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As clinical toxoplasmosis is not considered a problem in pigs, the main reason to implement a control strategy against Toxoplasma gondii (T. gondii) in this species is to reduce the establishment of T. gondii tissue cysts in pork, consequently reducing the risk of the parasite entering the human food chain. Consumption of T. gondii tissue cysts from raw or undercooked meat is one of the main sources of human infection, with infected pork being considered a high risk. This study incorporates a mouse bioassay with molecular detection of T. gondii DNA to study the effectiveness of vaccination (incomplete S48 strain) in its ability to reduce tissue cyst burden in pigs, following oocyst (M4 strain) challenge. Results from the mouse bioassay show that 100% of mice which had received porcine tissues from vaccinated and challenged pigs survived compared with 51.1% of mice which received tissues from non-vaccinated and challenged pigs. The presence (or absence) of T. gondii DNA from individual mouse brains also confirmed these results. This indicates a reduction in viable T. gondii tissue cysts within tissues from pigs which have been previously vaccinated with the S48 strain. In addition, the study demonstrated that the main predilection sites for the parasite were found to be brain and highly vascular muscles (such as tongue, diaphragm, heart and masseter) of pigs, while meat cuts used as human food such as chop, loin, left tricep and left semitendinosus, had a lower burden of T. gondii tissue cysts. These promising results highlight the potential of S48 strain tachyzoites for reducing the number of T. gondii tissues cysts in pork and thus improving food safety. ; This project was partially funded by a transnational access project funded through the European Union Seventh Framework Network of Animal Disease Infectiology Research Facilities (NADIR; reference number FP7-228394). AB was funded by the Moredun Foundation. FK, PMB, MN, JT, FC, EAI were supported by the Scottish Government, Rural and Environmental Sciences and Analytical Services (RESAS). JB was supported by CSIC and financed in part by European Social Fund (ESF). JLG was financed by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, BEX 10259-/12-0), Brazil and GC by the Instituto Nacional de Tecnologia Agropecuaria (INATA), Argentina. ; Peer reviewed

14 páginas, 2 tablas, 6 figuras. ; Bartley, Paul M. et al. ; 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. ; The authors would like to thank The Scottish Government's Rural and Environment Science and Analytical Services Division (RESAS) for supporting this work and the Bioservices Division of the Moredun Research Institute for care and management of the animals. ; Peer Reviewed

Cross-species transmission (CST) of bacterial pathogens has major implications for human health, livestock, and wildlife management because it determines whether control actions in one species may have subsequent effects on other potential host species. The study of bacterial transmission has benefitted from methods measuring two types of genetic variation: variable number of tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs). However, it is unclear whether these data can distinguish between different epidemiological sce-narios. We used a simulation model with two host species and known transmis-sion rates (within and between species) to evaluate the utility of these markers for inferring CST. We found that CST estimates are biased for a wide range of parameters when based on VNTRs and a most parsimonious reconstructed phy-logeny. However, estimations of CST rates lower than 5% can be achieved with relatively low bias using as low as 250 SNPs. CST estimates are sensitive to several parameters, including the number of mutations accumulated since introduction, stochasticity, the genetic difference of strains introduced, and the sampling effort. Our results suggest that, even with whole-genome sequences, unbiased estimates of CST will be difficult when sampling is limited, mutation rates are low, or for pathogens that were recently introduced. ; We thank Steven Kalinowsky, Pauline Kamath, Gina K. Hi-mes Boor and Aaron Foley for comments on previous versions of this manuscript. We also thank two anonymous reviewers for their helpful comments. This work was sup-ported by the National Science Foundation and National Institutes of Health Ecology of Infectious Disease (grant number DEB-1067129), the United States Geological Survey and WGFD. The views and opinions in this article are those of the authors and should not be construed to represent any views, determinations, or policies of the US Government. Also, any use of trade, product, or firm names is for descriptive purposes only and does not imply ...

14 páginas, 3 tablas, 11 figuras.--This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. ; Background: Stoat (Mustela erminea) and weasel (Mustela nivalis) populations in south-west England are declining whilst polecats (Mustela putorius), absent for over a century, are increasing. Little is known about the health status of these species nationally. This study aimed at investigating respiratory disease in specimens found dead in south-west England. Results: Trauma caused by road traffic, predator attack or being trapped was the predominant cause of death in 42 stoats, 31 weasels and 20 polecats; most were in good physical condition. Skrjabingylus nasicola was present in all species (weasels 37 %, polecats 39 %, stoats 41 %) and infected animals showed no evidence of loss of body condition. Even in carcases stored frozen L larvae were frequently alive and highly motile. Angiostrongylus vasorum infection was diagnosed in two stoats and one weasel: in stoats infections were patent and the lung lesions were likely of clinical significance. These are believed to be the first records of A. vasorum in small mustelids. Pleuritis and pyothorax was seen in two polecats, in one case due to a migrating grass awn. Histological examination of lungs showed granulomata in stoats (38 %), weasels (52 %) and polecats (50 %). Spherules consistent with Emmonsia spp. adiaspores were present in the granulomata of stoats (60 %), weasels (36 %) and polecats (29 %). Adiaspore diameter in all three species was similar (means: stoats 39 μm, weasels 30 μm, polecats 36 μm); these are markedly smaller than that normally recorded for E. crescens. Although they lie within the accepted range for spores of Emmonsia parva this arid-zone species is not found in Britain, thus raising a question over the identity of the fungus. Cases showing numerous granulomata but few or no adiaspores were Ziehl-Neelsen-stain negative for acid-fast bacilli and IHC negative for Mycobacterium spp. However, in some cases PCR analyses revealed mycobacteria, including Mycobacterium kumamotonense and Mycobacterium avium Complex. One stoat had numerous unidentified small organisms present centrally within granulomata. Conclusions: Stoats, weasels and polecats in south-west England share several respiratory diseases, often of high prevalence, but the pathology would appear insufficient to impact on the health status of the populations and other ultimate causes of death should be investigated when examining these species. ; The authors gratefully acknowledge the histological support given by Trevor Whitbread, Judith Hargreaves, Richard Fox, Lucy Oldroyd, Malcolm Silkstone, Sonja Rivers and Michelle Woodman at Abbey Veterinary Services. They also thank Nicholas Davison, Beverley Rule and Philip Booth, AHVLA Truro, Mark Wessels, Finn Pathologists, Luke Roberts and Eric Morgan, Bristol University, Marc Artois, Campus Vétérinaire de Lyon. Becki Lawson, Fieke Molenaar, Tamsyn Stephenson, Zoe Greatorex and Jane Simpson at Wildlife Veterinary Investigation Centre assisted with post-mortem-examinations. David Groves, Kate Stokes, Derek Lord and Cornwall Mammal Group and Cornwall Wildlife Trust members and staff, James Williams, Somerset Otter Group, and David Couper, Royal Society for the Prevention of Cruelty to Animals helped with carcase submissions. Andrew Borman, Mycology Reference Laboratory South West Health Protection Agency kindly commented on draft manuscripts. Eileen Harris and Rodney Bray at Natural History Museum are thanked for advice on parasites. Those parts of this study performed at AHVLA were funded under the Diseases of Wildlife Scheme and those performed at the Moredun Research Institute were funded by the Scottish Government Rural and Environment Science and Analytical Services Division. J. Benavides is supported by a "Ramón y Cajal" contract of the Spanish Ministry of Economy and Competitiveness. None of the authors received funding from other outside sources for this work. ; Peer Reviewed

9 páginas, 6 tablas. ; Fifty male Merino lambs (6 to 8 weeks, 14.1 kg; n=10 per group) were used to study the effect of feeding system: barley straw in long form and concentrate pellets in separate troughs (Control), ad libitum alfalfa supplemented with concentrate in separate troughs (Alfalfa) or including various levels of ground barley straw in concentrate pellets (B05, B15 and B25 for 50, 150 and 250 g barley straw/kg), on rumen characteristics, acid-base status, blood cell counts and lymphocyte stimulation. Alfalfa lambs had the heaviest digestive tract contents, highest rumen pH values, lowest volatile fatty acid concentration, highest papillae counts and best mucosa colour and the greatest blood pCO2 values, lowest sodium and chloride and highest potassium concentrations (P<0.05). Including ground barley straw in the concentrate pellet or providing straw in long form separately from the concentrate reduces rumen pH and darkens ruminal mucosa as compared with alfalfa-fed lambs, thus affecting acid-base status. © 2014 The Animal Consortium ; Spanish Ministry of Science and Innovation AGL2010-19094; Council of Castile and Leon ; European Social Fund; Scottish Government Rural & Environment Science & Analytical Services (RESAS) division ; BBSRC: BB/I019863/1 ;BB/I020519/1 ;AbD Serotec; Bio-Rad Company ; Peer Reviewed

Music is a powerful approach to engage communities and disseminate information. Specifically, health campaigns employing music have been used to promote behaviors that can prevent emerging infectious diseases (EIDs). For example, hip hop artists supported campaigns to prevent acquired immunodeficiency syndrome in the 70s in the United States, while Brazilian funk promoted vaccination to mitigate the ongoing COVID-19 pandemic. Similarly, we broadcast musical messages in local languages to increase community awareness and support prevention measures in Guinea and Liberia in response to the recent Ebola outbreak in 2021. Given the potential of music to promote both individual and population-level behavioral changes to prevent transmission, there is a need to consolidate information on music-based health interventions, and on how we can measure their effectiveness. In this perspective, we provide examples of relevant initiatives, discussing challenges and solutions associated with implementing interventions based on our experience with the 2021 Ebola outbreak. We recommend four steps for a successful music-based health intervention including (1) establishing a task force, (2) compose a "catchy" song including critical preventive measures, (3) deliver the song to the target audience, and (4) evaluate the campaign effectiveness. We argue that close interactions between scientists and musicians can produce rapid musical content for disease prevention. We also identify and discuss several methodological frameworks for testing the effectiveness of such interventions. We conclude that support from public health authorities, government media departments, and international agencies, is necessary to deliver wide outreach and long-term sustainability of musical messaging toward effective EID prevention.

Music is a powerful approach to engage communities and disseminate information. Specifically, health campaigns employing music have been used to promote behaviors that can prevent emerging infectious diseases (EIDs). For example, hip hop artists supported campaigns to prevent acquired immunodeficiency syndrome in the 70s in the United States, while Brazilian funk promoted vaccination to mitigate the ongoing COVID-19 pandemic. Similarly, we broadcast musical messages in local languages to increase community awareness and support prevention measures in Guinea and Liberia in response to the recent Ebola outbreak in 2021. Given the potential of music to promote both individual and population-level behavioral changes to prevent transmission, there is a need to consolidate information on music-based health interventions, and on how we can measure their effectiveness. In this perspective, we provide examples of relevant initiatives, discussing challenges and solutions associated with implementing interventions based on our experience with the 2021 Ebola outbreak. We recommend four steps for a successful music-based health intervention including (1) establishing a task force, (2) compose a "catchy" song including critical preventive measures, (3) deliver the song to the target audience, and (4) evaluate the campaign effectiveness. We argue that close interactions between scientists and musicians can produce rapid musical content for disease prevention. We also identify and discuss several methodological frameworks for testing the effectiveness of such interventions. We conclude that support from public health authorities, government media departments, and international agencies, is necessary to deliver wide outreach and long-term sustainability of musical messaging toward effective EID prevention.

11 páginas, 3 figuras, 2 tablas. ; The present study aimed to isolate and perform molecular and phenotypic characterization of Toxoplasma gondii strains infecting Iberian pigs bred under semi-free conditions and destined for human consumption. Blood and heart tissue samples from 361 fattening pigs from 10 various herds selected in the main areas of Iberian pig production were collected at a slaughterhouse; the sera were tested for anti-T. gondii antibodies using a commercial indirect ELISA kit, and a mouse bioassay was carried out using heart muscle of seropositive individual representatives from each geographical location. Seventy-nine (21.9%) of the 361 animals tested positive for anti-T. gondii antibodies according to the serology test. Fifteen samples of myocardial tissue were subjected to bioassay and 5 isolates (TgPigSp1 to TgPigSp5) were obtained. The isolates were characterized by using 11 PCR-RFLP genetic markers; three isolates had a ToxoDB #3 genotype (3/5) and two isolates had a ToxoDB #2 genotype (2/5). The TgPigSp1 and TgPigSp4 isolates were selected for virulence in mice characterization as instances of each different RFLP-genotype found. The TgPigSp1 isolate (#2 genotype) was virulent in mice with notable cumulative mortality (87.5%) and morbidity rates (100%); the TgPigSp4 (#3) was nonvirulent and triggered mild clinical signs in 42.1% of seropositive mice. Infection dynamics and organ distribution of both isolates were analyzed; the data revealed significant differences, including substantially higher parasite load in the lung during the acute phase of infection, in mice infected with TgPigSp1 than in the case of TgPigSp4 (median parasite load 7.6 vs. 0 zoites/mg, respectively; p < 0.05). Furthermore, degrees of severity of detected histopathological lesions appeared to be related to higher parasite burdens. Taking into account the unexpectedly high mortality rate and parasite load associated with the clonal genotype III, which is traditionally considered nonvirulent in mice, the need for further investigation and characterization of the T. gondii strains circulating in any host in Europe is emphasized. ; This research was supported by the projects funded by the Spanish Ministry of Science and Innovation (AGL2016- 75935-C2-R) and the Community of Madrid (PLATESA2-CMP2018/BAA-4370). MF-E was funded by UCM-Santander/2017 pre-doctoral grants. RC-B, EC-F, and LO-M are part of the TOXOSOURCES consortium supported by the funding from the European Union's Horizon 2020 Research and Innovation Programme under the grant agreement No 773830: One Health European Joint Programme.

Acknowledgements We are grateful to the staff of the Moredun Research Institute Bioservices Division for excellent support during the animal studies described in this work. Microarray hybridisation and data extraction were performed by the ARK-Genomics Centre for Comparative and Functional Genomics (www.ARKgenomics.org). This work was funded by the Scottish Government's Rural and Environment Science and Analytical Services Division and by the UK Biotechnology and Biological Sciences Research Council (BB/H008950) ; Peer reviewed ; Publisher PDF

15 páginas, 5 figuras, 2 tablas. ; Toxoplasma gondii is an important zoonotic agent with high genetic diversity, complex epidemiology, and variable clinical outcomes in animals and humans. In veterinary medicine, this apicomplexan parasite is considered one of the main infectious agents responsible for reproductive failure in small ruminants worldwide. The aim of this study was to phenotypically characterize 10 Spanish T. gondii isolates recently obtained from sheep in a normalized mouse model and in an ovine trophoblast cell line (AH-1) as infection target cells. The panel of isolates met selection criteria regarding such parameters as genetic diversity [types II (ToxoDB #1 and #3) and III (#2)], geographical location, and sample of origin (aborted foetal brain tissues or adult sheep myocardium). Evaluations of in vivo mortality, morbidity, parasite burden and histopathology were performed. Important variations between isolates were observed, although all isolates were classifed as "nonvirulent" (<30% cumulative mortality). The isolates TgShSp16 (#3) and TgShSp24 (#2) presented higher degrees of virulence. Signifcant diferences were found in terms of in vitro invasion rates and tachyzoite yield at 72 h post-inoculation (hpi) between TgShSp1 and TgShSp24 isolates, which exhibited the lowest and highest rates, respectively. The study of the CS3, ROP18 and ROP5 loci allelic profles revealed only type III alleles in ToxoDB #2 isolates and type II alleles in the #1 and #3 isolates included. We concluded that there are relevant intraand inter-genotype virulence diferences in Spanish T. gondii isolates, which could not be inferred by genetic charac‑terization using currently described molecular markers. ; This research was supported by projects funded by the Spanish Ministry of Science and Innovation (AGL2016-75935-C2-R) and the Community of Madrid (PLATESA2-CMP2018/BAA-4370). MF-E was funded by UCM-Santander/2017 pre-doctoral Grants. RC-B, EC-F, and LO-M are part of the TOXOSOURCES con‑ sortium supported by the funding from the European Union's Horizon 2020 Research and Innovation Programme under the Grant Agreement No 773830: One Health European Joint Programme.

Abstract Protection of cattle from alcelaphine herpesvirus-1 (AlHV-1)-induced malignant catarrhal fever (MCF) has been described previously, using an attenuated virus vaccine in an unlicensed adjuvant. The vaccine was hypothesised to induce a protective barrier of virus-neutralising antibody in the oro-nasal region, supported by the observation of high titre neutralising antibodies in nasal secretions of protected animals. Here we describe further analysis of this vaccine strategy, studying the effectiveness of the vaccine formulated with a licensed adjuvant; the duration of immunity induced; and the virus-specific antibody responses in plasma and nasal secretions. The results presented here show that the attenuated AlHV-1 vaccine in a licensed adjuvant protected cattle from fatal intranasal challenge with pathogenic AlHV-1 at three or six months. In addition, animals protected from MCF had significantly higher initial anti-viral antibody titres than animals that succumbed to disease; and these antibody titres remained relatively stable after challenge, while titres in vaccinated animals with MCF increased significantly prior to the onset of clinical disease. These data support the view that a mucosal barrier of neutralising antibody blocks infection of vaccinated animals and suggests that the magnitude of the initial response may correlate with long-term protection. Interestingly, the high titre virus-neutralising antibody responses seen in animals that succumbed to MCF after vaccination were not protective. ; This work was supported by the Scottish Government Rural and Environment Research and Analysis Directorate. ; Peer Reviewed

7 páginas, 3 figuras, 4 tablas. ; The experimental vaccine for bovine malignant catarrhal fever consists of viable attenuated alcelaphine herpesvirus 1 (AlHV-1) derived' by extensive culture passage, combined with an oil-in-water adjuvant, delivered intramuscularly. This immunisation strategy was over 80% effective in previous experimental and field trials and protection appeared to be associated with induction of virus-neutralising antibodies. Whether the vaccine virus is required to be viable at the point of immunisation and whether adjuvant is required to induce the appropriate immune responses remains unclear. To address these issues two studies were performed, firstly to analyse immune responses in the presence and absence of adjuvant and secondly, to investigate immune responses to vaccines containing adjuvant plus viable or inactivated AlHV-1. The first study showed that viable attenuated AlHV-1 in the absence of adjuvant induced virus-specific antibodies but the titres of virus-neutralising antibodies were significantly lower than those induced by vaccine containing viable virus and adjuvant, suggesting adjuvant was required for optimal responses. In contrast, the second study found that the vaccine containing inactivated (>99.9%) AlHV-1 induced similar levels of virus-neutralising antibody to the equivalent formulation containing viable AlHV-1. Together these studies suggest that the MCF vaccine acts as an antigen depot for induction of immune responses, requiring adjuvant and a suitable antigen source, which need not be viable virus. These obser- vations may help in directing the development of alternative MCF vaccine formulations for distribution in the absence of an extensive cold chain. ; This work was supported by the Scottish Government Rural and Environment Science and Analytical Services (RESAS) Strategic Research Programme; the Department for International Develop- ment and the Biotechnology and Biological Sciences Research Council, grant BB/H008950/1; and GALVmed with funding from Bill & Melinda Gates Foundation and UKAID, grant MRI-R34A0985 ; Peer reviewed

12 páginas, 4 figuras. ; The Spanish Ministry of Science and Innovation (Projects AGL2015-66540-C21-R and RTI2018-099496-B-I00) and Junta de Castilla y León (Project LE259P18) fnanced this work. This work was also fnancially supported by the University of León. Noive Arteche Villasol has been fnancially supported by a predoc‑ toral contract (BES-2016-076513) from the Spanish Ministry of Science and Innovation. Daniel Gutiérrez-Expósito and José Espinosa are the recipients of a postdoctoral contract from the Ministry of Science and Innovation (Grants No. FJCI-2017-32020 and FJC2019-042422-I, respectively). Iraia Ladero-Auñon holds a pre-doctoral grant from "Departamento de Economía e Infraestruc‑ turas" of the Basque Government. ; Peer reviewed