Recombinant Vaccine Strain ASFV-G-Δ9GL/ΔUK Produced in the IPKM Cell Line Is Genetically Stable and Efficacious in Inducing Protection in Pigs Challenged with the Virulent African Swine Fever Virus Field Isolate Georgia 2010.

We have previously reported that the recombinant African Swine Fever (ASF) vaccine candidate ASFV-G-Δ9GL/ΔUK efficiently induces protection in domestic pigs challenged with the virulent strain Georgia 2010 (ASFV-G). As reported, ASFV-G-Δ9GL/ΔUK induces protection, while intramuscularly (IM), administered at doses of 104 HAD50 or higher, prevents ASF clinical disease in animals infected with the homologous ASFV g strain. Like other recombinant vaccine candidates obtained from ASFV field isolates, ASFV-G-Δ9GL/ΔUK stocks need to be produced in primary cultures of swine macrophages, which constitutes an important limitation in the production of large virus stocks at the industrial level. Here, we describe the development of ASFV-G-Δ9GL/ΔUK stocks using IPKM (Immortalized Porcine Kidney Macrophage) cells, which are derived from swine macrophages. We show that ten successive passages of ASFV-G-Δ9GL/ΔUK in IPKM cells induced small changes in the virus genome. The produced virus, ASFV-G-Δ9GL/ΔUKp10, presented a similar level of replication in swine macrophages cultures to that of the original ASFV-G-Δ9GL/ΔUK (ASFV-G-Δ9GL/ΔUKp0). The protective efficacy of ASFV-G-Δ9GL/ΔUKp10 was evaluated in pigs that were IM-inoculated with either 104 or 106 HAD50 of ASFV-G-Δ9GL/ΔUKp10. While animals inoculated with 104 HAD50 present a partial protection against the experimental infection with the virulent parental virus ASFV-G, those inoculated with 106 HAD50 were completely protected. Therefore, as was just recently reported for another ASF vaccine candidate, ASFV-G-ΔI177L, IPKM cells are an effective alternative to produce stocks for vaccine strains which only grow in swine macrophages.

Deletion of the EP402R Gene from the Genome of African Swine Fever Vaccine Strain ASFV-G-∆I177L Provides the Potential Capability of Differentiating between Infected and Vaccinated Animals.

The African swine fever virus (ASFV) mutant ASFV-G-∆I177L is a safe and efficacious vaccine which induces protection against the challenge of its parental virus, the Georgia 2010 isolate. Although a genetic DIVA (differentiation between infected and vaccinated animals) assay has been developed for this vaccine, still there is not a serological DIVA test for differentiating between animals vaccinated with ASFV-G-∆I177L and those infected with wild-type viruses. In this report, we describe the development of the ASFV-G-∆I177L mutant having deleted the EP402R gene, which encodes for the viral protein responsible for mediating the hemadsorption of swine erythrocytes. The resulting virus, ASFV-G-∆I177L/∆EP402R, does not have a decreased ability to replicates in swine macrophages when compared with the parental ASFV-G-∆I177L. Domestic pigs intramuscularly (IM) inoculated with either 102 or 106 HAD50 of ASFV-G-∆I177L/∆EP402R remained clinically normal, when compared with a group of mock-vaccinated animals, indicating the absence of residual virulence. Interestingly, an infectious virus could not be detected in the blood samples of the ASFV-G-∆I177L/∆EP402R-inoculated animals in either group at any of the time points tested. Furthermore, while all of the mock-inoculated animals presented a quick and lethal clinical form of ASF after the intramuscular inoculation challenge with 102 HAD50 of highly virulent parental field isolate Georgia 2010 (ASFV-G), all of the ASFV-G-∆I177L/∆EP402R-inoculated animals were protected, remaining clinically normal until the end of the observational period. Most of the ASFV-G-∆I177L/∆EP402R-inoculated pigs developed strong virus-specific antibody responses against viral antigens, reaching maximum levels at 28 days post inoculation. Importantly, all of the sera collected at that time point in the ASFV-G-∆I177L/∆EP402R-inoculated pigs did not react in a direct ELISA coated with the recombinant EP402R protein. Conversely, the EP402R protein was readily recognized by the pool of sera from the animals immunized with recombinant live attenuated vaccine candidates ASFV-G-∆I177L, ASFV-G-∆MGF, or ASFV-G-∆9GL/∆UK. Therefore, ASFV-G-∆I177L/∆EP402R is a novel, safe and efficacious candidate with potential to be used as an antigenically DIVA vaccine.

African Swine Fever Vaccine Candidate ASFV-G-ΔI177L Produced in the Swine Macrophage-Derived Cell Line IPKM Remains Genetically Stable and Protective against Homologous Virulent Challenge.

ASFV vaccine candidate ASFV-G-ΔI177L has been shown to be highly efficacious in inducing protection against challenges with the parental virus, the Georgia 2010 isolate, as well as against field strains isolated from Vietnam. ASFV-G-ΔI177L has been shown to produce protection even when used at low doses (102 HAD50) and shows no residual virulence even when administered at high doses (106 HAD50) or evaluated for a relatively long period of time (6 months). ASFV-G-ΔI177L stocks can only be massively produced in primary cell macrophages. Alternatively, its modified version (ASFV-G-ΔI177L/ΔLVR) grows in a swine-derived cell line (PIPEC), acquiring significant genomic modifications. We present here the development of ASFV-G-ΔI177L stocks in a swine macrophage cell line, IPKM, and its protective efficacy when evaluated in domestic pigs. Successive passing of ASFV-G-ΔI177L in IPKM cells produces minimal genomic changes. Interestingly, a stock of ASFV-G-ΔI177L obtained after 10 passages (ASFV-G-ΔI177Lp10) in IPKM cells showed very small genomic changes when compared with the original virus stock. ASFV-G-ΔI177Lp10 conserves similar growth kinetics in primary swine macrophage cultures than the original parental virus ASFV-G-ΔI177L. Pigs infected with 103 HAD50 of ASFV-G-ΔI177Lp10 developed a strong virus-specific antibody response and were completely protected against the challenge with the parental virulent field isolate Georgia 2010. Therefore, IPKM cells could be an effective alternative for the production of ASFV vaccine stocks for those vaccine candidates exclusively growing in swine macrophages.

Confirming the absence of parental African swine fever virus as a potential contaminant of recombinant live attenuated ASF vaccines.

African swine fever (ASF) is a devastating disease that is currently producing a panzootic significantly impacting the swine industry worldwide. One of the major challenges for advancing the development of ASF vaccines has been the absence of international standards for ASF vaccine purity, potency, safety, and efficacy. To date, the most effective experimental vaccines have been live attenuated strains of viruses. Most of these promising vaccine candidates have been developed by deleting virus genes involved in the process of viral pathogenesis and disease production. This approach requires genomic modification of a parental virus field strain through a process of homologous recombination followed by purification of the recombinant attenuated virus. In this scenario, it is critical to confirm the absence of any parental virulent virus in the final virus stock used for vaccine production. We present here a protocol to establish the purity of virus stock using the live attenuated vaccine candidates ASFV-G-ΔMGF, ASFV-G-Δ9 GLΔUK and ASFV-G-ΔI177L. Procedures described here includes inoculation in susceptible pigs followed by the assessment of the obtained material by differential qPCRs that allows the identification of vaccine virus from ASFV field isolates. This protocol is proposed as a model to ensure that master seed virus stock used for vaccine production does not contain residual parental virulent virus. Procedures described here includes a passage in susceptible pigs followed by the assessment of the obtained material by differential qPCRs that allows the identification of vaccine virus from ASFV field isolates.

Orally delivered Bacillus subtilis expressing chicken NK-2 peptide stabilizes gut microbiota and enhances intestinal health and local immunity in coccidiosis-infected broiler chickens.

We recently reported a stable Bacillus subtilis-carrying chicken NK-lysin peptide (B. subtilis-cNK-2) as an effective oral delivery system of an antimicrobial peptide to the gut with therapeutic effect against Eimeria parasites in broiler chickens. To further investigate the effects of a higher dose of an oral B. subtilis-cNK-2 treatment on coccidiosis, intestinal health, and gut microbiota composition, 100 (14-day-old) broiler chickens were allocated into 4 treatment groups in a randomized design: 1) uninfected control (CON), 2) infected control without B. subtilis (NC), 3) B. subtilis with empty vector (EV), and 4) B. subtilis with cNK-2 (NK). All chickens, except the CON group, were infected with 5,000 sporulated Eimeria acervulina (E. acervulina) oocysts on d 15. Chickens given B. subtilis (EV and NK) were orally gavaged (1 × 1012 cfu/mL) daily from d 14 to 18. Growth performances were measured on d 6, 9, and 13 postinfection (dpi). Spleen and duodenal samples were collected on 6 dpi to assess the gut microbiota, and gene expressions of gut integrity and local inflammation makers. Fecal samples were collected from 6 to 9 dpi to enumerate oocyst shedding. Blood samples were collected on 13 dpi to measure the serum 3-1E antibody levels. Chickens in the NK group showed significantly improved (P < 0.05) growth performance, gut integrity, reduced fecal oocyst shedding and mucosal immunity compared to NC. Interestingly, there was a distinct shift in the gut microbiota profile in the NK group compared to that of NC and EV chickens. Upon challenge with E. acervulina, the percentage of Firmicutes was reduced and that of Cyanobacteria increased. In NK chickens, however, the ratio between Firmicutes and Cyanobacteria was not affected and was similar to that of CON chickens. Taken together, NK treatment restored dysbiosis incurred by E. acervulina infection and showed the general protective effects of orally delivered B. subtilis-cNK-2 on coccidiosis infection. This includes reduction of fecal oocyst shedding, enhancement of local protective immunity, and maintenance of gut microbiota homeostasis in broiler chickens.

Evaluation of the Safety Profile of the ASFV Vaccine Candidate ASFV-G-ΔI177L.

African swine fever (ASF) is the cause of a recent pandemic that is posing a threat to much of the world swine production. The etiological agent, ASF virus (ASFV), infects domestic and wild swine, producing a variety of clinical presentations depending on the virus strain and the genetic background of the pigs infected. No commercial vaccine is currently available, although recombinant live attenuated vaccine candidates have been shown to be efficacious. In addition to determining efficacy, it is paramount to evaluate the safety profile of a live attenuated vaccine. The presence of residual virulence and the possibility of reversion to virulence are two of the concerns that must be evaluated in the development of live attenuated vaccines. Here we evaluate the safety profile of an efficacious live attenuated vaccine candidate, ASFV-G-ΔI177L. Results from safety studies showed that ASFV-G-ΔI177L remains genetically stable and phenotypically attenuated during a five-passage reversion to virulence study in domestic swine. In addition, large-scale experiments to detect virus shedding and transmission confirmed that even under varying conditions, ASFV-G-ΔI177L is a safe live attenuated vaccine.

African swine fever virus vaccine candidate ASFV-G-ΔI177L efficiently protects European and native pig breeds against circulating Vietnamese field strain.

The recent spread of African swine fever (ASF) in the People's Republic of China and neighbouring countries in Asia has had significant economic consequences with an estimated direct cost of $55-$130 billion. This pandemic, originally detected in Republic of Georgia in 2007, has devastated the swine industry in large geographical areas of Southeast Asia with 14 countries reporting ASF outbreaks since the first documented case was confirmed in the city of Shenyang, Liaoning Province, China, on 3 August 2018. In the absence of any available vaccines, the control of ASF relies on the detection and culling of infected animals. The United States Department of Agriculture recently developed a recombinant experimental vaccine candidate, ASFV-G-ΔI177L, by deleting the I177L gene from the genome of the highly virulent pandemic ASFV strain Georgia, which efficaciouly protects pigs from the parental virus. Here, the initial studies were extended demonstrating that ASFV-G-ΔI177L is able to protect pigs against the virulent ASFV isolate currently circulating and producing disease in Vietnam with similar efficacy as reported against the Georgia strain. Comparative studies performed using a large number of pigs of European and Vietnamese origin demonstrated that a minimum protective dose of 102 HAD50 of ASFV-G-ΔI177L equally protects animals of both breeds. In concurrence with those results, the onset of immunity in these animal breed showed appearance of protection in approximately one-third of the animals by the second week post vaccination, with full protection achieved by the fourth week post vaccination. Therefore, results presented here demonstrated that ASFV-G-ΔI177L is able to induce protection against virulent Vietnameese ASFV field strains and is effective in protecting local breeds of pigs as efficiently as previously shown for European cross-bred pigs. To our knowledge, this is the first report showing the efficacy of a Georgia 2007 based vaccine candidate in Asian breed of pigs or challenged with an Asian ASFV strain.

Erratum: Callaway et al. Alternatives to Antibiotics: A Symposium on the Challenges and Solutions for Animal Health and Production. Antibiotics 2021, 10, 471.

The authors would like to make the following corrections to the published paper [...].

Oral Delivery of Bacillus subtilis Expressing Chicken NK-2 Peptide Protects Against Eimeria acervulina Infection in Broiler Chickens.

Chicken NK-lysin peptide 2 (cNK-2) is a natural lytic peptide with direct cytotoxicity against many apicomplexan parasites including Eimeria. Developing an effective oral delivery strategy to express cNK-2 in the intestine, where Eimeria parasites interact with the host's gut epithelial cells, may effectively reduce the fecundity of parasites and minimize intestinal damage. Furthermore, cNK-2 modulates gut immune responses to decrease local inflammation elicited by Eimeria infection in the intestine. Therefore, we developed a stable strain of Bacillus subtilis (B. subtilis) that carries cNK-2 to the gut to determine its effectiveness in ameliorating the negative impacts of coccidiosis and to replace the use of antibiotics in controlling coccidiosis in commercial broiler chicken production. Chickens were randomly allocated into eight treatment groups: two control groups (NC: E. acervulina infected non-B. subtilis control; CON: non-infected control); three B. subtilis-empty vector (EV) groups (EV6: 106 cfu/day/bird; EV8: 108 cfu/day/bird; EV10: 1010 cfu/day/bird), and three B. subtilis-cNK-2 groups (NK6: 106 cfu/day/bird; NK8: 108 cfu/day/bird; NK10: 1010 cfu/day/bird). All chickens, except those in the CON group, were challenged with 5,000 freshly sporulated E. acervulina oocysts through oral gavage on day 15. Chickens were given an oral dose of B. subtilis on days 14, 15, and 16. Body weight, weight gains, and fecal oocyst shedding were measured. To investigate the efficacy of oral B. subtilis-cNK-2 against coccidiosis, gene expression of gut health-related biomarkers was measured using RT-PCR. Markers included SOD1, CAT, and HMOX1 for oxidative stress in the spleen and intestinal mucosa, OCLN, ZO-1, and JAM2 for tight junction proteins, and MUC2 for mucin gene expression in the gut. The results showed that oral treatment of young chickens with B. subtilis-cNK-2 improved growth performance, enhanced gut integrity, and reduced fecal oocyst shedding. Altogether, these results confirm B. subtilis-cNK-2 treatment as a promising and effective alternative strategy to replace antibiotics against coccidiosis based on its ability to reduce parasite survival, to reduce coccidiosis-induced body weight loss, and to decrease gut damage based on the enhanced expression of proteins associated with gut integrity and intestinal health.

Alternatives to Antibiotics: A Symposium on the Challenges and Solutions for Animal Health and Production.

Antibiotics have improved the length and quality of life of people worldwide and have had an immeasurable influence on agricultural animal health and the efficiency of animal production over the last 60 years. The increased affordability of animal protein for a greater proportion of the global population, in which antibiotic use has played a crucial part, has resulted in a substantial improvement in human quality of life. However, these benefits have come with major unintended consequences, including antibiotic resistance. Despite the inherent benefits of restricting antibiotic use in animal production, antibiotics remain essential to ensuring animal health, necessitating the development of novel approaches to replace the prophylactic and growth-promoting benefits of antibiotics. The third International Symposium on "Alternatives to Antibiotics: Challenges and Solutions in Animal Health and Production" in Bangkok, Thailand was organized by the USDA Agricultural Research Service, Faculty of Veterinary Science, Chulalongkorn University and Department of Livestock Development-Thailand Ministry of Agriculture and Cooperative; supported by OIE World Organization for Animal Health; and attended by more than 500 scientists from academia, industry, and government from 32 nations across 6 continents. The focus of the symposium was on ensuring human and animal health, food safety, and improving food animal production efficiency as well as quality. Attendees explored six subject areas in detail through scientific presentations and panel discussions with experts, and the major conclusions were as follows: (1) defining the mechanisms of action of antibiotic alternatives is paramount to enable their effective use, whether they are used for prevention, treatment, or to enhance health and production; (2) there is a need to integrate nutrition, health, and disease research, and host genetics needs to be considered in this regard; (3) a combination of alternatives to antibiotics may need to be considered to achieve optimum health and disease management in different animal production systems; (4) hypothesis-driven field trials with proper controls are needed to validate the safety, efficacy, and return of investment (ROI) of antibiotic alternatives.

ASFV-G-∆I177L as an Effective Oral Nasal Vaccine against the Eurasia Strain of Africa Swine Fever.

The African swine fever virus (ASFV) is currently causing a pandemic affecting wild and domestic swine from Western Europe to Asia. No commercial vaccines are available to prevent African swine fever (ASF), resulting in overwhelming economic losses to the swine industry. We recently developed a recombinant vaccine candidate, ASFVG-ΔI177L, by deleting the I177L gene from the genome of the highly virulent ASFV strain Georgia (ASFV-G). ASFV-G-ΔI177L has been proven safe and highly efficacious in challenge studies using parental ASFV-G. Here, we present data demonstrating that ASFV-G-ΔI177L can be administered by the oronasal (ON) route to achieve a similar efficacy to that of intramuscular (IM) administration. Animals receiving ON ASFV-G-ΔI177L were completely protected against virulent ASFV-G challenge. As previously described, similar results were obtained when ASFV-G-ΔI177L was given intramuscularly. Interestingly, viremias induced in animals inoculated oronasally were lower than those measured in IM-inoculated animals. ASFV-specific antibody responses, mediated by IgG1, IgG2 and IgM, do not differ in animals inoculated by the ON route from that had IM inoculations. Therefore, the ASFV-G-ΔI177L vaccine candidate can be administered oronasally, a critical attribute for potential vaccination of wild swine populations.

Novel Foot-and-Mouth Disease Vaccine Platform: Formulations for Safe and DIVA-Compatible FMD Vaccines With Improved Potency.

Inactivated, wild-type foot-and-mouth disease virus (FMDV) vaccines are currently used to control FMD around the world. These traditional FMD vaccines are produced using large quantities of infectious, virulent, wild-type FMD viruses, with the associated risk of virus escape from manufacturing facilities or incomplete inactivation during the vaccine formulation process. While higher quality vaccines produced from wild-type FMDV are processed to reduce non-structural antigens, there is still a risk that small amounts of non-structural proteins may be present in the final product. A novel, antigenically marked FMD-LL3B3D vaccine platform under development by Zoetis, Inc. and the USDA-ARS, consists of a highly attenuated virus platform containing negative antigenic markers in the conserved non-structural proteins 3Dpol and 3B that render resultant vaccines fully DIVA compatible. This vaccine platform allows for the easy exchange of capsid coding sequences to create serotype-specific vaccines. Here we demonstrate the efficacy of the inactivated FMD-LL3B3D-A24 Cruzeiro vaccine in cattle against wild-type challenge with A24 Cruzerio. A proprietary adjuvant system was used to formulate the vaccines that conferred effective protection at low doses while maintaining the DIVA compatibility. In contrast to wild-type FMDV, the recombinant FMD-LL3B3D mutant viruses have been shown to induce no clinical signs of FMD and no shedding of virus in cattle or pigs when inoculated as a live virus. The FMD-LL3B3D vaccine platform, currently undergoing development in the US, provides opportunities for safer vaccine production with full DIVA compatibility in support of global FMDV control and eradication initiatives.

Strategic Priorities for Research on Antibiotic Alternatives in Animal Agriculture-Results From an Expert Workshop.

The emergence, spread, and expansion of antibiotic resistance and increasing restrictions on the use of antibiotics in animal agriculture have created a need for efficacious alternatives that remains unmet. Prioritizing research needs in the development of alternatives is key to ensuring that scarce research resources are dedicated to the most promising approaches. However, frameworks to enable a consistent, systematic, and transparent evaluation of antibiotic alternative candidates are lacking. Here, we present such an evaluation framework.

Vaccines as alternatives to antibiotics for food producing animals. Part 1: challenges and needs.

Vaccines and other alternative products can help minimize the need for antibiotics by preventing and controlling infectious diseases in animal populations, and are central to the future success of animal agriculture. To assess scientific advancements related to alternatives to antibiotics and provide actionable strategies to support their development, the United States Department of Agriculture, with support from the World Organisation for Animal Health, organized the second International Symposium on Alternatives to Antibiotics. It focused on six key areas: vaccines; microbial-derived products; non-nutritive phytochemicals; immune-related products; chemicals, enzymes, and innovative drugs; and regulatory pathways to enable the development and licensure of alternatives to antibiotics. This article, part of a two-part series, synthesizes and expands on the expert panel discussions regarding opportunities, challenges and needs for the development of vaccines that may reduce the need for use of antibiotics in animals; new approaches and potential solutions will be discussed in part 2 of this series. Vaccines are widely used to prevent infections in food animals. Various studies have demonstrated that their animal agricultural use can lead to significant reductions in antibiotic consumption, making them promising alternatives to antibiotics. To be widely used in food producing animals, vaccines have to be safe, effective, easy to use, and cost-effective. Many current vaccines fall short in one or more of these respects. Scientific advancements may allow many of these limitations to be overcome, but progress is funding-dependent. Research will have to be prioritized to ensure scarce public resources are dedicated to areas of potentially greatest impact first, and private investments into vaccine development constantly compete with other investment opportunities. Although vaccines have the potential to improve animal health, safeguard agricultural productivity, and reduce antibiotic consumption and resulting resistance risks, targeted research and development investments and concerted efforts by all affected are needed to realize that potential.

Vaccines as alternatives to antibiotics for food producing animals. Part 2: new approaches and potential solutions.

Vaccines and other alternative products are central to the future success of animal agriculture because they can help minimize the need for antibiotics by preventing and controlling infectious diseases in animal populations. To assess scientific advancements related to alternatives to antibiotics and provide actionable strategies to support their development, the United States Department of Agriculture, with support from the World Organisation for Animal Health, organized the second International Symposium on Alternatives to Antibiotics. It focused on six key areas: vaccines; microbial-derived products; non-nutritive phytochemicals; immune-related products; chemicals, enzymes, and innovative drugs; and regulatory pathways to enable the development and licensure of alternatives to antibiotics. This article, the second part in a two-part series, highlights new approaches and potential solutions for the development of vaccines as alternatives to antibiotics in food producing animals; opportunities, challenges and needs for the development of such vaccines are discussed in the first part of this series. As discussed in part 1 of this manuscript, many current vaccines fall short of ideal vaccines in one or more respects. Promising breakthroughs to overcome these limitations include new biotechnology techniques, new oral vaccine approaches, novel adjuvants, new delivery strategies based on bacterial spores, and live recombinant vectors; they also include new vaccination strategies in-ovo, and strategies that simultaneously protect against multiple pathogens. However, translating this research into commercial vaccines that effectively reduce the need for antibiotics will require close collaboration among stakeholders, for instance through public-private partnerships. Targeted research and development investments and concerted efforts by all affected are needed to realize the potential of vaccines to improve animal health, safeguard agricultural productivity, and reduce antibiotic consumption and resulting resistance risks.

Phytochemicals as antibiotic alternatives to promote growth and enhance host health.

There are heightened concerns globally on emerging drug-resistant superbugs and the lack of new antibiotics for treating human and animal diseases. For the agricultural industry, there is an urgent need to develop strategies to replace antibiotics for food-producing animals, especially poultry and livestock. The 2nd International Symposium on Alternatives to Antibiotics was held at the World Organization for Animal Health in Paris, France, December 12-15, 2016 to discuss recent scientific developments on strategic antibiotic-free management plans, to evaluate regional differences in policies regarding the reduction of antibiotics in animal agriculture and to develop antibiotic alternatives to combat the global increase in antibiotic resistance. More than 270 participants from academia, government research institutions, regulatory agencies, and private animal industries from >25 different countries came together to discuss recent research and promising novel technologies that could provide alternatives to antibiotics for use in animal health and production; assess challenges associated with their commercialization; and devise actionable strategies to facilitate the development of alternatives to antibiotic growth promoters (AGPs) without hampering animal production. The 3-day meeting consisted of four scientific sessions including vaccines, microbial products, phytochemicals, immune-related products, and innovative drugs, chemicals and enzymes, followed by the last session on regulation and funding. Each session was followed by an expert panel discussion that included industry representatives and session speakers. The session on phytochemicals included talks describing recent research achievements, with examples of successful agricultural use of various phytochemicals as antibiotic alternatives and their mode of action in major agricultural animals (poultry, swine and ruminants). Scientists from industry and academia and government research institutes shared their experience in developing and applying potential antibiotic-alternative phytochemicals commercially to reduce AGPs and to develop a sustainable animal production system in the absence of antibiotics.

Microbial-derived products as potential new antimicrobials.

Due to the continuing global concerns involving antibiotic resistance, there is a need for scientific forums to assess advancements in the development of antimicrobials and their alternatives that might reduce development and spread of antibiotic resistance among bacterial pathogens. The objectives of the 2nd International Symposium on Alternatives to Antibiotics were to highlight promising research results and novel technologies that can provide alternatives to antibiotics for use in animal health and production, assess challenges associated with their authorization and commercialization for use, and provide actionable strategies to support their development. The session on microbial-derived products was directed at presenting novel technologies that included exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials, probiotics development via fecal microbiome transplants among monogastric production animals such as chickens and mining microbial sources such as bacteria or yeast to identify new antimicrobial compounds. Other research has included continuing development of antimicrobial peptides such as newly discovered bacteriocins as alternatives to antibiotics, use of bacteriophages accompanied by development of unique lytic proteins with specific cell-wall binding domains and novel approaches such as microbial-ecology guided discovery of anti-biofilm compounds discovered in marine environments. The symposium was held at the Headquarters of the World Organisation for Animal Health (OIE) in Paris, France during 12-15 December 2016.

Innovative drugs, chemicals, and enzymes within the animal production chain.

The alarming number of recently reported human illnesses with bacterial infections resistant to multiple antibacterial agents has become a serious concern in recent years. This phenomenon is a core challenge for both the medical and animal health communities, since the use of antibiotics has formed the cornerstone of modern medicine for treating bacterial infections. The empirical benefits of using antibiotics to address animal health issues in animal agriculture (using therapeutic doses) and increasing the overall productivity of animals (using sub-therapeutic doses) are well established. The use of antibiotics to enhance profitability margins in the animal production industry is still practiced worldwide. Although many technical and economic reasons gave rise to these practices, the continued emergence of antimicrobial resistant bacteria is furthering the need to reduce the use of medically important antibiotics. This will require improving on-farm management and biosecurity practices, and the development of effective antibiotic alternatives that will reduce the dependence on antibiotics within the animal industry in the foreseeable future. A number of approaches are being closely scrutinized and optimized to achieve this goal, including the development of promising antibiotic alternatives to control bacterial virulence through quorum-sensing disruption, the use of synthetic polymers and nanoparticles, the exploitation of recombinant enzymes/proteins (such as glucose oxidases, alkaline phosphatases and proteases), and the use of phytochemicals. This review explores the most recent approaches within this context and provides a summary of practical mitigation strategies for the extensive use of antibiotics within the animal production chain in addition to several future challenges that need to be addressed.

Bacillus spp. as direct-fed microbial antibiotic alternatives to enhance growth, immunity, and gut health in poultry.

The increasing occurrence of antibiotic-resistant bacteria combined with regulatory pressure and consumer demands for foods produced without antibiotics has caused the agricultural industry to restrict its practice of using antibiotic growth promoters (AGP) in food animals. The poultry industry is not immune to this trend, and has been actively seeking natural alternatives to AGP that will improve the health and growth performance of commercial poultry flocks. Bacillus probiotics have been gaining in popularity in recent years as an AGP alternative because of their health-promoting benefits and ability to survive the harsh manufacturing conditions of chicken feed production. This review provides an overview of several modes of action of some Bacillus direct-fed microbials as probiotics. Among the benefits of these direct-fed microbials are their production of naturally synthesized antimicrobial peptides, gut flora modulation to promote beneficial microbiota along the gastrointestinal tract, and various immunological and gut morphological alterations. The modes of action for increased performance are not well defined, and growth promotion is not equal across all Bacillus species or within strains. Appropriate screening and characterization of Bacillus isolates prior to commercialization are necessary to maximize poultry growth to meet the ultimate goal of eliminating AGP usage in animal husbandry.