Virus-like particles of louping ill virus elicit potent neutralizing antibodies targeting multimers of viral envelope protein.

Louping ill virus (LIV) is a tick-borne flavivirus that predominantly causes disease in livestock, especially sheep in the British Isles. A preventive vaccine, previously approved for veterinary use but now discontinued, was based on an inactivated whole virion that likely provided protection by induction of neutralizing antibodies recognizing the viral envelope (E) protein. A major disadvantage of the inactivated vaccine was the need for high containment facilities for the propagation of infectious virus, as mandated by the hazard group 3 status of the virus. This study aimed to develop high-efficacy non-infectious protein-based vaccine candidates. Specifically, soluble envelope protein (sE), and virus-like particles (VLPs), comprised of the precursor of membrane and envelope proteins, were generated, characterized, and studied for their immunogenicity in mice. Results showed that the VLPs induced more potent virus neutralizing response compared to sE, even though the total anti-envelope IgG content induced by the two antigens was similar. Depletion of anti-monomeric E protein antibodies from mouse immune sera suggested that the neutralizing antibodies elicited by the VLPs targeted epitopes spanning the highly organized structure of multimer of the E protein, whereas the antibody response induced by sE focused on E monomers. Thus, our results indicate that VLPs represent a promising LIV vaccine candidate.

Identification and characterisation of Gamma-herpesviruses in zoo artiodactyla.

BACKGROUND: Viruses within the γ-herpesviruses subfamily include the causative agents of Malignant Catarrhal Fever (MCF) in several species of the order Artiodactyla. MCF is a usually fatal lymphoproliferative disease affecting non-adapted host species. In adapted host species these viruses become latent and recrudesce and transmit during times of stress or immunosuppression. The undetected presence of MCF-causing viruses (MCFVs) is a risk to non-adapted hosts, especially within non-sympatric zoological collections. This study investigated the presence of MCFVs in six different zoological collections in the UK, to evaluate the presence of subclinical/latent MCFVs in carrier animals. METHODS: One-hundred and thirty eight samples belonging to 54 different species of Artiodactyla were tested by Consensus Pan-herpes PCR. The positive samples were sequenced and subjected to phylogenetic analyses to understand their own evolutionary relationships and those with their hosts. RESULTS: Twenty-five samples from 18 different species tested positive. All viruses but one clustered in the γ-herpesvirus family and within the Macavirus as well as the non-Macavirus groups (caprinae and alcelaphinae/hippotraginae clusters, respectively). A strong association between virus and host species was evident in the Macavirus group and clustering within the caprinae group indicated potential pathogenicity. CONCLUSION: This study shows the presence of pathogenic and non-pathogenic MCFVs, as well as other γ-herpesviruses, in Artiodactyla species of conservation importance and allowed the identification of new herpesviruses in some non-adapted species.

Presence of Anaplasma phagocytophilum Ecotype I in UK Ruminants and Associated Zoonotic Risk.

Anaplasma phagocytophilum is the causative agent of tick-borne fever in sheep, pasture fever in cattle, and granulocytic anaplasmosis in humans. The increasing prevalence and transboundary spread of A. phagocytophilum in livestock, ticks, and wildlife in the UK poses a potential zoonotic risk that has yet to be estimated. Several ecotypes of A. phagocytophilum show variable zoonotic potential. To evaluate the possible risk associated with the transmission of A. phagocytophilum from ruminants to humans, the ecotype was determined by sequencing the groEL gene from 71 positive blood and tissue samples from UK ruminants. Thirty-four groEL sequences were obtained, fourteen of which were identified in multiple samples. Of the 13 nucleotide polymorphisms identified through pairwise comparison, all corresponded to synonymous substitutions. The subsequent phylogenetic estimation of the relationship with other European/world isolates indicated that all the groEL sequences clustered with other ecotype I sequences. The presence of ecotype I closely reflects that observed in ruminants in continental Europe and suggests a lower risk of zoonotic transmission from this reservoir.

Molecular Tools to Identify and Characterize Malignant Catarrhal Fever Viruses (MCFV) of Ruminants and Captive Artiodactyla.

The family Herpesviridae includes viruses identified in mammals, birds and reptiles. All herpesviruses share a similar structure, consisting of a large linear double-stranded DNA genome surrounded by a proteic icosahedral capsid further contained within a lipidic bilayer envelope. The continuous rise of genetic variability and the evolutionary selective pressure underlie the appearance and consolidation of novel viral strains. This applies also to several gamma(γ)-herpesviruses, whose role as primary pathogen has been often neglected and, among these to newly emerged viruses or virus variants responsible for the development of Malignant Catarrhal Fever (MCF) or MCF-like disease. The identification of γ-herpesviruses adapted to new zoological hosts requires specific molecular tools for detection and characterization. These viruses can cause MCF in livestock and wild animals, a disease generally sporadic but with serious welfare implications and which, in many cases, leads to death within a few days from the appearance of the clinical signs. In the absence of a vaccine, the first step to improve disease control is based on the improvement of molecular tools to identify and characterize these viruses, their phylogenetic relationships and evolutionary interaction with the host species. A Panherpes PCR-specific test, based on the conserved DNA polymerase gene, employing consensus/degenerate and deoxyinosine-substituted primers followed by sequencing, is still the preferred diagnostic test to confirm and characterize herpesviral infections. The drawback of this test is the amplification of a relatively short sequence, which makes phylogenetic analysis less stringent. Based on these diagnostic requirements, and with a specific focus on γ-herpesviruses, the present review aims to critically analyze the currently available methods to identify and characterize novel MCFV strains, to highlight advantages and drawbacks and to identify the gaps to be filled in order to address research priorities. Possible approaches for improving or further developing these molecular tools are also suggested.

Recent advances and public health implications for environmental exposure to Chlamydia abortus: from enzootic to zoonotic disease.

Environmental transmission of Chlamydia abortus as a result of enzootic disease or disease outbreaks and the threats posed by this pathogen has been previously reported, however a state-of-the-science review of these reports and the identification of future research priorities in this area is still lacking. This study provides an overview of the current knowledge of host-pathogen-environment interactions, addressing public health risks and identifying critical questions and research gaps. We performed a systematic PubMed and Web of Science search for publications related to Chlamydia abortus in the past four decades, and we reviewed and combined the evidence critically discussing and commenting the results. A total of 182 studies, 5 chapters of specific books and the "OIE terrestrial manual" were included in this review. There were substantial variations between the studies in topic addressed and experimental design. Overall, the literature largely supports the crucial role played by environmental exposure on the acquisition of zoonotic disease caused by Chlamydia abortus. We also identify the paucity of information related to interspecies transmission and pathogen adaptation in relation to environmental dissemination and zoonotic risk. This analysis further highlights the need for additional research given that environmental transmission represents a serious risk not only to susceptible patients (pregnant women and immunocompromised individuals), but also for other species including wildlife.

Occurrence of tick-borne pathogens in questing Ixodes ricinus ticks from Wester Ross, Northwest Scotland.

BACKGROUND: Lyme borreliosis and other tick-borne diseases emerge from increased interactions between humans, other animals, and infected ticks. The risk of acquiring a tick-borne infection varies across space and time, so knowledge of the occurrence and prevalence of pathogens in ticks can facilitate disease diagnosis in a specific area and the implementation of mitigation measures and awareness campaigns. Here we identify the occurrence and prevalence of several pathogens in Ixodes ricinus ticks in Wester Ross, Northwest Scotland, a region of high tourism and tick exposure, yet data-poor in terms of tick-borne pathogens. METHODS: Questing I. ricinus nymphs (n = 2828) were collected from 26 sites in 2018 and 2019 and tested for the presence of tick-borne pathogens using PCR-based methods. Prevalence was compared with other regions of Scotland, England, Wales, and the Netherlands. RESULTS: Anaplasma phagocytophilum (4.7% prevalence), Borrelia burgdorferi sensu lato (s.l.) (2.2%), Babesia from clade X (0.2%), Rickettsia helvetica (0.04%), and Spiroplasma ixodetis (0.4%) were detected, but no Neoehrlichia mikurensis, Borrelia miyamotoi, or Babesia microti. Typing of A. phagocytophilum using a fragment of the GroEL gene identified the presence of both ecotype I and ecotype II. Genospecies identification of Borrelia burgdorferi s.l. revealed B. afzelii (53% of infected nymphs), B. garinii (9%), B. burgdorferi sensu stricto (7%), and B. valaisiana (31%). We found similar prevalence of A. phagocytophilum in Wester Ross as in the Netherlands, but higher than in other parts of Great Britain. We found lower B. burgdorferi s.l. prevalence than in England or the Netherlands, and similar to some other Scottish studies. We found higher prevalence of B. valaisiana and lower prevalence of B. garinii than in other Scottish studies. We found S. ixodetis at much lower prevalence than in the Netherlands, and R. helvetica at much lower prevalence than in England and the Netherlands. CONCLUSIONS: As far as we know, this is the first description of S. ixodetis in Great Britain. The results are relevant for disease surveillance and management for public and veterinary health. The findings can also aid in designing targeted public health campaigns and in raising awareness among outdoor recreationists and professionals.

Efficacy of Two Chlamydia abortus Subcellular Vaccines in a Pregnant Ewe Challenge Model for Ovine Enzootic Abortion.

Chlamydia abortus, the aetiological agent of enzootic abortion of ewes, is a major cause of reproductive loss in small ruminants worldwide, accounting for significant economic losses to the farming industry. Disease can be managed through the use of commercial inactivated or live whole organism-based vaccines, although both have limitations particularly in terms of efficacy, safety and disease-associated outbreaks. Here we report a comparison of two experimental vaccines (chlamydial outer membrane complex (COMC) and octyl glucoside (OG)-COMC) based on detergent extracted outer membrane preparations of C. abortus and delivered as prime-boost immunisations, with the commercial live vaccine Cevac® Chlamydia in a pregnant sheep challenge model. No abortions occurred in either experimental vaccine group, while a single abortion occurred in the commercial vaccine group. Bacterial shedding, as a measure of potential risk of transmission of infection to naïve animals, was lowest in the COMC vaccinated group, with reductions of 87.5%, 86.4% and 74% observed for the COMC, OG-COMC and live commercial vaccine groups, respectively, compared to the unvaccinated challenge control group. The results show that the COMC vaccine performed the best and is a safer efficacious alternative to the commercial vaccines. However, to improve commercial viability, future studies should optimise the antigen dose and number of inoculations required.

First confirmed reports of the isolation of Brucella ceti from a Risso's dolphin Grampus griseus and a killer whale Orcinus orca.

Brucella ceti has been recovered from a number species of cetaceans worldwide over the last 25 yr. Here we report, for the first time, the recovery of B. ceti from a Risso's dolphin Grampus griseus and a killer whale Orcinus orca. Recovery from an abdominal mass in the dolphin provides further evidence of the systemic pathogenic potential for B. ceti infection in cetaceans. The isolation of B. ceti ST23 (porpoise cluster) from a killer whale from a group known to eat other marine mammals raises the possibility of infection via ingestion. This report takes the number of cetacean species in UK coastal waters from which B. ceti has been isolated to 11 and highlights the value of routine, comprehensive and specific screening for significant pathogens such as Brucella sp. by strandings networks.

Meningoencephalitis in a common minke whale Balaenoptera acutorostrata associated with Brucella pinnipedialis and gamma-herpesvirus infection.

Fatal marine Brucella infections with histologic lesions specific to the central nervous system (CNS), known as neurobrucellosis, have been described in 5 species of odontocete cetaceans in the UK: striped dolphins Stenella coeruleoalba, Atlantic white-sided dolphins Lagenorhynchus acutus, short-beaked common dolphins Delphinus delphis, long-finned pilot whale Globicephala melas and Sowerby's beaked whale Mesoplodon bidens. To date, these CNS lesions have only been associated with Brucella ceti ST26 and not with B. pinnipedialis, which is rarely isolated from cetaceans and, although commonly found in various seal species, has never been associated with any pathology. This paper describes the first report of neurobrucellosis in a common minke whale Balaenoptera acutorostrata which was associated with the isolation of Brucella pinnipedialis ST24 and co-infection with Balaenoptera acutorostrata gamma-herpesvirus 2. This is the first report of neurobrucellosis in any species of mysticete and the first report of Brucella pinnipedialis in association with any pathology in any species of marine mammal, which may be due to co-infection with a herpesvirus, as these are known to be associated with immunosuppression.

LOUPING-ILL VIRUS SEROSURVEY OF WILLOW PTARMIGAN (LAGOPUS LAGOPUS LAGOPUS) IN NORWAY.

In Norway, the Willow Ptarmigan (Lagopus lagopus lagopus) is experiencing population declines and is nationally Red Listed as Near Threatened. Although disease has not generally been regarded as an important factor behind population fluctuations for Willow Ptarmigan in Norway, disease occurrence has been poorly investigated. Both louping-ill virus (LIV) and the closely related tick-borne encephalitis virus are found along the southern part of the Norwegian coast. We assessed whether and where Norwegian Willow Ptarmigan populations have been infected with LIV. We expected to find infected individuals in populations in the southernmost part of the country. We did not expect to find infected individuals in populations further north and at higher altitudes because of the absence of the main vector, the sheep tick (Ixodes ricinus). We collected serum samples on Nobuto filter paper and used a hemagglutination inhibition assay for antibodies against LIV. We collected data at both local and country-wide levels. For local sampling, we collected and analyzed 87 hunter-collected samples from one of the southernmost Willow Ptarmigan populations in Norway. Of these birds, only three positives (3.4%) were found. For the country-wide sampling, we collected serum samples from 163 Willow Ptarmigan carcasses submitted from selected locations all over the country. Of these birds, 32% (53) were seropositive for LIV or a cross-reacting virus. Surprisingly, we found seropositive individuals from locations across the whole country, including outside the known distribution of the sheep tick. These results suggest that either LIV or a cross-reacting virus infects ptarmigan in large parts of Norway, including at high altitudes and latitudes.

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.

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.

Neurobrucellosis due to Brucella ceti ST26 in Three Sowerby's Beaked Whales (Mesoplodon bidens).

Fatal meningoencephalitis due to Brucella ceti infection has been described in striped dolphins (Stenella coeruleoalba), Atlantic white-sided dolphins (Lagenorhynchus acutus), short-beaked common dolphins (Delphinus delphis) and long-finned pilot whales (Globicephala melas), which are all within the family Delphinidae. We report B. ceti-associated neurobrucellosis in three juvenile male Sowerby's beaked whales (Mesoplodon bidens) that all had typical lesions of lymphocytic meningoencephalitis, which increased in severity from rostral to caudal regions of the brain. In two cases there was loss of ependymal cells lining the cerebral ventricular system, with large numbers of lymphocytes in the underlying neuropil. This finding suggests that B. ceti gains access to, and multiplies in, the cerebrospinal fluid, and confirms that this is the sample of choice for bacteriological recovery of the causative organism. These findings expand the increasing range of cetaceans susceptible to neurobrucellosis to members of the family Ziphiidae.

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.

Defining immune correlates during latent and active chlamydial infection in sheep.

Ovine enzootic abortion (OEA) caused by the obligate intracellular bacterial pathogen Chlamydia abortus (C. abortus), is an endemic disease in most sheep-rearing countries worldwide. Following infection, C. abortus establishes a complex host-pathogen interaction with a latent phase in non-pregnant sheep followed by an active disease phase in the placenta during pregnancy leading to OEA. Improved knowledge of the host-pathogen interactions at these different phases of disease will accelerate the development of new diagnostic tests and vaccines to control OEA. Current evidence indicates that cellular immunity is essential for controlling C. abortus infection. We have previously described a model of mucosal (intranasal) infection of non-pregnant sheep with C. abortus that replicates the latent and active phases of OEA. We have investigated antigen-specific recall responses of peripheral blood mononuclear cells (PBMC) in sheep infected with C. abortus via the intranasal route to determine how these change during the latent and active phases of disease. By analysing cytokines associated with the major CD4+ve Thelper (Th) cell subsets (Interferon-gamma (IFN-γ)/Th1; Interleukin (IL)-4/Th2; IL-17A/Th17; IL-10/Tregulatory), we show that there is selective activation of PBMC producing IFN-γ and/or IL-10 during the latent phase following infection. These cytokines are also elevated during the active disease phase and while they are produced by sheep that are protected from OEA, they are also produced by sheep that abort, highlighting the difficulties in finding specific cellular immunological correlates of protection for complex intracellular pathogens.

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.

Cervids as sentinel-species for tick-borne encephalitis virus in Norway - A serological study.

Tick-borne encephalitis virus (TBEV) is the causative agent of tick-borne encephalitis (TBE). TBEV is one of the most important neurological pathogens transmitted by tick bites in Europe. The objectives of this study were to investigate the seroprevalence of TBE antibodies in cervids in Norway and the possible emergence of new foci, and furthermore to evaluate if cervids can function as sentinel animals for the distribution of TBEV in the country. Serum samples from 286 moose, 148 roe deer, 140 red deer and 83 reindeer from all over Norway were collected and screened for TBE immunoglobulin G (IgG) antibodies with a modified commercial enzyme-linked immunosorbent assay (ELISA) and confirmed by TBEV serum neutralisation test (SNT). The overall seroprevalence against the TBEV complex in the cervid specimens from Norway was 4.6%. The highest number of seropositive cervids was found in south-eastern Norway, but seropositive cervids were also detected in southern- and central Norway. Antibodies against TBEV detected by SNT were present in 9.4% of the moose samples, 1.4% in red deer, 0.7% in roe deer, and nil in reindeer. The majority of the positive samples in our study originated from areas where human cases of TBE have been reported in Norway. The study is the first comprehensive screening of cervid species in Norway for antibodies to TBEV, and shows that cervids are useful sentinel animals to indicate TBEV occurrence, as supplement to studies in ticks. Furthermore, the results indicate that TBEV might be spreading northwards in Norway. This information may be of relevance for public health considerations and supports previous findings of TBEV in ticks in Norway.