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Insulin-like peptide 3 (INSL3) is a constitutive product of mature, adult-type Leydig cells of the testes and consequently in most mammals is an ideal biomarker with which to monitor pubertal development. A new heterologous time-resolved fluorescence immunoassay was developed and validated to measure circulating INSL3 in the blood of adult male dogs. Compared to other species, INSL3 concentration is low with marked variation between individuals, which appears to be independent of breed, age, or weight. A model system was then used in which a cohort of beagle dogs was subject to a GnRH-agonist implant to suppress the HPG axis and spermatogenesis, followed by implant removal and recovery. Unlike testosterone, INSL3 levels were not fully suppressed in all animals by the GnRH agonist, nor was the recovery of Leydig cell function following implant removal uniform or complete, even after several weeks. In dogs, and dissimilar from other species (including humans), Leydig-cell INSL3 appears to be quite variable between individual dogs and only weakly connected to the physiology of the HPG axis after its suppression by a GnRH-agonist implant and recovery. Consequently, INSL3 may be less useful in this species for the assessment of testis function.

Citrus greening, or huanglongbing (HLB), caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus (CaLas), threatens the global citrus industry. Field observations have demonstrated that some citrus cultivars are more tolerant to the CaLas pathogen than others. ‘Parson Brown’ is an early maturing sweet orange variety that has consistently exhibited minimal leaf and fruit drop in the field compared with the ‘Hamlin’ sweet orange under similar conditions. This study aimed to understand performance of the ‘Parson Brown’ cultivar in several locations across the citrus production regions of Florida. Results indicated that the CaLas bacterial titer in both cultivars were similar with the quantitative polymerase chain reaction cycle threshold values ranging between 24.99 and 28.61 in ‘Hamlin’ and between 25.48 and 30.89 in ‘Parson Brown’. Leaves from the ‘Parson Brown’ trees however demonstrated higher chlorophyll content and total phenolic compounds in most of the locations. We also detected higher relative expression of CsPR1 and CsPR2 transcripts in ‘Parson Brown’ leaves in the first sampling period (March) and the fourth period (November). Additionally, Phloem protein 2 transcripts were downregulated in ‘Parson Brown’ leaves compared with ‘Hamlin’ at all locations. The ‘Hamlin’ juice had higher acidity, whereas ‘Parson Brown’ juice demonstrated a higher Brix to acidity ratio and juice color. The oil content in the juice ranged between 0.020% and 0.042%, and there was variation in the oil content between the locations, which could indicate clonal differences. ‘Parson Brown’ juice however contained higher limonin and nomilin content than ‘Hamlin’ juice in most of the locations. Taken together, the current results confirmed the enhanced tolerance of ‘Parson Brown’ trees to HLB when compared with ‘Hamlin’ in all sampled locations.

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Abstract Though relatively rare in dogs, prostate cancer (PCa) is the most common non‐cutaneous cancer in men. Human and canine prostate glands share many functional, anatomical and physiological features. Due to these similarities, canine PCa has been proposed as a model for PCa in men. PCa is typically androgen‐dependent at diagnosis in men and for this reason, androgen deprivation therapies (ADT) are important treatments for advanced PCa in men. In contrast, there is some evidence that PCa is diagnosed more commonly in castrate dogs, at which point, limited therapeutic options are available. In men, a major limitation of current ADT is that progression to a lethal and incurable form of PCa, termed castrate‐resistant prostate cancer (CRPC), is common. There is, therefore, an urgent need for a better understanding of the mechanism of PCa initiation and progression to CRPC to enable the development of novel therapeutic approaches. This review focuses on the functional, physiological, endocrine and histopathological similarities and differences in the prostate gland of these species. In particular, we focus on common physiological roles for androgen signalling in the prostate of men and dogs, we review the short‐ and longer‐term effects of castration on PCa incidence and progression in the dog and relate how this knowledge may be relevant to understanding the mechanisms of CRPC in men.

The toll-like receptor (TLR) family comprises both cell-surface and intracellular receptors that recognize different types of pathogen-associated molecular patterns (PAMPs) leading to the production of pro-inflammatory cytokines and subsequent development of adaptive immunity. TLR2 is a cell-surface receptor initially thought to act as a bacterial sentinel but also shown to recognize a number of viral glycoproteins. In this study, we sought to characterize the role of TLR2 in the activation of the immune response by peste des petits ruminants virus (PPRV), a morbillivirus of the Paramixoviridae family that causes an acute, highly contagious disease in goats and sheep. Using human embryonic kidney (HEK) 293 cells stably expressing human (h)TLR2 but lacking any other TLR, we found that PPRV induces IL-8 production in a dose-dependent manner. That activation is only observed in cells expressing hTLR2 and is greatly reduced when the receptor is blocked by pretreatment with specific antibody. We identified hemagglutinin (H) as the viral protein responsible of TLR2 activation by performing the same assays with purified recombinant mammalian-expressed H protein. Exogenous addition of recombinant H protein to cell culture induces high levels of interleukin (IL)-8 only in TLR2-expressing cells. Moreover, H engagement on TLR2 in the monocytic cell line THP-1 activates extracellular-signal-regulated kinase (ERK) signaling. Stimulation of primary ovine dendritic cells with either inactivated PPRV or purified recombinant H protein results in transcription of pro-inflammatory cytokines and the secretion of the Th1-polarizing cytokine IL-12. The role of these host immune mechanisms in the control of PPR is discussed.

Many dogs and cats are affected by chronic diseases that significantly impact their health and welfare and relationships with humans. Some of these diseases can be challenging to treat, and a better understanding of early-life risk factors for diseases occurring in adulthood is key to improving preventive veterinary care and husbandry practices. This article reviews early-life risk factors for obesity and chronic enteropathy, and for chronic behavioral problems, which can also be intractable with life-changing consequences. Aspects of early life in puppies and kittens that can impact the risk of adult disorders include maternal nutrition, establishment of the gut microbiome, maternal behavior, weaning, nutrition during growth, growth rate, socialization with conspecifics and humans, rehoming and neutering. Despite evidence in some species that the disorders reviewed here reflect the developmental origins of health and disease (DOHaD), developmental programming has rarely been studied in dogs and cats. Priorities and strategies to increase knowledge of early-life risk factors and DOHaD in dogs and cats are discussed. Critical windows of development are proposed: preconception, gestation, the suckling period, early growth pre-neutering or pre-puberty, and growth post-neutering or post-puberty to adult size, the durations of which depend upon species and breed. Challenges to DOHaD research in these species include a large number of breeds with wide genetic and phenotypic variability, and the existence of many mixed-breed individuals. Moreover, difficulties in conducting prospective lifelong cohort studies are exacerbated by discontinuity in pet husbandry between breeders and subsequent owners, and by the dispersed nature of pet ownership.

Plant microbiomes are critical components to plant health and can influence disease outcomes. We provide empirical data describing disease-induced shifts within the citrus microbiome at different levels of huanglongbing (HLB) disease severity. HLB is associated with an invasive phloem-limited bacterium, ‘Candidatus Liberibacter asiaticus’, that is introduced into the aerial portions of the tree by an insect vector. Disease manifests as aboveground foliar and fruit symptoms and significant root decline belowground. During the early phase of disease, there were depletions of putative keystone taxa in leaves and roots, followed by enrichments of putative beneficial taxa, suggesting a microbially derived immune response involved in plant protection that is ancillary to immune components encoded in the plant’s genome. In the late phase of disease, we observed enrichments of parasitic and saprophytic microorganisms, particularly in the roots. The community shifts within the root compartment are emblematic of a disease-induced dysbiosis where pathogens other than ‘Ca. L. asiaticus’ begin to dominate the community. Furthermore, we define key taxa enriched in trees with a slower rate of disease development, referred to as survivor trees, that are hallmarks of those found in trees in the early phase of disease that may be drivers of the survivor tree phenotype. We propose a disease ecology model that illustrates the relationship between the pathogen, the microbiome, and the host plant that highlights microorganisms that may serve as disease facilitators or antagonists.

Abstract 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.

This new 9-page publication of the UF/IFAS Horticultural Sciences Department focuses on the nitrogen fertilizer best management practices (BMP) for green fresh-market and processing cabbage head production in Florida. This publication aims to provide management strategies that comply with statewide BMP guidelines to optimize economic yield while minimizing nitrogen losses to the environment. Written by L. Zotarelli, C. E. Barrett, A. L. B. R. da Silva, C. T. Christensen, and G. K. England. https://edis.ifas.ufl.edu/hs1428

This new 11-page publication focuses on the nitrogen (N) fertilizer best management practices (BMP) for potato crop in Florida. The aim is to provide management strategies that comply with statewide BMP guidelines to maximize yield and economic return while minimizing N losses to the environment. Written by Lincoln Zotarelli, Tara Wade, Gary K. England, and Christian T. Christensen and published by the UF/IFAS Horticultural Sciences Department. https://edis.ifas.ufl.edu/hs1429

Ovine footrot is a complex multifactorial infectious disease, causing lameness in sheep with major welfare and economic consequences. Dichelobacter nodosus is the main causative bacterium; however, footrot is a polymicrobial disease with Fusobacterium necrophorum, Mycoplasma fermentans and Porphyromonas asaccharolytica also associated. There is limited understanding of the host response involved. The proinflammatory mediators, interleukin (IL)-1β and C-X-C Motif Chemokine Ligand 8 (CXCL8), have been shown to play a role in the early response to D. nodosus in dermal fibroblasts and interdigital skin explant models. To further understand the response of ovine skin to bacterial stimulation, and to build an understanding of the role of the cytokines and chemokines identified, primary ovine interdigital fibroblasts and keratinocytes were isolated, cultured and stimulated. The expression of mRNA and protein release of CXCL8 and IL-1β were measured after stimulation with LPS, D. nodosus or F. necrophorum, which resulted in increased transcript levels of IL-1β and CXCL8 in the M. fermentans-free cells. However, only an increase in the CXCL8 protein release was observed. No IL-1β protein release was detected, despite increases in IL-1β mRNA, suggesting the signal for intracellular pre-IL-1β processing may be lacking when culturing primary cells in isolation. The keratinocytes and fibroblasts naturally infected with M. fermentans showed little response to the LPS, a range of D. nodosus preparations or heat-inactivated F. necrophorum. Primary single cell culture models complement ex vivo organ culture models to study different aspects of the host response to D. nodosus. The ovine keratinocytes and fibroblasts infected with M. fermentans had a reduced response to the experimental bacterial stimulation. However, in the case of footrot where Mycoplasma spp. are associated with diseased feet, this natural infection gives important insights into the impact of multiple pathogens on the host response.

Potato (Solanum tuberosum L.) has low phosphorus (P) use efficiency as compared with other vegetable crops. This study was conducted at two commercial chipping potato farms (A and B) in Northeast Florida to evaluate different P rates for potato production. Plot size was 0.62 and 0.49 hectares for Farms A and B, respectively. The total trial area was 13.32 hectares per growing season for three consecutive years from 2018 to 2020. The randomized complete block design (RCBD) was employed with four replications per P rate. The chipping potato variety, ‘Atlantic’ was cultivated with three P rates: 0, 24.5, and 48.9 kg P ha−1 in 2018, and 12.2, 24.5, and 48.9 kg P ha−1 in 2019 and 2020. The soil of Farm A contained 497 mg kg−1 Mehlich-3 extractable P and 946 mg kg−1 aluminum (Al), and that of Farm B had 220 mg kg−1 Mehlich-3 extractable P and 253 mg kg−1 Al. The results showed that a P rate of 48.9 kg P ha−1 significantly improved tuber yield as compared to 0 in 2018 or 12.2 kg P ha−1 in 2019 and 2020. Application of 48.9 kg P ha−1 fertilizer P significantly increased the soil P level in 2018 and 2020, while the application of 24.5 kg P ha−1 fertilizer P increased the soil P level significantly in 2018 only. Tubers with 48.9 kg P ha−1 showed significantly lower external quality issues than 0 or 12.2 and 24.5 kg P ha−1. However, there were no significant differences in specific gravity, internal tuber quality, and tuber size among the different P rates. The tuber yield data show that potato plants grown on soil with high legacy P still require approximately 50 kg ha−1 P application for sustainable potato production in the area. This high P requirement results from the combination of high concentrations of active metals (Al and iron (Fe)) and a decrease in pH of one unit in the growing season. New P-fertilization programs with post-plant applications rather than with pre-plant application are urgently needed for minimizing P-immobilization by Al and Fe and improving P-use efficiency for potato production in the state.

It is well-recognized that research capability in veterinary species is restricted by a lack of immunological reagents relative to the extensive toolboxes for small rodent biomedical model species and humans. This creates a barrier to the strategic development of disease control solutions for livestock, companion animals and wildlife that not only affects animal health but can affect human health by increasing the risk of transmission of zoonotic pathogens. There have been a number of projects aimed at reducing the capability gaps in the veterinary immunological toolbox, the majority of these focusing on livestock species. Various approaches have been taken to veterinary immunological reagent development across the globe and technological advances in molecular biology and protein biochemistry have accelerated toolbox development. While short-term funding initiatives can address specific gaps in capability, they do not account for long-term sustainability of reagents and databases that requires a different funding model. We review the past, present and future of the veterinary immunological toolbox with specific reference to recent developments discussed at the International Union of Immunological Societies (IUIS) Veterinary Immunology Committee (VIC) Immune Toolkit Workshop at the 12th International Veterinary Immunology Symposium (IVIS) in Seattle, USA, 16–19 August 2019. The future availability of these reagents is critical to research for improving animal health, responses to infectious pathogens and vaccine design as well as for important analyses of zoonotic pathogens and the animal /human interface for One Health initiatives.

Chlamydia abortus is one of the most commonly diagnosed causes of infectious abortion in small ruminants worldwide. Control of the disease (Enzootic Abortion of Ewes or EAE) is achieved using the commercial live, attenuated C. abortus 1B vaccine strain, which can be distinguished from virulent wild-type (wt) strains by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Published studies applying this typing method and whole-genome sequence analyses to cases of EAE in vaccinated and non-vaccinated animals have provided strong evidence that the 1B strain is not attenuated and can infect the placenta causing disease in some ewes. Therefore, the objective of this study was to characterise the lesions found in the placentas of ewes vaccinated with the 1B strain and to compare these to those resulting from a wt infection. A C. abortus-free flock of multiparous adult ewes was vaccinated twice, over three breeding seasons, each before mating, with the commercial C. abortus 1B vaccine strain (Cevac® Chlamydia, Ceva Animal Health Ltd.). In the second lambing season following vaccination, placentas (n = 117) were collected at parturition and analysed by C. abortus-specific real-time quantitative PCR (qPCR). Two placentas, from a single ewe, which gave birth to live twin lambs, were found to be positive by qPCR and viable organisms were recovered and identified as vaccine type (vt) by PCR-RFLP, with no evidence of any wt strain being present. All cotyledons from the vt-infected placentas were analysed by histopathology and immunohistochemistry and compared to those from wt-infected placentas. Both vt-infected placentas showed lesions typical of those found in a wt infection in terms of their severity, distribution, and associated intensity of antigen labelling. These results conclusively demonstrate that the 1B strain can infect the placenta, producing typical EAE placental lesions that are indistinguishable from those found in wt infected animals.

Objective: To perform a scoping review of the current evidence on the horse-human relationship. Background: The horse-human relationship has a significant impact on how horse owners care for and make decisions for their horse. Evidentiary value: Identification of consensus and gaps in current evidence. Methods: A literature search was performed in CAB Abstracts and Medline using search terms relating to the nature of the horse-human relationship in horses used for pleasure riding. Publications were reviewed against inclusion and exclusion criteria. Original qualitative or observational research studies relating to the relationship between a horse and owner were analysed. Data were extracted on study method and population characteristics. Results: There were 4,481 studies identified; 27 studies were included in the final data extraction. The studies covered 11 different areas, the most frequent were effect of humans on equine behaviour (5/27), equine training methods and behaviour (4/27) and horses within sport and leisure (4/27). A range of methodologies were used, with the most frequent being thematic analysis (6/27 studies), use of an instrument, tool or scale (3/27) and behavioural scoring (4/27). The majority of studies considered the human’s perspective (20/27), six considered the horse perspective and one considered both the horse and human perspective. No studies investigated the same or similar aims or objectives. Conclusion: The current evidence on the horse-human relationship is diverse and heterogenous, which limits the strength of evidence for any particular area. Application: Future research should focus on developing reliable and repeatable tools to assess owner motivations and horse-human relationship, to develop a body of evidence.

Citrus production throughout the world is being severely threatened by Huanglongbing (HLB), which is a disease associated with the bacteria ‘Candidatus Liberibacter asiaticus’ (CLas), africanus, and americanus. This Resource Announcement provides amplicon-based next generation sequencing (NGS) datasets of the bacterial and fungal rRNA internal transcribed spacer (ITS) region from CLas-infected citrus budwood, leaves, and roots from five orchards located in different geographical regions in Florida (USA). To our knowledge, this is the first amplicon-based NGS study (i) that describes the fungal taxa associated with citrus and (ii) that provides comparative analyses of the bacterial and fungal taxa associated with budwood, leaves, and roots from the same citrus trees. This report also provides the sample metadata linked to these sequence datasets including HLB severity rating, tissue type, citrus rootstock, citrus scion, geographical region, and year trees were planted. When analyzed with other similar datasets, we anticipate that researchers will be able to obtain a greater understanding of the factors that shape the citrus microbiome as well as identify individual microorganisms or consortia of microorganisms that play a role in HLB suppression or exacerbation.

Tubal ectopic pregnancies are a leading cause of global maternal morbidity and mortality. Previous infection with Chlamydia trachomatis is a major risk factor for tubal embryo implantation but the biological mechanism behind this association is unclear. Successful intra-uterine embryo implantation is associated with increased expression of endometrial “receptivity” integrins (cell adhesion molecules). We examined integrin expression in Fallopian tubes of women with previous C. trachomatis infection, in mice experimentally infected with C. trachomatis, in immortalised human oviductal epithelial cells (OE-E6/E7) and in an in vitro model of human embryo attachment (trophoblast spheroid-OE-E6/7 cell co-culture). Previous exposure with C. trachomatis increased Fallopian tube/oviduct integrin-subunit beta-1 (ITGB1) in women and mice compared to controls. C. trachomatis increased OE-E6/E7 cell ITGB1 expression and promoted trophoblast attachment to OE-E6/E7 cells which was negated by anti-ITGB1-antibody. We demonstrate that infection with C. trachomatis increases tubal ITGB1 expression, predisposing to tubal embryo attachment and ectopic pregnancy. Keywords: Ectopic pregnancy, Chlamydia trachomatis, Integrins, Embryo implantation, Fallopian tube

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.

A clinical study was performed to evaluate the diagnostic methods for ovarian cysts (OC), and to determine the metabolic profiles of animals with OC in the region of Mitidja in the North of Algeria. A total of 504 non-pregnant lactating cows were used in this study. Ultrasonography was performed by EXAGO scanner and was combined with assessment of serum P4. Biochemical serum parameters were assayed by spectrophotometry and insulin and cortisol serum measurement was performed by electrochemiluminescence. The results showed an overall incidence of 11.9% of OC. The incidence of OC was higher among cows in third lactation. Holstein breed was the most affected by OC compared with other breeds (P0.05). OC were single in 91% of cases. They were found mainly on the right ovary (66.66%). Seasonality had a significant influence on incidence rate of OC with higher incidence rates during winter and spring (71.66%); while, 28.33% of OC were detected during the summer and autumn (P

Abstract Miscarriage affects ~20% of pregnancies and maternal infections account for ~15% of early miscarriages. Chlamydia trachomatis (Ct) has been associated with miscarriage but the underlying mechanisms are unknown. Successful implantation requires endometrial stromal cell (ESC) decidualisation. Maintenance of pregnancy requires angiogenesis, establishment of the correct cellular milieu and trophoblast invasion, all of which involve the action of chemokines. Our objective was to determine whether Ct infection impacts upon ESC decidualisation and chemokine secretion. Human primary ESC were decidualised in-vitro, infected with Ct serovar E, and changes in expression of genes of interest were measured using RT-PCR, proteomic array and ELISA. We demonstrate for the first time that Ct can infect and proliferate in ESC. Expression of the decidualisation marker prolactin was decreased in Ct-infected ESC at both mRNA and protein levels. Ct infection altered the chemokine profile of decidualised ESC as shown by proteomic array. Chemokines CXCL12 and CXCL16, important for trophoblast invasion, were analysed further and expression was reduced in infected decidualised cells at mRNA and protein levels. Our data indicate that Ct infection of ESC impairs decidualisation and alters chemokine release. These findings at least partially explain how Ct infection could result in adverse pregnancy outcomes.

Abstract The development of methods to detect cytokine expression by T cell subsets in ruminants is fundamental to strategic development of new livestock vaccines for prevention of infectious diseases. It has been possible to detect T cell expression of IFN-γ, IL-4 and IL-10 in ruminants for many years but methods to detect expression of IL-17A are relatively limited. To address this gap in capability we have cloned bovine and ovine IL-17A cDNAs and expressed biologically-active recombinant proteins in Chinese Hamster Ovary (CHO) cells. We used the transfected CHO cells to screen commercially-available antibodies for their ability to detect IL-17A expression intracellularly and in culture supernates. We demonstrate that an ELISA for bovine IL-17A detects native ovine IL-17A. Moreover, the constituent polyclonal antibodies (pabs) in the ELISA were used to enumerate peripheral blood mononuclear cells (PBMC) expressing IL-17A from cattle and sheep by ELISpot. We identified two monoclonal antibodies (mabs) that detect recombinant intracellular IL-17A in CHO cells by flow cytometry. One of these mabs was used to detect native intracellular IL-17A expression in PBMC in conjunction with cell surface phenotyping mabs [CD4+ve, CD8+ve and Workshop Cluster 1 (WC-1)+ve gamma-delta (γδ)] we show that distinct T cell subsets in cattle (defined as CD4+ve, CD8+ve or WC-1+ve) and sheep (defined as CD4+ve or WC-1+ve) can express IL-17A following activation. These novel techniques provide a solid basis to investigate IL-17A expression and define specific CD4+ve T cell subset activation in ruminants.

Objective: The aim of this study was to evaluate equine livery yard owners’ opinions of emergency conditions, and to identify factors influencing emergency decision making in the horse.Background: There is limited data on horse owners’ opinions and decision making in emergency conditions.Evidentiary value: An online questionnaire was distributed to UK livery yard owners accredited with the British Horse Society. There were 104 survey participants, who represented experienced owners with responsibility for care of a number of horses.Methods: The questionnaire consisted of open and closed questions on participants’ demographics, their experiences and opinions of the most common equine emergency conditions, and emergency decision making. Descriptive data analysis included frequency ranking and categorisation of free text responses.Results: The majority of respondents had kept horses for more than 10 years (97%), and reported previous experience of emergency conditions (99%), predominantly colic (96%) and wounds (92%). Participants considered that the most common emergency conditions were colic (98%), wounds (49%) and fractures (22%), and the most concerning conditions were colic (94%), lameness (36%) and wounds (21%). Factors believed to be important in emergency decision making were: degree of pain, likelihood of condition resolving, and severity of disease.Conclusion: This study highlights the importance of colic and wounds as emergency conditions in the horse, and describes factors considered important in emergency decision making.Application: The outcomes identify where research and clinical resources should be targeted to improve emergency care for horses. The results were used to inform a survey of the wider horse population.

Successful mammalian pregnancies are a result of complex physiological, endocrinological, and immunological processes that combine to create an environment where the mother is tolerant to the semi-allogeneic fetus. Our knowledge of the mechanisms that contribute to maternal tolerance is derived mainly from human and murine studies of haemochorial placentation. However, as this is the most invasive type of placentation it cannot be assumed that identical mechanisms apply to the less invasive epitheliochorial placentation found in other species such as ruminants. Here, we examine three features associated with reproductive immune regulation in a transformed ovine trophoblast cell line and ex-vivo ovine reproductive tissues collected at term, namely: major histocompatibility complex (MHC) expression, Indoleamine 2,3 dioxygenase-1 (IDO-1) expression, and Natural Killer (NK) cell infiltration. High levels of MHC class I protein expression were detected at the surface of the trophoblast cell line using a pan-MHC class I specific monoclonal antibody. The majority of MHC class I transcripts isolated from the cell line clustered with classical MHC alleles. Transcriptional analysis of placental tissues identified only classical MHC class I transcripts. We found no evidence of constitutive transcription of IDO-1 in either the trophoblast cell line or placental tissues. Ex-vivo tissues collected from the materno-fetal interface were negative for cells expressing NKp46/NCR1. Collectively, these observations suggest that the relatively non-invasive synepitheliochorial placentation found in sheep has a more limited requirement for local immunoregulation compared to the more invasive haemochorial placentation of primates and rodents.

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.

Objective: To assess whether conversion from twin to singleton pregnancy following the demise of a co-twin influences survival. Methods: This retrospective study compared the risk for neonatal, post-neonatal and infant death for converted co-twins versus unconverted co-twins using the US matched multiple file dataset for the period 1995–2000. We also examined the same risks for converted versus same-quantile co-twins, hazard ratios (HR) and 95% confidence intervals (CI) were computed using Cox Proportional Hazards models. Results: The risk for neonatal (HR = 0.18, 95% CI: 0.09–0.34 and HR = 0.69, 95% CI: 0.50–0.96) and infant death (HR = 0.22, 95% CI: 0.12–0.42 and HR = 0.57, 95% CI: 0.42–0.77) were significantly lower for converted twins than for unconverted twins and same-quantile twins, respectively. For black compared to white, the risk for post-neonatal death increased by 89% (HR = 1.89, 95% CI = 1.03, 3.48), and 79% (HR = 1.79, 95% CI = 1.53, 2.09) for converted vs. unconverted and converted vs. same-quantile, respectively. For converted black, the risk for neonatal death decreased by 17% (HR = 0.83, 95% CI = 0.73–0.93) as compared to unconverted. Conclusions: Risks for all mortality types were lower among converted co-twins than their unconverted or same-quantile counterparts. The lower neonatal and higher post-neonatal mortality among black require future research.

Skin infection studies are often limited by financial and ethical constraints, and alternatives, such as monolayer cell culture, do not reflect many cellular processes limiting their application. For a more functional replacement, 3D skin culture models offer many advantages such as the maintenance of the tissue structure and the cell types present in the host environment. A 3D skin culture model can be set up using tissues acquired from surgical procedures or post slaughter, making it a cost effective and attractive alternative to animal experimentation. The majority of 3D culture models have been established for aerobic pathogens, but currently there are no models for anaerobic skin infections. Footrot is an anaerobic bacterial infection which affects the ovine interdigital skin causing a substantial animal welfare and financial impact worldwide. Dichelobacter nodosus is a Gram-negative anaerobic bacterium and the causative agent of footrot. The mechanism of infection and host immune response to D. nodosus is poorly understood. Here we present a novel 3D skin ex vivo model to study anaerobic bacterial infections using ovine skin explants infected with D. nodosus. Our results demonstrate that D. nodosus can invade the skin explant, and that altered expression of key inflammatory markers could be quantified in the culture media. The viability of explants was assessed by tissue integrity (histopathological features) and cell death (DNA fragmentation) over 76 h showing the model was stable for 28 h. D. nodosus was quantified in all infected skin explants by qPCR and the bacterium was visualized invading the epidermis by Fluorescent in situ Hybridization. Measurement of pro-inflammatory cytokines/chemokines in the culture media revealed that the explants released IL1β in response to bacteria. In contrast, levels of CXCL8 production were no different to mock-infected explants. The 3D skin model realistically simulates the interdigital skin and has demonstrated that D. nodosus invades the skin and triggered an early cellular inflammatory response to this bacterium. This novel model is the first of its kind for investigating an anaerobic bacterial infection.

This study investigated the pathogenesis of two variant strains (LLG and POS) of Chlamydia abortus, in comparison to a typical wild-type strain (S26/3) which is known to be responsible for late term abortion in small ruminants. Challenge with the three strains at mid-gestation resulted in similar pregnancy outcomes, with abortion occurring in approximately 50-60% of ewes with the mean gestational lengths also being similar. However, differences were observed in the severity of placental pathology, with infection appearing milder for strain LLG, which was reflected in the lower number of organisms shed in vaginal swabs post-partum and less gross pathology and organisms present in placental smears. Results for strain POS were somewhat different than LLG with a more focal restriction of infection observed. Post-abortion antibody responses revealed prominent differences in seropositivity to the major outer membrane protein (MOMP) present in elementary body (EB) preparations under denaturing conditions, most notably with anti-LLG and anti-POS convalescent sera where there was no or reduced detection of MOMP present in EBs derived from the three strains. These results and additional analysis of whole EB and chlamydial outer membrane complex preparations suggest that there are conformational differences in MOMP for the three strains. Overall, the results suggest that gross placental pathology and clinical outcome is not indicative of bacterial colonization and the severity of infection. The results also highlight potential conformational differences in MOMP epitopes that perhaps impact on disease diagnosis and the development of new vaccines.

Citrus trees are evergreen, never become fully dormant, and cannot withstand temperatures as low as those tolerated by deciduous trees. But citrus trees can become preconditioned or acclimated to cool air temperatures that occur in late fall and winter. One of the best ways to lessen cold injury and to hasten recovery from cold damage is to maintain healthy trees. This 5-page fact sheet discusses the symptoms of freeze damage and ways to help recover trees that have been damaged. Written by Mongi Zekri, Chris Oswalt, Steve Futch, Gary England, Camille McAvoy, Laurie Hurner, and Parker Platts, and published by the Horticultural Sciences Department, January 2016. HS1275/HS1275: Freeze Damage Symptoms and Recovery for Citrus (ufl.edu)

Two bone graft extenders differing in chemical composition were implanted contralaterally in 27 consecutive patients undergoing instrumented posterolateral lumbar fusion as standard of care. Bone marrow aspirate and autogenous bone graft were equally combined either with β-tricalcium phosphate (β-TCP) or a hybrid biomaterial (containing hyaluronic acid but lacking a calcium salt) and implanted between the transverse processes. Fusion status on each side of the vertebrae was retrospectively graded (1-5 scale) on AP planar X-ray at multiple visits as available, through approximately month 12. Additionally, consolidation or resorption since prior visit for each treatment was recorded. Sides receiving β-TCP extender showed marked resorption prior to bone consolidation during the first 6 months. By contrast, sides receiving the hybrid biomaterial containing integrated hyaluronic acid showed rapid bone consolidation by week 6-8, with maintenance of initial bone volume through month 12. Fusion grade was superior for the hybrid biomaterial, differing significantly from β-TCP at day 109 and beyond. Fusion success at >month 12 was 92.9% vs 67.9% for the hybrid biomaterial and β-TCP-treated sides, respectively. The hybrid biomaterial extender demonstrated a shortened time to fusion compared to the calcium-based graft. Mode of action has been demonstrated in the literature to differ between these compositions. Therefore, choice of synthetic biomaterial composition may significantly influence mode of action of cellular events regulating appositional bone growth.

Introduction: Non-invasive measurements of brain activity have an important role to play in understanding driving ability. The current study aimed to identify the neural underpinnings of human driving behavior by visualizing the areas of the brain involved in driving under different levels of demand, such as driving while distracted or making left turns at busy intersections. Methods: To capture brain activity during driving, we placed a driving simulator with a fully functional steering wheel and pedals in a 3.0 Tesla functional magnetic resonance imaging (fMRI) system. To identify the brain areas involved while performing different real-world driving maneuvers, participants completed tasks ranging from simple (right turns) to more complex (left turns at busy intersections). To assess the effects of driving while distracted, participants were asked to perform an auditory task while driving analogous to speaking on a hands-free device and driving. Results: A widely distributed brain network was identified, especially when making left turns at busy intersections compared to more simple driving tasks. During distracted driving, brain activation shifted dramatically from the posterior, visual and spatial areas to the prefrontal cortex. Conclusions: Our findings suggest that the distracted brain sacrificed areas in the posterior brain important for visual attention and alertness to recruit enough brain resources to perform a secondary, cognitive task. The present findings offer important new insights into the scientific understanding of the neuro-cognitive mechanisms of driving behavior and lay down an important foundation for future clinical research.

Latency is a key feature of the animal pathogen Chlamydia abortus, where infection remains inapparent in the non-pregnant animal and only becomes evident during a subsequent pregnancy. Often the first sign that an animal is infected is abortion occurring late in gestation. Despite this, little is understood of the underlying mechanisms that control latency or the recrudescence of infection that occurs during subsequent pregnancy. The aim of this study was to develop an experimental model of latency by mimicking the natural route of infection through the intranasal inoculation of non-pregnant sheep with C. abortus.Three groups of sheep (groups 1, 2 and 3) were experimentally infected with different doses of C. abortus (5×10(3), 5×10(5) and 5×10(7) inclusion forming units (IFU), respectively) prior to mating and monitored over 2 breeding cycles for clinical, microbiological, pathological, immunological and serological outcomes. Two further groups received either negative control inoculum (group 4a,b) or were inoculated subcutaneously on day 70 of gestation with 2×10(6) IFU C. abortus (group 5). Animals in groups 1, 2 and 5 experienced an abortion rate of 50-67%, while only one animal aborted in group 3 and none in group 4a,b. Pathological, microbiological, immunological and serological analyses support the view that the maternal protective immune response is influenced by initial exposure to the bacterium.The results show that intranasal administration of non-pregnant sheep with a low/medium dose of C. abortus results in a latent infection that leads in a subsequent pregnancy to infection of the placenta and abortion. In contrast a high dose stimulates protective immunity, resulting in a much lower abortion rate. This model will be useful in understanding the mechanisms of infection underlying latency and onset of disease, as well as in the development of novel therapeutics and vaccines for controlling infection.

Sepsis leads to multiple organ failure by engaging catastrophic feedback loops in which stressed tissue evokes an inflammatory response and, in turn, inflammation damages tissue. Manifestations of this maladaptive inflammatory response include cardio-respiratory dysfunction that may be reflected in reduced heart rate and ventilatory patterns variabilities. We have developed signal-processing algorithms that quantify non-linear deterministic characteristics of variability in biologic signals. Now, coalescing under the aegis of the NIH Computational Biology Program and the Society for Complexity in Acute Illness, two research teams performed iterative experiments and computational modeling on inflammation and cardio-pulmonary dysfunction in sepsis as well as on neural control of respiration and ventilatory pattern variability. These teams, with additional collaborators, have recently formed a multi-institutional, interdisciplinary consortium, whose goal is to delineate the fundamental interrelationship among the inflammatory response, heart rate and ventilatory pattern variability. Multiscale mathematical modeling and complementary physiological experiments will be combined to gain insight into the physiological control structures

To increase nutrient and water use efficiencies and reduce nutrient leaching and environmental concerns, fertigation is recommended for commercial blueberry production. In fact, successful fertigation can enhance sustainability and maximize profitability for commercial blueberry enterprises. One of the key factors in fertigation is the correct calculation of fertilizer injection rate and time for the acreage. This publication helps blueberry growers correctly calculate fertigation injection rates and times and provides reference tables for checking injection rates and times needed for a variety of production scenarios. This 3-page fact sheet was written by Guodong Liu, Jeffrey Williamson, Gary England, and Alicia Whidden, and published by the UF Department of Horticultural Sciences, March 2012. HS1197/HS1197: How to Calculate Fertigation Injection Rates for Commercial Blueberry Production (ufl.edu)

HS1165, a 7-page fact sheet by Timothy M. Spann, Ryan A. Atwood, Megan M. Dewdney, Robert C. Ebel, Reza Ehsani, Gary England, Stephen H. Futch, Tim Gaver, Tim Hurner, Chris Oswalt, Michael E. Rogers, Fritz M. Roka, Mark A. Ritenour, Mongi Zekri, Brian J. Boman, Kuang-Ren Chung, Michelle D. Danyluk, Renee Goodrich-Schneider, Kelly T. Morgan, Robert A. Morris, Ronald P. Muraro, Pamela Roberts, Robert E. Rouse, Arnold W. Schumann, Philip A. Stansly, and Lukasz L. Stelinski, provides guidance to the Florida citrus industry in making management decisions regarding huanglongbing (HLB, citrus greening). Published by the UF Department of Horticultural Sciences, June 2010.

COVID-19 remains a major public health concern. Monoclonal antibodies have received emergency use authorization (EUA) for pre-exposure prophylaxis against COVID-19 among high-risk groups for treatment of mild to moderate COVID-19. In addition to recombinant biologics, engineered synthetic DNA-encoded antibodies (DMAb) are an important strategy for direct in vivo delivery of protective mAb. A DMAb cocktail was synthetically engineered to encode the immunoglobulin heavy and light chains of two different two different Fc-engineered anti-SARS-CoV-2 antibodies. The DMAbs were designed to enhance in vivo expression and delivered intramuscularly to cynomolgus and rhesus macaques with a modified in vivo delivery regimen. Serum levels were detected in macaques, along with specific binding to SARS-CoV-2 spike receptor binding domain protein and neutralization of multiple SARS-CoV-2 variants of concern in pseudovirus and authentic live virus assays. Prophylactic administration was protective in rhesus macaques against signs of SARS-CoV-2 (USA-WA1/2020) associated disease in the lungs. Overall, the data support further study of DNA-encoded antibodies as an additional delivery mode for prevention of COVID-19 severe disease. These data have implications for human translation of gene-encoded mAbs for emerging infectious diseases and low dose mAb delivery against COVID-19.

Host cell infection by SARS-CoV-2, similar to that by HIV-1, is driven by a conformationally metastable and highly glycosylated surface entry protein complex, and infection by these viruses has been shown to be inhibited by the mannose-specific lectins cyanovirin-N (CV-N) and griffithsin (GRFT). We discovered in this study that CV-N not only inhibits SARS-CoV-2 infection but also leads to irreversibly inactivated pseudovirus particles. The irreversibility effect was revealed by the observation that pseudoviruses first treated with CV-N and then washed to remove all soluble lectin did not recover infectivity. The infection inhibition of SARS-CoV-2 pseudovirus mutants with single-site glycan mutations in spike suggested that two glycan clusters in S1 are important for both CV-N and GRFT inhibition: one cluster associated with the RBD (receptor binding domain) and the second with the S1/S2 cleavage site. We observed lectin antiviral effects with several SARS-CoV-2 pseudovirus variants, including the recently emerged omicron, as well as a fully infectious coronavirus, therein reflecting the breadth of lectin antiviral function and the potential for pan-coronavirus inactivation. Mechanistically, observations made in this work indicate that multivalent lectin interaction with S1 glycans is likely a driver of the lectin infection inhibition and irreversible inactivation effect and suggest the possibility that lectin inactivation is caused by an irreversible conformational effect on spike. Overall, lectins' irreversible inactivation of SARS-CoV-2, taken with their breadth of function, reflects the therapeutic potential of multivalent lectins targeting the vulnerable metastable spike before host cell encounter.

Despite numerous clinically available vaccines and therapeutics, aged patients remain at increased risk for COVID-19 morbidity. Furthermore, various patient populations, including the aged can have suboptimal responses to SARS-CoV-2 vaccine antigens. Here, we characterized vaccine-induced responses to SARS-CoV-2 synthetic DNA vaccine antigens in aged mice. Aged mice exhibited altered cellular responses, including decreased IFNγ secretion and increased TNFα and IL-4 secretion suggestive of T H 2-skewed responses. Aged mice exhibited decreased total binding and neutralizing antibodies in their serum but significantly increased T H 2-type antigen-specific IgG1 antibody compared to their young counterparts. Strategies to enhance vaccine-induced immune responses are important, especially in aged patient populations. We observed that co-immunization with plasmid-encoded adenosine deaminase (pADA)enhanced immune responses in young animals. Ageing is associated with decreases in ADA function and expression. Here, we report that co-immunization with pADA enhanced IFNγ secretion while decreasing TNFα and IL-4 secretion. pADA expanded the breadth and affinity SARS-CoV-2 spike-specific antibodies while supporting T H 1-type humoral responses in aged mice. scRNAseq analysis of aged lymph nodes revealed that pADA co-immunization supported a T H 1 gene profile and decreased FoxP3 gene expression. Upon challenge, pADA co-immunization decreased viral loads in aged mice. These data support the use of mice as a model for age-associated decreased vaccine immunogenicity and infection-mediated morbidity and mortality in the context of SARS-CoV-2 vaccines and provide support for the use of adenosine deaminase as a molecular adjuvant in immune-challenged populations., Competing Interests: DW has received grant funding, participates in industry collaborations, has received speaking honoraria, and has received fees for consulting, including serving on scientific review committees. Remunerations received by DW include direct payments and equity/options. DW also discloses the following associations with commercial partners: Geneos consultant/advisory board, AstraZeneca advisory board, speaker, Inovio board of directors, consultant, Sanofi advisory board, BBI advisory board, Pfizer advisory Board, Flagship consultant, and Advaccine consultant. SW is a scientific founder of Avamab Pharma Inc., a pre-clinical, pre-revenue stage company dedicated to research and development of AAV gene therapies for the treatment and prevention of infectious diseases. SW is a co-founder and Chief Scientific Officer of Inspire Biotherapeutics, a pre-clinical, pre-revenue stage gene therapy company developing AAV-based therapies for monogenic lung diseases. SW is an inventor on issued patents in Canada and US for the AAV6.2FF capsid, which are owned by the University of Guelph, and licensed to Avamab Pharma Inc., Inspire Biotherapeutics, and Cellastra Inc. From 2020 to February 2023, SW was an unpaid scientific advisor for Cellastra Inc., which is dedicated to research and development of gene therapies targeting root causes of scarring. SW is a co-inventor on a pending US and Canadian patent for the Engineered Newcastle disease virus vector and uses thereof, filed/owned by the University of Guelph. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gary, Tursi, Warner, Cuismano, Connors, Parzych, Griffin, Bell, Ali, Frase, Hojecki, Canziani, Chaiken, Kannan, Moffat, Embury-Hyatt, Wooton, Kossenkov, Patel, Kobasa, Kutzler, Haddad and Weiner.)

Glioblastoma multiforme (GBM) is among the most difficult cancers to treat with a 5-year survival rate less than 5%. An immunotherapeutic vaccine approach targeting GBM-specific antigen, EGFRvIII, previously demonstrated important clinical impact. However, immune escape variants were reported in the trial, suggesting that multivalent approaches targeting GBM-associated antigens may be of importance. Here we focused on multivalent in vivo delivery of synthetic DNA-encoded bispecific T cell engagers (DBTEs) targeting two GBM-associated antigens, EGFRvIII and HER2. We designed and optimized an EGFRvIII-DBTE that induced T cell-mediated cytotoxicity against EGFRvIII-expressing tumor cells. In vivo delivery in a single administration of EGFRvIII-DBTE resulted in durable expression over several months in NSG mice and potent tumor control and clearance in both peripheral and orthotopic animal models of GBM. Next, we combined delivery of EGFRvIII-DBTEs with an HER2-targeting DBTE to treat heterogeneous GBM tumors. In vivo delivery of dual DBTEs targeting these two GBM-associated antigens exhibited enhanced tumor control and clearance in a heterogeneous orthotopic GBM challenge, while treatment with single-target DBTE ultimately allowed for tumor escape. These studies support that combined delivery of DBTEs, targeting both EGFRvIII and HER2, can potentially improve outcomes of GBM immunotherapy, and such multivalent approaches deserve additional study., Competing Interests: D.B.W. has received grant funding, participates in industry collaborations, has received speaking honoraria, and has received fees for consulting, including serving on scientific review committees. Remunerations received by D.B.W. include direct payments and equity/options. D.B.W. also discloses the following associations with commercial partners: Geneos (consultant/advisory board), AstraZeneca (advisory board, speaker), Inovio (board of directors, consultant), Sanofi (advisory board), BBI (advisory board), Pfizer (advisory board), Flagship (consultant), and Advaccine (consultant)., (© 2023 The Authors.)

Monoclonal antibody therapy has played an important role against SARS-CoV-2. Strategies to deliver functional, antibody-based therapeutics with improved in vivo durability are needed to supplement current efforts and reach underserved populations. Here, we compare recombinant mAbs COV2-2196 and COV2-2130, which compromise clinical cocktail Tixagevimab/Cilgavimab, with optimized nucleic acid-launched forms. Functional profiling of in vivo-expressed, DNA-encoded monoclonal antibodies (DMAbs) demonstrated similar specificity, broad antiviral potency and equivalent protective efficacy in multiple animal challenge models of SARS-CoV-2 prophylaxis compared to protein delivery. In PK studies, DNA-delivery drove significant serum antibody titers that were better maintained compared to protein administration. Furthermore, cryo-EM studies performed on serum-derived DMAbs provide the first high-resolution visualization of in vivo-launched antibodies, revealing new interactions that may promote cooperative binding to trimeric antigen and broad activity against VoC including Omicron lineages. These data support the further study of DMAb technology in the development and delivery of valuable biologics., (© 2022. The Author(s).)

The global coronavirus disease 2019 (COVID-19) pandemic has claimed more than 5 million lives. Emerging variants of concern (VOCs) continually challenge viral control. Directing vaccine-induced humoral and cell-mediated responses to mucosal surfaces may enhance vaccine efficacy. Here we investigate the immunogenicity and protective efficacy of optimized synthetic DNA plasmids encoding wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (pS) co-formulated with the plasmid-encoded mucosal chemokine cutaneous T cell-attracting chemokine (pCTACK; CCL27). pCTACK-co-immunized animals exhibit increased spike-specific antibodies at the mucosal surface and increased frequencies of interferon gamma (IFNγ) + CD8 + T cells in the respiratory mucosa. pCTACK co-immunization confers 100% protection from heterologous Delta VOC challenge. This study shows that mucosal chemokine adjuvants can direct vaccine-induced responses to specific immunological sites and have significant effects on heterologous challenge. Further study of this unique chemokine-adjuvanted vaccine approach in the context of SARS-CoV-2 vaccines is likely important., Competing Interests: Declarations of interests D.B.W. has received grant funding, participates in industry collaborations, has received speaking honoraria, and has received fees for consulting, including serving on scientific review committees and board series. Remuneration received by D.B.W. includes direct payments and stock or stock options. D.B.W. discloses the following paid associations with commercial partners: GeneOne (consultant), Geneos (advisory board), AstraZeneca (advisory board, speaker), Inovio (BOD, SRA, stock), Sanofi (advisory board), and BBI (advisory board)., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines have demonstrated strong immunogenicity and protection against severe disease, concerns about the duration and breadth of these responses remain. In this study, we show that codelivery of plasmid-encoded adenosine deaminase-1 (pADA) with SARS-CoV-2 spike glycoprotein DNA enhances immune memory and durability in vivo. Coimmunized mice displayed increased spike-specific IgG of higher affinity and neutralizing capacity as compared with plasmid-encoded spike-only-immunized animals. Importantly, pADA significantly improved the longevity of these enhanced responses in vivo. This coincided with durable increases in frequencies of plasmablasts, receptor-binding domain-specific memory B cells, and SARS-CoV-2-specific T follicular helper cells. Increased spike-specific T cell polyfunctionality was also observed. Notably, animals coimmunized with pADA had significantly reduced viral loads compared with their nonadjuvanted counterparts in a SARS-CoV-2 infection model. These data suggest that pADA enhances immune memory and durability and supports further translational studies., (Copyright © 2022 by The American Association of Immunologists, Inc.)

The enhanced transmissibility and immune evasion associated with emerging SARS-CoV-2 variants demands the development of next-generation vaccines capable of inducing superior protection amid a shifting pandemic landscape. Since a portion of the global population harbors some level of immunity from vaccines based on the original Wuhan-Hu-1 SARS-CoV-2 sequence or natural infection, an important question going forward is whether this immunity can be boosted by next-generation vaccines that target emerging variants while simultaneously maintaining long-term protection against existing strains. Here, we evaluated the immunogenicity of INO-4800, our synthetic DNA vaccine candidate for COVID-19 currently in clinical evaluation, and INO-4802, a next-generation DNA vaccine designed to broadly target emerging SARS-CoV-2 variants, as booster vaccines in nonhuman primates. Rhesus macaques primed over one year prior with the first-generation INO-4800 vaccine were boosted with either INO-4800 or INO-4802 in homologous or heterologous prime-boost regimens. Both boosting schedules led to an expansion of T cells and antibody responses which were characterized by improved neutralizing and ACE2 blocking activity across wild-type SARS-CoV-2 as well as multiple variants of concern. These data illustrate the durability of immunity following vaccination with INO-4800 and additionally support the use of either INO-4800 or INO-4802 in prime-boost regimens., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kate Broderick reports financial support was provided by Coalition for Epidemic Preparedness Innovations. David B. Weiner reports a relationship with Inovio Pharmaceuticals Inc that includes: board membership, consulting or advisory, and equity or stocks. A.P., E.L.R., E.P., E.N.G., M.P., D.F., F.I.Z, A.K., declare no competing interests. J.N.W., B.S., K.S., I.M., Z.E., A.D., D.E., A.G., V.M.A., J.J.K., L.M.H., S.J.R., T.R.F.S., K.E.B. are employees of Inovio Pharmaceuticals and as such receive salary and benefits, including ownership of stock and stock options, from the company. D.B.W. discloses the following paid associations with commercial partners: GeneOne (Consultant), Geneos (Advisory Board), AstraZeneca (Advisory Board, Speaker), Inovio (BOD, SRA, Stock), Pfizer (Speaker), Merck (Speaker), Sanofi (Advisory Board), BBI (Advisory Board)., (Copyright © 2022. Published by Elsevier Ltd.)

To advance a novel concept of debulking virus in the oral cavity, the primary site of viral replication, virus-trapping proteins CTB-ACE2 were expressed in chloroplasts and clinical-grade plant material was developed to meet FDA requirements. Chewing gum (2 g) containing plant cells expressed CTB-ACE2 up to 17.2 mg ACE2/g dry weight (11.7% leaf protein), have physical characteristics and taste/flavor like conventional gums, and no protein was lost during gum compression. CTB-ACE2 gum efficiently (>95%) inhibited entry of lentivirus spike or VSV-spike pseudovirus into Vero/CHO cells when quantified by luciferase or red fluorescence. Incubation of CTB-ACE2 microparticles reduced SARS-CoV-2 virus count in COVID-19 swab/saliva samples by >95% when evaluated by microbubbles (femtomolar concentration) or qPCR, demonstrating both virus trapping and blocking of cellular entry. COVID-19 saliva samples showed low or undetectable ACE2 activity when compared with healthy individuals (2,582 versus 50,126 ΔRFU; 27 versus 225 enzyme units), confirming greater susceptibility of infected patients for viral entry. CTB-ACE2 activity was completely inhibited by pre-incubation with SARS-CoV-2 receptor-binding domain, offering an explanation for reduced saliva ACE2 activity among COVID-19 patients. Chewing gum with virus-trapping proteins offers a general affordable strategy to protect patients from most oral virus re-infections through debulking or minimizing transmission to others., Competing Interests: Declaration of interests All authors declare no competing interests except the corresponding author (H.D.), who is a patentee in this field but has no specific financial conflict of interest to disclose., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Latent Epstein-Barr virus (EBV) infection is associated with several types of cancer. Several clinical studies have targeted EBV antigens as immune therapeutic targets with limited efficacy of EBV malignancies, suggesting that additional targets might be important. Bam HI-A rightward frame 1 (BARF1) is an EBV antigen that is highly expressed in EBV + nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma (EBVaGC). BARF1 antigen can transform human epithelial cells in vivo. BARF1-specific antibodies and cytotoxic T cells were detected in some EBV + NPC patients. However, BARF1 has not been evaluated as an antigen in the context of therapeutic immunization. Its possible importance in this context is unclear. Here, we developed a synthetic-DNA-based expression cassette as immunotherapy targeting BARF1 (pBARF1). Immunization with pBARF1 induced potent antigen-specific humoral and T cell responses in vivo . Immunization with pBARF1 plasmid impacted tumor progression through the induction of CD8 + T cells in novel BARF1 + carcinoma models. Using an in vivo imaging system, we observed that pBARF1-immunized animals rapidly cleared cancer cells. We demonstrated that pBARF1 can induce antigen-specific immune responses that can impact cancer progression. Further study of this immune target is likely important as part of therapeutic approaches for EBV + malignancies., Competing Interests: D.B.W. has received grant funding, participates in industry collaborations, has received speaking honoraria, and has received fees for consulting, including serving on scientific review committees and board series. Remuneration received by D.B.W. includes direct payments, stock, or stock options, and in the interest of disclosure, D.B.W. discloses the following paid associations with commercial partners: Geneos (advisory board), Astrazeneca (advisory board, speaker), Inovio (BOD, SRA, Stock), Sanofi (advisory board), and BBI (advisory board). All other authors declare no competing interests., (© 2022 The Authors.)

Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has had a dramatic global impact on public health and social and economic infrastructures. Here, we assess the immunogenicity and anamnestic protective efficacy in rhesus macaques of an intradermal (i.d.)-delivered SARS-CoV-2 spike DNA vaccine, INO-4800, currently being evaluated in clinical trials. Vaccination with INO-4800 induced T cell responses and induced spike antigen and RBD binding antibodies with ADCP and ADCD activity. Sera from the animals neutralized both the D614 and G614 SARS-CoV-2 pseudotype viruses. Several months after vaccination, animals were challenged with SARS-CoV-2 resulting in rapid recall of anti-SARS-CoV-2 spike protein T cell and neutralizing antibody responses. These responses were associated with lower viral loads in the lung. These studies support the immune impact of INO-4800 for inducing both humoral and cellular arms of the adaptive immune system, which are likely important for providing durable protection against COVID-19 disease., Competing Interests: A.P., E.L.R., E.P., E.N.G., M.P., S.N.W., P.B., Z.X., S.M.R., K.Y.K., N.C., E.T-R., J.C., N.J.T., K.M., and D.K.W. declare no competing interests. J.N.W., K.S., I.M., Z.E., D.G., A.D., D.E., A.G., V.M.A., J.J.K., L.M.H., S.J.R., T.R.F.S., and K.E.B. are employees of Inovio Pharmaceuticals and as such receive salary and benefits, including ownership of stock and stock options, from the company. D.B.W. discloses the following paid associations with commercial partners: GeneOne (Consultant), Geneos (Advisory Board), Astrazeneca (Advisory Board, Speaker), Inovio (BOD, SRA, Stock), Pfizer (Speaker), Merck (Speaker), Sanofi (Advisory Board), BBI (Advisory Board)., (© 2021 The Author(s).)

More than 100 million people have been infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Common laboratory mice are not susceptible to wild-type SARS-CoV-2 infection, challenging the development and testing of effective interventions. Here, we describe the development and testing of a mouse model for SARS-CoV-2 infection based on transduction of the respiratory tract of laboratory mice with an adeno-associated virus vector ( AAV6 ) expressing human ACE-2 ( AAV6.2FF-hACE2 ). We validated this model using a previously described synthetic DNA vaccine plasmid, INO-4800 ( pS ). Intranasal instillation of AAV6.2FF-hACE2 resulted in robust hACE2 expression in the respiratory tract. pS induced robust cellular and humoral responses. Vaccinated animals were challenged with 10 5 TCID 50 SARS-CoV-2 (hCoV-19/Canada/ON-VIDO-01/2020) and euthanized four days post-challenge to assess viral load. One immunization resulted in 50% protection and two immunizations were completely protective. Overall, the AAV6.2FF-hACE2 mouse transduction model represents an easily accessible, genetically diverse mouse model for wild-type SARS-CoV-2 infection and preclinical evaluation of potential interventions., Competing Interests: D.B.W. has received grant funding, participates in industry collaborations, has received speaking honoraria, and has received fees for consulting, including serving on scientific review committees and board series. Remuneration received by D.B.W. includes direct payments, stock or stock options, and in the interest of disclosure D.B.W. discloses the following paid associations with commercial partners: GeneOne (Consultant), Geneos (Advisory Board), Astrazeneca (Advisory Board, Speaker), Inovio (BOD, SRA, Stock), Sanofi (Advisory Board), BBI (Advisory Board)., (© 2021 The Author(s).)

S evere a cute r espiratory s yndrome co rona v irus 2 (SARS-CoV-2) has caused a global pandemic of COVID-19, resulting in cases of mild to severe respiratory distress and significant mortality. The global outbreak of this novel coronavirus has now infected >20 million people worldwide, with >5 million cases in the United States (11 August 2020). The development of diagnostic and research tools to determine infection and vaccine efficacy is critically needed. We have developed multiple serologic assays using newly designed SARS-CoV-2 reagents for detecting the presence of receptor-binding antibodies in sera. The first assay is surface plasmon resonance (SPR) based and can quantitate both antibody binding to the SARS-CoV-2 spike protein and blocking to the Angiotensin-converting enzyme 2 (ACE2) receptor in a single experiment. The second assay is enzyme-linked immunosorbent assay (ELISA) based and can measure competition and blocking of the ACE2 receptor to the SARS-CoV-2 spike protein with antispike antibodies. The assay is highly versatile, and we demonstrate the broad utility of the assay by measuring antibody functionality of sera from small animals and nonhuman primates immunized with an experimental SARS-CoV-2 vaccine. In addition, we employ the assay to measure receptor blocking of sera from SARS-CoV-2-infected patients. The assay is shown to correlate with pseudovirus neutralization titers. This type of rapid, surrogate neutralization diagnostic can be employed widely to help study SARS-CoV-2 infection and assess the efficacy of vaccines., (Copyright © 2020 American Society for Microbiology.)

Recently newer synthetic DNA vaccines have been rapidly advanced to clinical study and have demonstrated an impressive degree of immune potency and tolerability. Improvements in DNA delivery over prior needle and syringe approaches include jet delivery, gene gun delivery, among others. Among the most effective of these new delivery methods, advanced electroporation (EP), combined with other advances, induces robust humoral and cellular immunity in both preventative as well as therapeutic studies. Advancements in the design of the DNA inserts include leader sequence changes, RNA and codon optimizations, improved insert designs, increased concentrations of DNA, and skin delivery, appear to complement newer delivery strategies. These advances also provide a framework for the in vivo production of synthetic DNA biologics. In this review, we focus on recent studies of synthetic DNA vaccines in the clinic for the prevention or treatment of infectious diseases with a focus on adaptive electroporation for delivery, and briefly summarize novel preclinical data advancing the in vivo delivery of DNA-encoded antibody-like biologics., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The coronavirus family member, SARS-CoV-2 has been identified as the causal agent for the pandemic viral pneumonia disease, COVID-19. At this time, no vaccine is available to control further dissemination of the disease. We have previously engineered a synthetic DNA vaccine targeting the MERS coronavirus Spike (S) protein, the major surface antigen of coronaviruses, which is currently in clinical study. Here we build on this prior experience to generate a synthetic DNA-based vaccine candidate targeting SARS-CoV-2 S protein. The engineered construct, INO-4800, results in robust expression of the S protein in vitro. Following immunization of mice and guinea pigs with INO-4800 we measure antigen-specific T cell responses, functional antibodies which neutralize the SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and biodistribution of SARS-CoV-2 targeting antibodies to the lungs. This preliminary dataset identifies INO-4800 as a potential COVID-19 vaccine candidate, supporting further translational study.

An effective prophylactic vaccine targeting HIV must induce a robust humoral response and must direct the bulk of this response to the mucosa-the primary site of HIV transmission. The chemokine, CCL28, is secreted by epithelial cells at mucosal surfaces and recruits' cells expressing its receptor CCR10. CCR10 is predominantly expressed by IgA + ASCs. We hypothesized that co-immunization with plasmid DNA encoding consensus envelope antigens with plasmid-encoded CCL28 would enhance anti-HIV IgA responses at mucosal surfaces. Indeed, animals receiving pCCL28 and pEnvA/C had significantly increased HIV-specific IgA in fecal extract. Surprisingly, CCL28 co-immunization induced a significant increase in anti-HIV IgG in the serum in mice compared to those receiving pEnvA/C alone. These robust antibody responses were not associated with changes in the frequency of germinal center B cells but depended upon the expression of CCR10, as these responses we abolished in CCR10-deficient animals. Finally, immunization with CCL28 led to increased frequencies in HIV-specific CCR10 + and CCR10 + IgA + B cells in the small intestine and Peyer's patches of vaccinated animals as compared to those receiving pEnvA/C alone. These data indicate that CCL28 administration can enhance antigen-specific humoral responses systemically and at mucosal surfaces., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)