grade colorectal cancer (CRC). Phosphatase and tensin homolog deleted on chromosome ten (PTEN) regulates morphogenic growth of benign MDCK (Madin Darby Canine Kidney) cells through effects on the Rho-like GTPase cdc42 (cell division cycle 42). This study investigates PTEN-dependent morphogenesis in a CRC model. Stable short hairpin RNA knockdown of PTEN in Caco-2 cells influenced expression or localization of cdc42 guanine nucleotide exchange factors and inhibited cdc42 activation. Parental Caco-2 cells formed regular hollow gland-like structures (glands) with a single central lumen, in three-dimensional (3D) cultures. Conversely, PTEN-deficient Caco-2 ShPTEN cells formed irregular glands with multiple abnormal lumens as well as intra- and/or intercellular vacuoles evocative of the high-grade CRC phenotype. Effects of targeted treatment were investigated. Phosphatidinylinositol 3-kinase (PI3K) modulating treatment did not affect gland morphogenesis but did influence gland number, gland size and/or cell size within glands. As PTEN may be regulated by the nuclear receptor peroxisome proliferator-activated receptor-? (PPAR?), cultures were treated with the PPAR? ligand rosiglitazone. This treatment enhanced PTEN expression, cdc42 activation and rescued dysmorphogenesis by restoring single lumen formation in Caco-2 ShPTEN glands. Rosiglitazone effects on cdc42 activation and Caco-2 ShPTEN gland development were attenuated by cotreatment with GW9662, a PPAR? antagonist. Taken together, these studies show PTEN-cdc42 regulation of lumen formation in a 3D model of human CRC glandular morphogenesis. Treatment by the PPAR? ligand rosiglitazone, but not PI3K modulators, rescued colorectal glandular dysmorphogenesis of PTEN deficiency.

Morbillivirus cross-species infection:is there a risk for humans?

The World Health Organisation (WHO) has set regional elimination goals for Measles (MV) eradication to be achieved by 2020 or earlier. A major question is whether an opportunity for veterinary virus infection of humans may arise when MV is eradicated and if vaccination is discontinued. Lessons have been learned from animal to human virus transmission i.e. human immunodeficiency virus (HIV) and more recently from severe acute respiratory syndrome (SARS) and avian influenza virus infections. We are therefore alerted to the risk of zoonosis from the veterinary morbilliviruses. In this review the evidence from viral genomics, animal studies and cell culture experiments will be explored to evaluate the possibility of cross infection of humans with these viruses.

Multicenter Blinded Analysis of RT-PCR Detection Methods for Paramyxoviruses in Relation to Paget's Disease of Bone

Conflicting results have been reported on the detection of paramyxovirus transcripts in Paget's disease, and a possible explanation is differences in the sensitivity of RT-PCR methods for detecting virus. In a blinded study, we found no evidence to suggest that laboratories that failed to detect viral transcripts had less sensitive RT-PCR assays, and we did not detect measles or distemper transcripts in Paget's samples using the most sensitive assays evaluated.

Introduction: There is conflicting evidence on the possible role of persistent paramyxovirus infection in Paget's disease of bone (PDB). Some workers have detected measles virus (MV) or canine distemper virus (CDV) transcripts in cells and tissues from patients with PDB, but others have failed to confirm this finding. A possible explanation might be differences in the sensitivity of RT-PCR methods for detecting virus. Here we performed a blinded comparison of the sensitivity of different RT-PCR-based techniques for MV and CDV detection in different laboratories and used the most sensitive assays to screen for evidence of viral transcripts in bone and blood samples derived from patients with PDB.

Materials and Methods: Participating laboratories analyzed samples spiked with known amounts of MV and CDV transcripts and control samples that did not contain viral nucleic acids. All analyses were performed on a blinded basis.

Results: The limit of detection for CDV was 1000 viral transcripts in three laboratories (Aberdeen, Belfast, and Liverpool) and 10,000 transcripts in another laboratory (Manchester). The limit of detection for MV was 16 transcripts in one laboratory (NIBSC), 1000 transcripts in two laboratories (Aberdeen and Belfast), and 10,000 transcripts in two laboratories (Liverpool and Manchester). An assay previously used by a U.S.-based group to detect MV transcripts in PDB had a sensitivity of 1000 transcripts. One laboratory (Manchester) detected CDV transcripts in a negative control and in two samples that had been spiked with MV. None of the other laboratories had false-positive results for MV or CDV, and no evidence of viral transcripts was found on analysis of 12 PDB samples using the most sensitive RT-PCR assays for MV and CDV.

Conclusions: We found that RT-PCR assays used by different laboratories differed in their sensitivity to detect CDV and MV transcripts but found no evidence to suggest that laboratories that previously failed to detect viral transcripts had less sensitive RT-PCR assays than those that detected viral transcripts. False-positive results were observed with one laboratory, and we failed to detect paramyxovirus transcripts in PDB samples using the most sensitive assays evaluated. Our results show that failure of some laboratories to detect viral transcripts is unlikely to be caused by problems with assay sensitivity and highlight the fact that contamination can be an issue when searching for pathogens by sensitive RT-PCR-based techniques.

Characteristics of a cetacean morbillivirus isolated from a porpoise (Phocoena phocoena)

A virus isolated from a porpoise during the 1988 seal epizootic was shown to be a morbillivirus. In order to determine the relationship of the virus to phocine distemper virus (PDV) a battery of monoclonal antibodies raised against canine distemper virus (CDV), PDV or the porpoise isolate were assessed for their ability to bind to CDV, PDV or porpoise virus epitopes in indirect immunofluorescence assays and ELISAs. The porpoise isolate contained several unique epitopes and several epitopes present on CDV and PDV were absent on the porpoise isolate. The data presented in this study indicate that the porpoise virus is an antigenically distinct morbillivirus and as such has been tentatively named as delphinoid distemper virus (DDV).

Use of SLAM and PVRL4 and Identification of Pro-HB-EGF as Cell Entry Receptors for Wild Type Phocine Distemper Virus

Signalling lymphocyte activation molecule (SLAM) has been identified as an immune cell receptor for the morbilliviruses, measles (MV), canine distemper (CDV), rinderpest and peste des petits ruminants (PPRV) viruses, while CD46 is a receptor for vaccine strains of MV. More recently poliovirus like receptor 4 (PVRL4), also known as nectin 4, has been identified as a receptor for MV, CDV and PPRV on the basolateral surface of polarised epithelial cells. PVRL4 is also up-regulated by MV in human brain endothelial cells. Utilisation of PVRL4 as a receptor by phocine distemper virus (PDV) remains to be demonstrated as well as confirmation of use of SLAM. We have observed that unlike wild type (wt) MV or wtCDV, wtPDV strains replicate in African green monkey kidney Vero cells without prior adaptation, suggesting the use of a further receptor. We therefore examined candidate molecules, glycosaminoglycans (GAG) and the tetraspan proteins, integrin β and the membrane bound form of heparin binding epithelial growth factor (proHB-EGF),for receptor usage by wtPDV in Vero cells. We show that wtPDV replicates in Chinese hamster ovary (CHO) cells expressing SLAM and PVRL4. Similar wtPDV titres are produced in Vero and VeroSLAM cells but more limited fusion occurs in the latter. Infection of Vero cells was not inhibited by anti-CD46 antibody. Removal/disruption of GAG decreased fusion but not the titre of virus. Treatment with anti-integrin β antibody increased rather than decreased infection of Vero cells by wtPDV. However, infection was inhibited by antibody to HB-EGF and the virus replicated in CHO-proHB-EGF cells, indicating use of this molecule as a receptor. Common use of SLAM and PVRL4 by morbilliviruses increases the possibility of cross-species infection. Lack of a requirement for wtPDV adaptation to Vero cells raises the possibility of usage of proHB-EGF as a receptor in vivo but requires further investigation.

Behavioural and physiological responses of shelter dogs to long-term confinement

In Italy, National Law (281/1991) prohibits euthanasia of shelter dogs if they are not dangerous or suffering seriously. Adoption rates in rescue shelters are often lower than entrance rates, leading inevitably to overcrowded facilities where animals are likely to spend the rest of their lives in kennels. In this situation, housing conditions (i.e. space provided, environmental, and social stimulation) may have an impact on canine welfare. In this research project, the effects of two different forms of housing (group- and pair housing) on long-term shelter dogs were compared using behavioural and physiological parameters. Observational data and saliva samples were collected from dogs exposed to both experimental settings; behaviour and cortisol concentration levels were used as welfare indicators. Pair housing offered fewer social and environmental stimuli and behavioural analysis showed a significant decrease in locomotor, exploratory, and social behaviour. Cortisol levels show that this parameter varied independently of housing conditions. Although this study found no evidence suggesting that one form of confinement reduced animal welfare more than the other (e.g. in terms of abnormal behaviour, or higher cortisol concentrations), the type of confinement did affect the expression of a variety of behaviours and these variations should not be ignored with respect to housing decisions for long-term shelter dogs.

Comparing lateral bias in dogs and humans using the KongTM ball test

The KongTM ball test has been used extensively to assess lateral bias in the domestic dog. Implicit in this challenge is the assumption that dogs use their dominant paw to stabilise the ball. This study examined whether or not this is the case. A comparative approach was adopted, exploring limb use in dogs and humans. In Experiment 1, the paw preference of 48 dogs was assessed on the KongTM ball test. Analysis revealed an equal distribution of paw use, although significantly more dogs were paw-preferent than ambilateral. Significantly more male dogs were classified as right-pawed, while more females were ambilateral. There was no significant effect of canine sex or castration status on the dogs’ paw preferences. In Experiment 2, 94 adult humans were assessed on their ability to remove a piece of paper from a KongTM ball with their mouth, using their left, right or both hands to stabilise the ball. 76% of the right-handed people used their left hand, and 82% of the left-handed participants used their right hand, to hold the KongTM steady. It is concluded that dogs, like humans, are most likely using their non-dominant limb to stabilise the KongTM ball and their dominant side for postural support. This has potential applied implications from an animal welfare perspective.