Tick-Borne Encephalitis Virus, United Kingdom.

During February 2018-January 2019, we conducted large-scale surveillance for the presence and prevalence of tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) in sentinel animals and ticks in the United Kingdom. Serum was collected from 1,309 deer culled across England and Scotland. Overall, 4% of samples were ELISA-positive for the TBEV serocomplex. A focus in the Thetford Forest area had the highest proportion (47.7%) of seropositive samples. Ticks collected from culled deer within seropositive regions were tested for viral RNA; 5 of 2,041 ticks tested positive by LIV/TBEV real-time reverse transcription PCR, all from within the Thetford Forest area. From 1 tick, we identified a full-length genomic sequence of TBEV. Thus, using deer as sentinels revealed a potential TBEV focus in the United Kingdom. This detection of TBEV genomic sequence in UK ticks has important public health implications, especially for undiagnosed encephalitis.

Inflammatory infiltration into placentas of Neospora caninum challenged cattle correlates with clinical outcome of pregnancy.

Infection with Neospora caninum stimulates host cell-mediated immune responses, which may be responsible for placental damage leading to bovine abortion. The aim of this study was to compare immune responses in the bovine placenta, following experimental infection in different stages of pregnancy. Placentomes were examined by immunohistochemistry and inflammation in early gestation was generally moderate to severe, particularly in the placentas carrying non-viable foetuses, whereas it was milder in later stages, mainly characterised by the presence of CD3+, CD4+ and γδ T-cells. This distinctive cellular immune response may explain the milder clinical outcome observed when animals are infected in later gestation.

Development of maternal and foetal immune responses in cattle following experimental challenge with Neospora caninum at day 210 of gestation.

This study examined the immunological responses of pregnant cattle and their foetuses following an experimental challenge with live Neospora caninum tachyzoites at day 210 of gestation. Animals were bled prior to and weekly throughout the experiment and sacrificed at 14, 28, 42 and 56 days post inoculation (dpi). At post mortem examination, samples of lymph nodes and spleen were collected from both dam and foetus for immunological analysis. Subcutaneous (sc) inoculation over the left prefemoral (LPF) lymph node of pregnant cattle at day 210 of gestation, led to the vertical transmission of parasites by 14 dpi, however no foetal deaths were observed in the infected animals. Foetuses from infected dams mounted Neospora-specific humoral and cell-mediated immune (CMI) responses by 14 dpi. These responses involved anti-Neospora IgG, antigen-specific lymphocyte proliferation, and the production of the cytokines IFN-γ, interleukin (IL)-4 and IL-10. There was also evidence of innate immunity during the response against Neospora from infected dams, with statistically significant (p < 0.05) increases in mean expression of toll like receptors (TLR)-2 on 56 dpi in maternal spleen, LPF, right prefemoral (RPF), left uterine (LUL) and right uterine (RUL) lymph nodes and TLR-9 in retropharyngeal (RLN), LPF and RPF lymph nodes from 28 dpi. Statistically significant (p < 0.05) increases in mean TLR-9 were detected in spleen samples from foetuses of infected dams, compared to the foetuses from control animals. Our results show that vertical transmission of the parasite occurred in all infected dams, with their foetuses showing effective Neospora-specific cell mediated, humoral and innate immune responses.

High rate of transplacental infection and transmission of Neospora caninum following experimental challenge of cattle at day 210 of gestation.

In order to investigate the pathogenesis of neosporosis following a primary infection in late pregnancy, cattle were subcutaneously challenged with 5 × 108Neospora caninum (NC1 isolate) tachyzoites at day 210 of gestation and serial necropsies were then carried out at 14, 28, 42 and 56 days post-infection (dpi). No abortions occurred and all the foetuses were viable at the time of euthanasia. There was a high rate of vertical transmission, as parasites were detected by immunohistochemical labelling and PCR in all the foetuses from 28 dpi. Focal necrotic lesions were observed in the placentomes of the placenta from 28 dpi and showed resolution during later time points, denoted by infiltration of inflammatory cells at 42 dpi and fibrosis at 56 dpi. Foetuses at 28 and 42 dpi showed scarce and isolated lesions which are unlikely to represent a threat to foetal viability. No lesions were observed in the foetuses at 14 or 56 dpi suggesting control of the infection and resolution of the lesions by maternal and foetal immune responses. Once infection was established, it could not be cleared from the host and vertical transmission of the parasite occurred in all infected hosts. Parasite was detected in the placenta at 28 dpi, while in previous experimental infections of cattle at day 70 and 140 of gestation using the same challenge model, it was already present at day 14 post infection. This suggests that a change in the maternal immune response plays a crucial role in limiting the initial infection during the last term of pregnancy.

Selection of Neospora caninum antigens stimulating bovine CD4+ve T cell responses through immuno-potency screening and proteomic approaches.

Neospora caninum is recognised worldwide as a major cause of bovine infectious abortion. There is a real need to develop effective strategies to control infection during pregnancy which may lead to either abortion or congenital transmission. Due to the intracellular nature of the parasite, cell-mediated immune (CMI) responses involving CD4(+ve), CD8(+ve), γ/δ TCR(+ve) T cells and NK cells, as well as production of IFN-γ, are thought to be important for protective immunity. In this study we applied a combination of proteomic and immunological approaches to identify antigens of N. caninum that are recognized by CD4(+ve) T cell lines derived from infected cattle. Initially, N. caninum tachyzoite Water Soluble Antigens (NcWSA) were fractionated by size-exclusion HPLC and then screened for immune-potency using CD4(+ve) T cell lines. LC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry) was employed to catalogue and identify the proteins comprising three immunologically selected fractions and led to the identification of six N. caninum target proteins as well as sixteen functional orthologues of Toxoplasma gondii. This approach allows the screening of biologically reactive antigenic fractions by the immune cells responsible for protection (such as bovine CD4(+ve) cells) and the subsequent identification of the stimulating components using tandem mass spectrometry.

Novel Dermatitis and Relative Viral Nucleic Acid Tissue Loads in a Fin Whale (Balaenoptera physalus) with Systemic Cetacean Morbillivirus Infection.

Cetacean morbilliviruses (CeMVs) are significant causes of mortality in many cetacean species in epizootics and smaller outbreaks. Despite the prominence of skin lesions in seals and terrestrial animals, including humans, affected by other morbilliviruses, they have not been reported in CeMV-infected cetaceans. Here we report CeMV-associated skin lesions in a fin whale (Balaenoptera physalus) with subacute, systemic CeMV infection that live-stranded in Scotland, UK. Grossly, the skin was sloughing in large sheets, presumed due to autolysis, but histological examination showed syncytia, below the dermoepidermal junction, that were strongly immunopositive for morbillivirus antigen, as were syncytia in other organs. By polymerase chain reaction (PCR), the relative load of CeMV-specific RNA was largest in the liver and urinary bladder, even in formalin-fixed, paraffin-wax embedded samples. Levels were low in skin and only detectable in frozen samples. Genetic comparison of the CeMV revealed close alignment with isolates from fin whales from the North Atlantic Ocean and Mediterranean Sea, but that it was distinct from the porpoise CeMV clade. These findings show skin samples can be used to diagnose CeMV infection in cetaceans, highlighting the potential of ante-mortem sampling for monitoring disease in current populations and assessment of changes in host and pathogen genetics.

Novel Presentation of DMV-Associated Encephalitis in a Long-Finned Pilot Whale (Globicephala melas).

Cetacean morbillivirus (CeMV) is an important global cause of morbidity and mortality in cetacean populations, with four pathological presentations including non-suppurative encephalitis. We describe an unusual case of dolphin morbillivirus (DMV)-associated non-suppurative encephalitis in a long-finned pilot whale (Globicephala melas), in which the lesions were orientated on the periventricular white matter and comprised prominent multifocal syncytia formation in the absence of systemic lesions. DMV RNA was detected in brain tissue by qRT-PCR and immunohistochemistry for morbillivirus antigen yielded intense labelling of syncytia in periventricular sites, with sparse involvement of the deeper neuroparenchyma. The pattern of lesions raises the possibility of viral dissemination through the cerebrospinal fluid, as described for canine distemper virus, suggesting that similar pathogenic mechanisms may be implicated in lesion development. Further investigation is required to establish the pathogenesis of CeMV encephalitis and the behaviour of the virus within the central nervous system of cetaceans.

Anatomical distribution of respiratory tract leukocyte cell subsets in neonatal calves.

Neonatal calves are highly susceptible to a number of diseases including those that infect via the mucosal surfaces of the respiratory and gastrointestinal tracts. In order to determine appropriate vaccine design and delivery systems, or to identify suitable immunostimulatory methods to combat these infections, a detailed understanding of the immune cell populations present at clinically relevant sites is key. Few studies have assessed the immune cell composition of the neonatal calf lung and comparisons with circulating immune cells in the blood are lacking. We describe immune cell populations present in the peripheral blood, bronchoalveolar lavage (BAL) fluid and lung tissue of young disease-free calves. Flow cytometric analysis revealed significant differences in cell subset distribution between the peripheral blood and respiratory tract, and between compartments within the respiratory tract. Notably, whereas WC1+ γδ TCR + T lymphocytes dominate the peripheral blood, both the BAL fluid and lung tissue contained a high proportion of myeloid cells which expressed CD14 and CD172a (SIRPα). Very low numbers of tissue myeloid cells expressed MHC Class II in comparison to circulating myeloid cells in the blood. Respiratory tract tissues had low frequencies of CD4+ and CD8 + T lymphocytes, which were significantly lower than in the blood. Differences in the proportion of NKp46+ natural killer cells were also observed between tissue compartments. In order to target vaccines or immunostimulatory therapeutics appropriately, these differences in immune cell populations in tissue compartments should be taken into consideration.

Protective adaptive immunity to Chlamydophila abortus infection and control of ovine enzootic abortion (OEA).

Ovine enzootic abortion (OEA) remains a major problem in sheep-rearing countries despite the availability of protective vaccines. The causative agent, Chlamydophila abortus, is a Gram-negative bacterium that can induce a persistent, subclinical infection in non-pregnant sheep. The development of a new safe, effective and practical vaccine requires a detailed understanding of host-pathogen interactions and the identification of clear correlates of protection. Since disease (abortion) is only observed during pregnancy, the nature of host immunity to C. abortus and the specialised immunological features that permit maternal acceptance of the semi-allogeneic fetus are central to the pathogenesis of OEA. We review the current literature on persistence of C. abortus, host immunity to infection and mechanisms of abortion. We identify the key outstanding questions surrounding OEA and discuss the current knowledge gaps with a view to developing improved control strategies.

Immunophenotyping of Sheep Paraffin-Embedded Peripheral Lymph Nodes.

Sheep are not only a major livestock species globally, they are also an important large animal model for biomedical research and have contributed to our understanding of the ontogeny and architecture of the mammalian immune system. In this study, we applied immunohistochemistry and multicolor immunofluorescence in fixed and paraffin-embedded lymph nodes to phenotype the key populations of antigen presenting cells, lymphocytes, and stromal cells that orchestrate the host adaptive immune response. We used an extensive panel of antibodies directed against markers associated with dendritic cells (MHC class II, CD83, and CD208), macrophages (CD11b, CD163, and CD169), stromal cells (CNA.42, S100, and CD83), and lymphocytes (CD3, Pax5, CD4, CD8). Using different methods of tissue fixation and antigen retrieval, we provide a detailed immunophenotyping of sheep lymph nodes including the identification of potential subpopulations of antigen presenting cells and stromal cells. By characterizing cells expressing combinations of these markers in the context of their morphology and location within the lymph node architecture, we provide valuable new tools to investigate the structure, activation, and regulation of the sheep immune system in health and disease.

Three-colour flow cytometric detection of PrP in ovine leukocytes.

PrP(c) (cellular prion protein, CD230) expression by subpopulations of lymphoid cells has been widely investigated in a variety of species, possibly because of the possible link between transmissible spongiform encephalopathies (TSE) transmission and blood transfusion. However, the role of the immune cells in the transmission of the disease is still unclear. Here we describe the optimisation and standardisation of a three-colour staining procedure to detect PrP in association with phenotypic and activation markers in ovine immune cells. We demonstrate a reproducible, flexible and sensitive method and that the combination of isotype-specific antibodies and Fab fragments is feasible. To our knowledge, this is the first report of such labelling of ovine cells. Using this method, we were able to detect differences in levels of PrP expression between blood and lymph node cells of the same animal, and considerable variability between animals. Moreover, we were able to explore possible associations between PrP expression and cellular activation and to identify cell subsets with different labelling patterns. We are currently employing this approach to evaluate variations in immunological parameters during experimental infection in sheep.

The kinetics of Theileria parva infection and lymphocyte transformation in vitro.

Theileriaparva is an intracellular protozoan parasite that causes a fatal lymphoproliferative disease of cattle known as East Coast Fever. The parasite infects host lymphocytes causing their transformation and uncontrolled proliferation. Infiltration of major organs with parasitized lymphoblasts results in most cases in death within 3 weeks. Although both T and B lymphocytes are susceptible to infection, the majority of cell lines arising from infection of peripheral blood mononuclear cells in vitro are of T cell lineage. To explore the basis of this phenotypic bias we have followed the very early stages of parasite development in vitro at the single cell level. Peripheral blood mononuclear cells were infected and stained for both surface phenotype and intracellular parasite antigen and analysed by flow cytometry. Although the parasite antigen was detected intracellularly as early as 6h p.i., our data indicate that parasite infection does not lead to cell transformation in all instances. Rather, specific cell types appear to undergo selection very early after infection and expansion of particular cell subsets results in survival and growth of only a small proportion of the cells originally parasitized.

Reproduction of bovine neonatal pancytopenia (BNP) by feeding pooled colostrum reveals variable alloantibody damage to different haematopoietic lineages.

Bovine neonatal pancytopenia (BNP) is a recently described haemorrhagic disease of calves characterised by thrombocytopenia, leucopenia and bone marrow depletion. Feeding colostrum from cows that have previously produced a BNP affected calf has been shown to induce the disease in some calves, leading to the hypothesis that alloantibodies in colostrum from dams of affected calves mediate destruction of blood and bone marrow cells in the recipient calves. The aims of the current experimental study were first to confirm the role of colostrum-derived antibody in mediating the disease and second to investigate the haematopoietic cell lineages and maturation stages depleted by the causative antibodies. Clinical, haematological and pathological changes were examined in 5 calves given a standardised pool of colostrum from known BNP dams, and 5 control calves given an equivalent pool of colostrum from non-BNP dams. All calves fed challenge colostrum showed progressive depletion of bone marrow haematopoietic cells and haematological changes consistent with the development of BNP. Administration of a standardised dose of the same colostrum pool to each calf resulted in a consistent response within the groups, allowing detailed interpretation of the cellular changes not previously described. Analyses of blood and serial bone marrow changes revealed evidence of differential effects on different blood cell lineages. Peripheral blood cell depletion was confined to leucocytes and platelets, while bone marrow damage occurred to the primitive precursors and lineage committed cells of the thrombocyte, lymphocyte and monocyte lineages, but only to the more primitive precursors in the neutrophil, erythrocyte and eosinophil lineages. Such differences between lineages may reflect cell type-dependent differences in levels of expression or conformational nature of the target antigens.

Demonstration of early functional compromise of bone marrow derived hematopoietic progenitor cells during bovine neonatal pancytopenia through in vitro culture of bone marrow biopsies.

BACKGROUND: Bovine neonatal pancytopenia (BNP) is a syndrome characterised by thrombocytopenia associated with marked bone marrow destruction in calves, widely reported since 2007 in several European countries and since 2011 in New Zealand. The disease is epidemiologically associated with the use of an inactivated bovine virus diarrhoea (BVD) vaccine and is currently considered to be caused by absorption of colostral antibody produced by some vaccinated cows ("BNP dams"). Alloantibodies capable of binding to the leukocyte surface have been detected in BNP dams and antibodies recognising bovine MHC class I and ß-2-microglobulin have been detected in vaccinated cattle. In this study, calves were challenged with pooled colostrum collected from BNP dams or from non-BNP dams and their bone marrow hematopoietic progenitor cells (HPC) cultured in vitro from sternal biopsies taken at 24 hours and 6 days post-challenge. RESULTS: Clonogenic assay demonstrated that CFU-GEMM (colony forming unit-granulocyte/erythroid/macrophage/megakaryocyte; pluripotential progenitor cell) colony development was compromised from HPCs harvested as early as 24 hour post-challenge. By 6 days post challenge, HPCs harvested from challenged calves failed to develop CFU-E (erythroid) colonies and the development of both CFU-GEMM and CFU-GM (granulocyte/macrophage) was markedly reduced. CONCLUSION: This study suggests that the bone marrow pathology and clinical signs associated with BNP are related to an insult which compromises the pluripotential progenitor cell within the first 24 hours of life but that this does not initially include all cell types.

Identification of CD4+CD25 high Foxp3+ T cells in ovine peripheral blood.

Regulatory T cells (Treg) are an important subset of T lymphocytes which play a key role in maintaining peripheral immunological tolerance. The most studied subpopulation of Treg in mice and humans are natural Treg, which differentiate in the thymus and are identified by expression of CD4, high levels of IL-2Rα (CD25), and forkhead box P3 (Foxp3), a transcription factor intimately associated with Treg function. We and others have previously identified Foxp3(+) T cells in ovine tissue, suggesting that Treg exist in this species. However, the existence of putative natural Treg in sheep, as identified by co-expression of CD4, CD25 and Foxp3, has yet to be determined. In this study we demonstrate that the anti-rat/mouse Foxp3 monoclonal antibody FJK-16s cross-reacts with ovine Foxp3. Using a transfected Chinese hamster ovary cell line that constitutively expresses recombinant ovine Foxp3 as a positive control, we have developed a sensitive triple-labelling flow cytometry protocol to simultaneously label CD4, CD25 and Foxp3. We demonstrate that Foxp3(+) T lymphocytes exist in ovine peripheral blood, and that the majority of Foxp3 expression occurs within the CD4(+)CD25(hi) population. These results are consistent with those seen in other mammalian species and indicate that putative natural Treg exist in sheep.

Trans-species polymorphism and selection in the MHC class II DRA genes of domestic sheep.

Highly polymorphic genes with central roles in lymphocyte mediated immune surveillance are grouped together in the major histocompatibility complex (MHC) in higher vertebrates. Generally, across vertebrate species the class II MHC DRA gene is highly conserved with only limited allelic variation. Here however, we provide evidence of trans-species polymorphism at the DRA locus in domestic sheep (Ovis aries). We describe variation at the Ovar-DRA locus that is far in excess of anything described in other vertebrate species. The divergent DRA allele (Ovar-DRA*0201) differs from the sheep reference sequences by 20 nucleotides, 12 of which appear non-synonymous. Furthermore, DRA*0201 is paired with an equally divergent DRB1 allele (Ovar-DRB1*0901), which is consistent with an independent evolutionary history for the DR sub-region within this MHC haplotype. No recombination was observed between the divergent DRA and B genes in a range of breeds and typical levels of MHC class II DR protein expression were detected at the surface of leukocyte populations obtained from animals homozygous for the DRA*0201, DRB1*0901 haplotype. Bayesian phylogenetic analysis groups Ovar-DRA*0201 with DRA sequences derived from species within the Oryx and Alcelaphus genera rather than clustering with other ovine and caprine DRA alleles. Tests for Darwinian selection identified 10 positively selected sites on the branch leading to Ovar-DRA*0201, three of which are predicted to be associated with the binding of peptide antigen. As the Ovis, Oryx and Alcelaphus genera have not shared a common ancestor for over 30 million years, the DRA*0201 and DRB1*0901 allelic pair is likely to be of ancient origin and present in the founding population from which all contemporary domestic sheep breeds are derived. The conservation of the integrity of this unusual DR allelic pair suggests some selective advantage which is likely to be associated with the presentation of pathogen antigen to T-cells and the induction of protective immunity.

CD21 B cell populations are altered following subcutaneous scrapie inoculation in sheep.

In order to gain a better understanding of the pathogenesis of scrapie in sheep an experimental model was developed to characterise immune system cells in the minutes following inoculation with scrapie-brain homogenate. Four 1-year-old susceptible (ARQ/ARQ) sheep were inoculated via the subcutaneous route at four different peripheral lymph node (LNs) drainage sites, at specific time points, prior to euthanasia of the sheep. The LNs were removed post-mortem at 30, 90, 180 and 300min after inoculation. Flow cytometric triple-labelling was carried out on the LN cells and indicated that inoculation of scrapie-brain homogenate adjacent to a lymph node may delay or even inhibit the number of host CD21(+) B cells expressed within the first 5h. Immunohistochemistry was used to attempt detection of the abnormal form of prion protein (PrP(sc)) in draining LNs adjacent to inoculation sites, with negative results at those time points.