One Health in the context of medical and veterinary education.

This paper discusses how best to develop the educational platforms that can foster a wider appreciation of the importance of the One Health concept in medical and veterinary education. There are many compelling examples, from genetics to infectious diseases, where significant advances have been made in medicine and veterinary medicine by applying the principles of One Health, i.e. by recognising the interconnectedness between medicine, veterinary medicine and related sciences. In the medical and veterinary curriculum the objective should be to ensure that all opportunities are taken throughout preclinical and clinical teaching to incorporate the lessons which have been learned from the success stories in One Health. This will ensure that advances continue to be made and that a more pervasive and forward-looking scientific culture sustains One Health in the future.

E-cadherin expression in canine cutaneous histiocytomas.

Canine cutaneous histiocytoma is a common skin tumour of Langerhans cell origin. Langerhans cells are members of the dendritic cell family of antigen-presenting cells and are located in the epidermis. They are unique among the dendritic cell lineage in that they express high levels of the adhesion molecule E-cadherin. The expression of E-cadherin by the neoplastic Langerhans cells in 37 dogs with cutaneous histiocytoma was studied by flow cytometry and immunohistochemistry. In all the cases, these cells expressed E-cadherin, whereas the infiltrating lymphocytes did not.

CCH cells are potent stimulators in the allogeneic mixed leucocyte reaction.

Canine cutaneous histiocytoma (CCH) has been identified as a tumour of epidermal Langerhans cells (LCs) on the basis of immunophenotypic studies. Neoplastic Langerhans cells (CCH-LCs) were isolated from lesions of canine cutaneous histiocytoma. The CCH-LC cells expressed CD1b, CD11/18, CD45, MHC-I, and MHC-II. The CCH-LC cells were potent stimulators of the mixed leucocyte reaction (MLR) in vitro when compared to PBMCs from the tumour-bearing animals. This provides evidence that the neoplastic cells in CCH have functional as well as immunophenotypic characteristics of Langerhans cells.

Cloning and sequencing of ovine Flt3 ligand.

A cDNA (879 bp) containing the complete open reading frame of ovine Fms-related tyrosine kinase 3 ligand (Flt3-L) was amplified by reverse transcriptase polymerase chain reaction (RT-PCR), cloned and sequenced. The deduced amino acid sequence has 97.6% similarity with bovine Flt3-L isoform 1 and shares the long cytoplasmic domain observed in bovine Flt3-L but not in human Flt3-L.

A quantitative assessment of the carbonic anhydrase activity in photosystem II.

Using a carbonic anhydrase assay based on membrane inlet mass spectrometry (MIMS), we have extended our earlier investigations of Photosystem II (PSII)-associated carbonic anhydrase activity in spinach PSII preparations (W. Hillier, I. McConnell, M. R. Badger, A. Boussac, V.V. Klimov G. C. Dismukes, T. Wydrzynski Biochemistry 2006, 45:2094). The relationship between the carbonic anhydrase activity and O(2) evolution has been evaluated in terms of the effects of metal ion addition, preparation type, light, and response to specific inhibitors. The results indicate that the PSII-associated carbonic anhydrase activity is variable and appears not to be associated specifically with the oxygen evolving activity nor the 33 kDa extrinsic manganese stabilising protein.

Oxidative and nitrosative responses of the chicken macrophage cell line MQ-NCSU to experimental Salmonella infection.

Phagocytes limit replication or kill ingested organisms by producing toxic reactive oxygen and nitrogen species via NADPH oxidase and inducible nitric oxide synthase (iNOS). The present experiments were to investigate the production and the possible roles of superoxide, hydrogen peroxide (H2O2) and nitric oxide (NO) in the MQ-NCSU chicken macrophage cell line infected with Salmonella in vitro. After infection, intracellular Salmonella viable counts remained constant until 24 h post infection (PI) and started to decline from 48 h PI. Infection of cells with S. Typhimurium, S. Enteritidis and S. Gallinarum, as well as exposure to S. Enteritidis LPS induced low, but significant concentrations of superoxide 1 to 2 h PI, as determined by reduction of ferricytochrome c. There was no difference in superoxide production in infected cells and control cells after 4 h. Increased H2O2 was observed from cells infected with all the different Salmonella species between 2 and 3 h of infection. Nitrite was always greater in infected cells compared to uninfected cells at all times. However, Salmonella was not completely eliminated from the cells though these cells are capable of eliciting a noticeable oxidative burst response and great nitrosative responses, indicating that a strong oxidative burst (and other mechanism/s) is essential for the elimination of intracellular Salmonella.

Detection of variant Creutzfeldt-Jakob disease infectivity in extraneural tissues.

Abnormal accumulations of prion protein (PrP) can be detected in the spleen, lymph nodes, and tonsils of patients with variant Creutzfeldt-Jakob disease (vCJD). Therefore, it has been assumed, but not shown, that these tissues harbour infectivity, which in turn presents the potential for iatrogenic spread through surgery. Here, we show and measure levels of infectivity in spleen and tonsil from two patients with vCJD, by bioassay in intracerebrally inoculated RIII mice. Similar bioassays failed to detect infectivity in buffy coat and plasma.

Genetic and environmental factors modify bovine spongiform encephalopathy incubation period in mice.

The incubation period (IP) and the neuropathology of transmissible spongiform encephalopathies (TSEs) have been extensively used to distinguish prion isolates (or strains) inoculated into panels of inbred mouse strains. Such studies have shown that the bovine spongiform encephalopathy (BSE) agent is indistinguishable from the agent causing variant Creutzfeldt-Jakob disease (vCJD), but differs from isolates of sporadic CJD, reinforcing the idea that the vCJD epidemic in Britain results from consumption of contaminated beef products. We present a mouse model for genetic and environmental factors that modify the incubation period of BSE cross-species transmission. We have used two mouse strains that carry the same prion protein (PrP) allele, but display a 100-day difference in their mean IP following intracerebral inoculation with primary BSE isolate. We report genetic effects on IP that map to four chromosomal regions, and in addition we find significant factors of host environment, namely the age of the host's mother, the age of the host at infection, and an X-cytoplasm interaction in the host.

From genes to proteins: high-throughput expression and purification of the human proteome.

The development of high-throughput methods for gene discovery has paved the way for the design of new strategies for genome-scale protein analysis. Lawrence Livermore National Laboratory and Onyx Pharmaceuticals, Inc., have produced an automatable system for the expression and purification of large numbers of proteins encoded by cDNA clones from the IMAGE (Integrated Molecular Analysis of Genomes and Their Expression) collection. This high-throughput protein expression system has been developed for the analysis of the human proteome, the protein equivalent of the human genome, comprising the translated products of all expressed genes. Functional and structural analysis of novel genes identified by EST (Expressed Sequence Tag) sequencing and the Human Genome Project will be greatly advanced by the application of this high-throughput expression system for protein production. A prototype was designed to demonstrate the feasibility of our approach. Using a PCR-based strategy, 72 unique IMAGE cDNA clones have been used to create an array of recombinant baculoviruses in a 96-well microtiter plate format. Forty-two percent of these cDNAs successfully produced soluble, recombinant protein. All of the steps in this process, from PCR to protein production, were performed in 96-well microtiter plates, and are thus amenable to automation. Each recombinant protein was engineered to incorporate an epitope tag at the amino terminal end to allow for immunoaffinity purification. Proteins expressed from this system are currently being analyzed for functional and biochemical properties.

Infection of dendritic cells by the Maedi-Visna lentivirus.

The early stages of lentivirus infection of dendritic cells have been studied in an in vivo model. Maedi-visna virus (MVV) is a natural pathogen of sheep with a tropism for macrophages, but the infection of dendritic cells has not been proven, largely because of the difficulties of definitively distinguishing the two cell types. Afferent lymphatic dendritic cells from sheep have been phenotypically characterized and separated from macrophages. Dendritic cells purified from experimentally infected sheep have been demonstrated not only to carry infectious MVV but also to be hosts of the virus themselves. The results of the in vivo infection experiments are supported by infections of purified afferent lymph dendritic cells in vitro, in which late reverse transcriptase products are demonstrated by PCR. The significance of the infection of afferent lymph dendritic cells is discussed in relation to the initial spread of lentivirus infection and the requirement for CD4 T cells.

Cutaneous T cell lymphoma mimicking cutaneous histiocytosis: differentiation by flow cytometry.

A two-year-old, neutered female cross-bred labrador had multiple cutaneous nodules, biopsies of which revealed pathological changes consistent with cutaneous histiocytosis. During a period of one month the dog developed multicentric lymphadenopathy, a retrobulbar mass and masses within the quadriceps and cervical muscles. Fine needle aspiration cytology of the cutaneous nodules and lymph nodes and histological examination of the cutaneous nodules and muscle masses suggested the presence of lymphoblastic lymphoma. A definitive diagnosis of CD8+ T cell lymphoma was achieved by immunophenotyping the tumour cells by flow cytometry.

Closely similar values obtained when the ME7 strain of scrapie agent was titrated in parallel by two individuals in separate laboratories using two sublines of C57BL mice.

A single C57BL mouse-brain infected with the ME7 strain of mouse-passaged scrapie agent was used to carry out four parallel infectivity titrations in mice. These were carried out by two individuals in two laboratories using two sublines of C57BL mice. The titre values obtained by the four assays were very similar, and showed no significant differences between the two different operatives, the two different laboratories or the two different sublines of C57BL mice. The data confirm the validity of comparing these types of transmission data generated in different laboratories when a common methodology is used.

A single amino acid alteration in murine PrP dramatically alters TSE incubation time.

In order to investigate mutations linked to human TSEs, we have used the technique of gene targeting to introduce specific mutations into the endogenous murine PrP gene which resulted in a P101L substitution (Prnp(a101L)) in the murine PrP gene. This mutation is equivalent to the 102L mutation in the human PrP gene which is associated with Gerstmann-Sträussler syndrome. Since the mutated gene is in the correct chromosomal location and control of the mutant gene expression is identical to that of the wild type murine PrP gene, the precise effect of the 101L mutation in the uninfected and TSE infected mouse can be investigated in this transgenic model. Mice homozygous for this mutation (101LL) while showing no spontaneous TSE disease were more susceptible to TSE disease than wild type mice following inoculation with GSS infectivity. Disease was transmitted from these mice to mice both with and without the Prnp(a101L) allele. The 101L mutation does not therefore produce spontaneous genetic disease in mice but does dramatically alter incubation periods following TSE infection. Additionally, a rapid TSE transmission was demonstrated associated with extremely low amounts of PrP(Sc).

A single amino acid alteration (101L) introduced into murine PrP dramatically alters incubation time of transmissible spongiform encephalopathy.

A mutation equivalent to P102L in the human PrP gene, associated with Gerstmann-Straussler syndrome (GSS), has been introduced into the murine PrP gene by gene targeting. Mice homozygous for this mutation (101LL) showed no spontaneous transmissible spongiform encephalopathy (TSE) disease, but had incubation times dramatically different from wild-type mice following inoculation with different TSE sources. Inoculation with GSS produced disease in 101LL mice in 288 days. Disease was transmitted from these mice to both wild-type (226 days) and 101LL mice (148 days). In contrast, 101LL mice infected with ME7 had prolonged incubation times (338 days) compared with wild-type mice (161 days). The 101L mutation does not, therefore, produce any spontaneous genetic disease in mice but significantly alters the incubation time of TSE infection. Additionally, a rapid TSE transmission was demonstrated despite extremely low levels of disease-associated PrP.

In vivo depletion of CD8+ cells does not affect primary maedi visna virus infection in sheep.

T-cells have been implicated both, in promoting and reducing viral replication during lentivirus infection. CD8+ lymphocytes are believed to be important in controlling viral load through direct killing of virus-infected cells and by secretion of inhibitory chemokines and cytokines. To evaluate the role of CD8+ T-cells in the induction and control of the primary phase of a lentivirus infection, we have used a non-T-cell tropic lentivirus, maedi-visna virus (MVV), to study the initial pathogenesis and subsequent immune responses in sheep depleted in vivo of CD8+ cells. Sheep were depleted of CD8+ cells in both blood and efferent lymph for up to 14 days. No difference in MVV replication was observed in either the draining efferent lymph or lymph node of these sheep. Surprisingly, these animals displayed a normal induction of pCTL whereas the virus-specific proliferative responses were reduced. This could reflect either that a proportion of functional CD8+ lymphocytes remained in these animals, as suggested by the appearance of pCTLs, or that CD8+ cells are not required for control of primary MVV infection.

Survival of scrapie agent after exposure to sodium dodecyl sulphate and heat.

Fifty mg aliquots of macerated mouse-brain infected with the 22A strain of scrapie agent were treated by exposing them without mechanical mixing to (a) distilled water for 2 h, (b) 5% sodium dodecyl sulphate (SDS) for 2 h, (c) autoclaving at 121 degrees C for 15 min in distilled water, (d) autoclaving at 121 degrees C for 15 min in 5% SDS, or (e) boiling in 5% SDS for 15 min. Prior to injection into mice, all samples were washed by a procedure that is described and was shown not to reduce infectivity titres. Although the infectivity titre of the sample that was autoclaved in SDS was reduced considerably, infectivity was present in all of the samples exposed to cold or hot SDS.

CD4(+) T-cells are required for the establishment of maedi-visna virus infection in macrophages but not dendritic cells in vivo.

The role of CD4(+) lymphocytes in the establishment of lentivirus infection in macrophages has been studied in an in vivo system of lentivirus infection where CD4(+) lymphocytes are not the targets for infection. Using the non-T-cell-tropic lentivirus, maedi-visna virus (MVV), in CD4-depleted sheep, we have found that CD4(+) T cells were required for MVV infection in macrophages but not dendritic cells. CD4-depleted sheep had significantly lower levels of MVV-infected cells in lymph nodes and efferent lymph after MVV challenge in the drainage area of the lymph node. Due to the absence of virus in combination with the lack of CD4(+) T helper cells, virus-specific immune responses were reduced. There was delayed induction of cytotoxic T cell precursors, a marked reduction in virus-specific in vitro proliferative responses, and a delay in the appearance of MVV-specific antibodies. By contrast, CD4 depletion had no effect on the establishment of MVV infection in afferent lymph dendritic cells migrating from the skin infection site to the lymph node.

Solvent extraction as an adjunct to rendering: the effect on BSE and scrapie agents of hot solvents followed by dry heat and steam.

The study was designed to determine the effect on bovine spongiform encephalopathy (BSE) and scrapie agents of the solvent extraction processes used in the past by British renderers. The raw material was mouse spleen infected with either the 22A strain of scrapie agent or the 301V strain of BSE agent. Samples were exposed to hexane, heptane, petroleum spirit or perchlorethylene at the relevant temperatures for the appropriate times. Control samples were exposed to the same range of temperatures for the same range of times in saline. Other samples were exposed to the hot solvents, followed by treatment with dry heat at 100 degrees C for 30 minutes and steam at 100 degrees C for 30 minutes. Further samples were exposed only to the dry heat and steam cycles. No single complete process was significantly more effective than any of the others, and they all produced only slight inactivation, less than one log on average for both strains of agent. The average degree of inactivation produced by exposure to hot saline was generally comparable to that produced by exposure to the hot solvents. This was also true for the samples exposed only to dry heat and steam compared with those exposed to hot solvent before treatment with dry heat and steam, and suggests that the slight inactivation was caused by the heat rather than by the solvents. It is concluded that the solvent extraction processes used by renderers in Britain had little capacity to inactivate BSE and scrapie agents.

Two B cell subpopulations have distinct recirculation characteristics.

This report describes two subpopulations of B cells in sheep. These subpopulations have distinct recirculation characteristics and tissue distributions. Phenotypically the populations are distinguished by their differential expression of the complement receptors, CD21 (CR2) and CD11b/CD18 (CR3). CD11b+ B cells are surface (s)IgMhi, co-express CD11c but are L-selectin negative. They populate the splenic marginal zone but are absent from splenic and ileal Peyer's patch (IPP) follicles and both afferent and efferent lymph compartments. Fluorescent tracing experiments showed that the CD11b+ B cells are non-recirculating as they did not appear in lymph after intravenous inoculation but are restricted to the blood and spleen. The CD11b-negative population expresses a conformational determinant of CD21 that is recognized by the monoclonal antibody Du 2-74. These cells are sIgMlo and co-express L-selectin. They populate the splenic and IPP follicles, are absent from the splenic marginal zone and are the only B cells in afferent lymph, efferent lymph and all lymph nodes. Fluorescence tracing experiments showed that the CD21 B cells are recirculating cells with their entry into efferent lymph being detectable by 16 h and peaking at 24-30 h. These data suggest that there are at least two lineages of B cells in the sheep with different phenotypic, functional and recirculation characteristics.