Screening and selection of essential oils for an intranasal spray against bovine respiratory pathogens based on antimicrobial, antiviral, immunomodulatory, and antibiofilm activities.

Introduction: The rise in antibiotic resistant pathogens associated with bovine respiratory disease (BRD) poses a serious challenge, particularly to the beef feedlot industry, as they currently depend on antibiotics to prevent BRD to mitigate the financial burden (approx. $1 billion annual loss) inflicted by BRD-associated high mortality and morbidity in feedlot cattle. Thus, there is an impetus need for the development of antimicrobial alternative strategies against BRD. This study aimed to screen and select candidate essential oils (EOs) for the development of an intranasal EO spray that can inhibit BRD pathogens and promote microbiota-mediated respiratory health. Methods: The effects of selected EOs (ajowan, cinnamon leaf, citronella, grapefruit, fennel, and thyme) on a bovine nasopharyngeal microbiota culture were evaluated using 16S rRNA gene sequencing. The microbiota culture was enriched by incubating nasopharyngeal swabs obtained from finishing beef heifers in brain heart infusion broth with and without EOs (0.025%, v/v). These EOs were then also evaluated for their immunomodulatory effects on bovine turbinate (BT) cells by analyzing the concentrations of 15 cytokines and chemokines in cell culture after 24 h incubation. The crystal violet assay was done to assess the antibiofilm activity of EOs against Escherichia coli UMN026 strain. Finally, 15 EOs were screened for their antiviral activity against the bovine viral diarrhea virus 1 (BVDV-1) using BT cells and a fluorescence-based method. Results: Ajowan, fennel, and thyme resulted in a moderate reduction of overall nasopharyngeal microbiota growth with significant alterations of both alpha and beta diversity, and the relative abundance of predominant bacterial families (e.g., increasing Enterobacteriaceae and decreasing Moraxellaceae) compared to the control (p < 0.05). Co-incubation of BT cells with selected EOs resulted in minimal alterations in cytokine and chemokine levels (p > 0.05). Ajowan, thyme, fennel, and cinnamon leaf exhibited antibiofilm activity at concentrations of 0.025 and 0.05%. Reduction of BVDV-1 replication in BT cells was observed with thyme (strong), and ajowan and citronella (moderate) at 0.0125% concentration. Discussion: Accordingly, ajowan, thyme, fennel, cinnamon leaf, and citronella EOs were selected for further development as an intranasal EO spray to prevent and control of BRD pathogens in feedlot cattle.

Harnessing the Genetic Plasticity of Porcine Circovirus Type 2 to Target Suicidal Replication.

Porcine circovirus type 2 (PCV2), the causative agent of a wasting disease in weanling piglets, has periodically evolved into several new subtypes since its discovery, indicating that the efficacy of current vaccines can be improved. Although a DNA virus, the mutation rates of PCV2 resemble RNA viruses. The hypothesis that recoding of selected serine and leucine codons in the PCV2b capsid gene could result in stop codons due to mutations occurring during viral replication and thus result in rapid attenuation was tested. Vaccination of weanling pigs with the suicidal vaccine constructs elicited strong virus-neutralizing antibody responses. Vaccination prevented lesions, body-weight loss, and viral replication on challenge with a heterologous PCV2d strain. The suicidal PCV2 vaccine construct was not detectable in the sera of vaccinated pigs at 14 days post-vaccination, indicating that the attenuated vaccine was very safe. Exposure of the modified virus to immune selection pressure with sub-neutralizing levels of antibodies resulted in 5 of the 22 target codons mutating to a stop signal. Thus, the described approach for the rapid attenuation of PCV2 was both effective and safe. It can be readily adapted to newly emerging viruses with high mutation rates to meet the current need for improved platforms for rapid-response vaccines.

Comparative immunopathogenesis and biology of recently discovered porcine circoviruses.

Porcine circoviruses are important pathogens of production swine. Porcine circovirus type 1 (PCV1) is non-pathogenic, and discovered as a contaminant of a porcine kidney cell line, PK-15. The discovery of pathogenic variant, PCV2, occurred in the late 90s in association with post-weaning multi-systemic wasting disease syndrome (PMWS), which is characterized by wasting, respiratory signs and lymphadenopathy in weanling pigs. A new PCV type, designated as PCV3, was discovered in 2016, in pigs manifesting porcine dermatitis and nephropathy syndrome (PDNS), respiratory distress and reproductive failure. Pathological manifestations of PCV3 Infections include systemic inflammation, vasculitis and myocarditis. A fourth PCV type, PCV4, was identified in 2020 in pigs with PDNS, respiratory and enteric signs. All the pathogenic PCV types are detected in both healthy and morbid pigs. They cause chronic, systemic infections with various clinical manifestations. Dysregulation of the immune system homeostasis is a pivotal trigger for pathogenesis in porcine circoviral infections. While the study of PCV3 immunobiology is still in its infancy lessons learned from PCV2 and other circular replication-associated protein (Rep)-encoding single stranded (ss) (CRESS) DNA viruses can inform the field of exploration for PCV3. Viral interactions with the innate immune system, interference with dendritic cell function coupled with the direct loss of lymphocytes compromises both innate and adaptive immunity in PCV2 infections. Dysregulated immune responses leading to the establishment of a pro-inflammatory state, immune complex associated hypersensitivity, and the necrosis of lymphocytes and immune cells are key features of PCV3 immunopathogenesis. A critical overview of the comparative immunopathology of PCV2 and PCV3/4, and directions for future research in the field are presented in this review.

Targeted Alteration of Antibody-Based Immunodominance Enhances the Heterosubtypic Immunity of an Experimental PCV2 Vaccine.

Despite the availability of commercial vaccines which can effectively prevent clinical signs, porcine circovirus type 2 (PCV2) continues to remain an economically important swine virus, as strain drift, followed by displacement of new subtypes, occurs periodically. We had previously determined that the early antibody responses to the PCV2 capsid protein in infected pigs map to immunodominant but non-protective, linear B cell epitopes. In this study, two of the previously identified immunodominant epitopes were mutated in the backbone of a PCV2b infectious clone, to rationally restructure the immunogenic capsid protein. The rescued virus was used to immunize 3-week-old weanling piglets, followed by challenge with a virulent heterologous PCV2d strain. As expected, immunodominant antibody responses to the targeted epitopes were abrogated in vaccinated pigs, while a broadening of the virus neutralization responses was detected. Vaccinated pigs were completely protected against challenge viral replication, had reduced microscopic lesions in lymphoid organs and gained significantly more body weight when compared to unvaccinated pigs. Thus, the experimental PCV2 vaccine developed was highly effective against challenge, and, if adopted commercially, can potentially slow down or eliminate new strain creation.

Torque teno viruses in health and disease.

Torque teno viruses (TTVs) are small, ubiquitous, viruses with a highly diverse, single-stranded, negative sense DNA genome and wide host range. They are detected at high rates in both healthy and diseased individuals and are considered a significant part of the mammalian virome. Similar to human TTVs, swine TTVs (TTSuVs) are epidemiologically linked to several coinfections including porcine circovirus types 2 and 3 and the porcine reproductive and respiratory disease syndrome virus. Experimental infection of gnotobiotic pigs with TTSuVs resulted in lesions in multiple organs and exacerbation of coinfections, making TTSuVs the only members of the Anelloviridae family with experimental evidence for pathogenicity. However, due to the lack of reliable cell culture and animal models, mechanistic studies on viral immunity and pathogenesis are limited. The objective of this review is to summarize the current status of knowledge regarding the biology, detection, pathogenesis and public health significance of TTSuVs, while identifying gaps in knowledge which limit the field.

Efficacy of a Commercial PCV2a Vaccine with a Two-Dose Regimen Against PCV2d.

Porcine circovirus type 2, the causative agent of porcine circovirus associated diseases (PCVAD), consists of three major genotypes PCV2a, 2b and 2d. Current commercial vaccines contain the first-identified PCV2a's capsid protein or whole virions. Outbreaks of PCVAD, caused by the recently identified PCV2d in vaccinated herds have raised concerns regarding the efficacy of current PCV2a vaccines against PCV2d. Thus, the primary objective of this study was to assess the efficacy of a two-dose regimen for the recently reformulated Fostera PCV MetaStim vaccine, to determine if reformulation with the squalene oil adjuvant and two-dose regimen improves the threshold of protection enough to eliminate viremia in a vaccination and challenge model. Two groups of seven pigs each were vaccinated with the commercial vaccine or PBS, and challenged with the PCV2d virus. Strong pre-challenge virus neutralizing responses were detected against all three genotypes. Post-challenge viremia was not completely eliminated as expected but a 2 log10 mean reduction in viral load was achieved in vaccinated pigs. Vaccinated pigs had a mean score of 0 for pathological evaluation, while unvaccinated pigs had a score of 6.6. In conclusion, the reformulated Fostera PCV MetaStim PCV2a-based vaccine provided significant heterologous protection and was effective against PCV2d.