The Presence of Virus Neutralizing Antibodies Is Highly Associated with Protection against Virulent Challenge in Domestic Pigs Immunized with ASFV live Attenuated Vaccine Candidates.

African swine fever virus (ASFV) is currently producing a pandemic affecting a large area of Eurasia, and more recently, the Dominican Republic in the Western Hemisphere. ASFV is a large and structurally complex virus with a large dsDNA genome encoding for more than 150 genes. Live attenuated virus strains can induce protection in domestic swine against disease produced by homologous virulent parental viruses. The roles of the different immune mechanisms induced by the attenuated strains in protection still need to be understood. In particular, the role of ASFV neutralizing antibody in protection still is an important controversial issue to be elucidated. Here we present the development of a novel methodology to detect virus neutralizing antibodies based on the reduction of virus infectivity in a Vero cell adapted ASFV strain. The described method was used to assess levels of virus neutralizing antibodies in domestic swine inoculated with live attenuated ASFV. Results demonstrated a high association between the presence of virus neutralizing antibodies and protection in 84 animals immunized with the recombinant vaccine candidates ASFV-G-Δ9GL/ΔUK or ASFV-G-ΔI177L. To our knowledge, this is the first report demonstrating an association between virus neutralizing antibodies and protection against virulent challenge in such a large number of experimental individuals.

Contact Challenge of Cattle with Foot-and-Mouth Disease Virus Validates the Role of the Nasopharyngeal Epithelium as the Site of Primary and Persistent Infection.

The pathogenesis of foot-and-mouth disease virus (FMDV) in cattle was investigated through early and late stages of infection by use of an optimized experimental model for controlled contact exposure. Time-limited exposure of cattle to FMDV-infected pigs led to primary FMDV infection of the nasopharyngeal mucosa in both vaccinated and nonvaccinated cattle. In nonvaccinated cattle, the infection generalized rapidly to cause clinical disease, without apparent virus amplification in the lungs prior to establishment of viremia. Vaccinated cattle were protected against clinical disease and viremia; however, all vaccinated cattle were subclinically infected, and persistent infection occurred at similarly high prevalences in both animal cohorts. Infection dynamics in cattle were consistent and synchronous and comparable to those of simulated natural and needle inoculation systems. However, the current experimental model utilizes a natural route of virus exposure and is therefore superior for investigations of disease pathogenesis and host response. Deep sequencing of viruses obtained during early infection of pigs and cattle indicated that virus populations sampled from sites of primary infection were markedly more diverse than viruses from vesicular lesions of cattle, suggesting the occurrence of substantial bottlenecks associated with vesicle formation. These data expand previous knowledge of FMDV pathogenesis in cattle and provide novel insights for validation of inoculation models of bovine FMD studies.IMPORTANCE Foot-and-mouth disease virus (FMDV) is an important livestock pathogen that is often described as the greatest constraint to global trade in animal products. The present study utilized a standardized pig-to-cow contact exposure model to demonstrate that FMDV infection of cattle initiates in the nasopharyngeal mucosa following natural virus exposure. Furthermore, this work confirmed the role of the bovine nasopharyngeal mucosa as the site of persistent FMDV infection in vaccinated and nonvaccinated cattle. The critical output of this study validates previous studies that have used simulated natural inoculation models to characterize FMDV pathogenesis in cattle and emphasizes the importance of continued research of the unique virus-host interactions that occur within the bovine nasopharynx. Specifically, vaccines and biotherapeutic countermeasures designed to prevent nasopharyngeal infection of vaccinated animals could contribute to substantially improved control of FMDV.

Effects of orally administered cortisol and norepinephrine on weanling piglet gut microbial populations and Salmonella passage.

Stress and anxiety have been associated with changes in the microbiota of the gut and ultimately diminished resistance to pathogens. The objective of this study was to observe intestinal microbiota and susceptibility to Salmonella associated with stress hormones, cortisol (CORT), and norepinephrine (NE), in piglets. At weaning, 90 piglets (15 for a Salmonella challenge) were trained to take the carrier (apple juice) orally. At 2 wk after weaning, pens of piglets were assigned randomly to 1 of 3 treatments: control (CNT), NE, or CORT. Blood samples were collected prior to treatment, then piglets were dosed orally with treatments twice on day 0; at 0800 and 1600 h. Control piglets were administered 6.1 mL of the carrier only, NE pigs were administered 40 mg/mL of NE-bitartrate salt dissolved in the carrier, and CORT pigs were administered 12 mg/mL of hydrocortisone acetate dissolved in the carrier. Jugular blood samples were collected prior to necropsies (n = 5/treatment) at 0800 and 1600 h on day 1, and at 0800 h on days 2, 7, and 14 after treatments were started. A subset of pigs were subjected to a 24-h Salmonella challenge. Jejunal and ileal tissues and jejunal, ileal, cecal, and rectal contents were collected and colonies were counted. Microbial data and blood samples were analyzed using mixed models with fixed effects of treatment and day. Cortisol-treated piglets exhibited a spike in plasma CORT concentrations at 0800 h day 1 (P = 0.001) accompanied by greater concentrations of cecal Escherichia coli (P < 0.05) and a shift in intestinal environment to favor coliforms on day 2 (P < 0.05). Salmonella concentrations from rectal contents tended (P = 0.07) to be suppressed by CORT. Lactic acid-producing bacteria rectal concentrations were greater (P = 0.03) in CORT pigs on 0800 h on day 1 then NE pigs and tended to be greater than CNT (P = 0.09) and were greater on day 14 for both CNT (P = 0.003) and NE (P = 0.02). Norepinephrine spiked in NE piglets at 0800 h on day 1 (P = 0.001), 1600 h day 1 (P = 0.004), through day 2 (P = 0.04). Intestinal environment of NE pigs shifted to favor ileal anaerobes (P ≤ 0.05) and facultative anaerobes (E. coli; P = 0.01) compared to CNT. However, Salmonella concentrations in rectal contents were suppressed by NE compared to CNT (P = 0.05). Oral administration of NE and CORT had the desired effect of increasing concentrations of stress hormones and resulted in microbiome shifts throughout the intestines.

A partial deletion within foot-and-mouth disease virus non-structural protein 3A causes clinical attenuation in cattle but does not prevent subclinical infection.

Deletions within the 3A coding region of foot-and-mouth disease virus (FMDV) are associated with decreased virulence in cattle; however, the mechanisms are unknown. We compared experimental infection of cattle with virulent FMDV O1Campos (O1Ca) and a mutant derivative (O1Ca∆3A) lacking residues 87-106 of 3A. Unexpectedly, primary infection of the nasopharyngeal mucosa was similar for both viruses. However, while O1Ca caused viremia and fulminant clinical disease, O1Ca∆3A infection was subclinical and aviremic. There were no differences in expression of anti-viral cytokines in nasopharyngeal tissues between the groups, suggesting attenuation by O1Ca∆3A was a consequence of reduced replication efficiency in bovine cells, rather than a difference in the host response. These results demonstrated that although deletion in 3A of FMDV confers a clinically attenuated phenotype in cattle, the deletion does not prevent subclinical infection. These findings have implications for field scenarios involving outbreaks with apparently host-limited strains of FMDV.

Correlates of immune protection following cutaneous immunization with an attenuated Burkholderia pseudomallei vaccine.

Infections with the Gram-negative bacterium Burkholderia pseudomallei (melioidosis) are associated with high mortality, and there is currently no approved vaccine to prevent the development of melioidosis in humans. Infected patients also do not develop protective immunity to reinfection, and some individuals will develop chronic, subclinical infections with B. pseudomallei. At present, our understanding of what constitutes effective protective immunity against B. pseudomallei infection remains incomplete. Therefore, we conducted a study to elucidate immune correlates of vaccine-induced protective immunity against acute B. pseudomallei infection. BALB/c and C57BL/6 mice were immunized subcutaneously with a highly attenuated, Select Agent-excluded purM deletion mutant of B. pseudomallei (strain Bp82) and then subjected to intranasal challenge with virulent B. pseudomallei strain 1026b. Immunization with Bp82 generated significant protection from challenge with B. pseudomallei, and protection was associated with a significant reduction in bacterial burden in lungs, liver, and spleen of immunized mice. Humoral immunity was critically important for vaccine-induced protection, as mice lacking B cells were not protected by immunization and serum from Bp82-vaccinated mice could transfer partial protection to nonvaccinated animals. In contrast, vaccine-induced protective immunity was found to be independent of both CD4 and CD8 T cells. Tracking studies demonstrated uptake of the Bp82 vaccine strain predominately by neutrophils in vaccine-draining lymph nodes and by smaller numbers of dendritic cells (DC) and monocytes. We concluded that protection following cutaneous immunization with a live attenuated Burkholderia vaccine strain was dependent primarily on generation of effective humoral immune responses.

Development of Burkholderia mallei and pseudomallei vaccines.

Burkholderia mallei and Burkholderia pseudomallei are Gram-negative bacteria that cause glanders and melioidosis, respectively. Inhalational infection with either organism can result in severe and rapidly fatal pneumonia. Inoculation by the oral and cutaneous routes can also produce infection. Chronic infection may develop after recovery from acute infection with both agents, and control of infection with antibiotics requires prolonged treatment. Symptoms for both meliodosis and glanders are non-specific, making diagnosis difficult. B. pseudomallei can be located in the environment, but in the host, B. mallei and B. psedomallei are intracellular organisms, and infection results in similar immune responses to both agents. Effective early innate immune responses are critical to controlling the early phase of the infection. Innate immune signaling molecules such as TLR, NOD, MyD88, and pro-inflammatory cytokines such as IFN-γ and TNF-α play key roles in regulating control of infection. Neutrophils and monocytes are critical cells in the early infection for both microorganisms. Both monocytes and macrophages are necessary for limiting dissemination of B. pseudomallei. In contrast, the role of adaptive immune responses in controlling Burkholderia infection is less well understood. However, T cell responses are critical for vaccine protection from Burkholderia infection. At present, effective vaccines for prevention of glanders or meliodosis have not been developed, although recently development of Burkholderia vaccines has received renewed attention. This review will summarize current and past approaches to develop B. mallei and B. pseudomalllei vaccines, with emphasis on immune mechanisms of protection and the challenges facing the field. At present, immunization with live attenuated bacteria provides the most effective and durable immunity, and it is important therefore to understand the immune correlates of protection induced by live attenuated vaccines. Subunit vaccines have typically provided less robust immunity, but are safer to administer to a wider variety of people, including immune compromised individuals because they do not reactivate or cause disease. The challenges facing B. mallei and B. pseudomalllei vaccine development include identification of broadly protective antigens, design of efficient vaccine delivery and adjuvant systems, and a better understanding of the correlates of protection from both acute and chronic infection.