Domestic pigs are a vital component of the global food supply, with a population nearing 780 million worldwide, making them one of the most commonly raised livestock. As pig production intensifies, the associated practices and environmental conditions may elevate the risk of emergence and spread of zoonotic agents, including ebolaviruses. Previously, we demonstrated that experimentall infection with Orthoebolavirus bundibugyoense and Orthoebolavirus restonense in pigs caused sub-clinical signs, with only a few animals exhibiting elevated temperatures and limited signs of acute respiratory distress. In this study, we sought to describe immune-related gene exression changes following those viral infections in pigs. Our findings revealed no significant changes in infection- and inflammation-related cytokines, but a strong adaptive immune response was observed in the lungs and tracheobronchial lymph nodes. Comparative analysis with a study in which non-human primates were experimentally infected with Orthoebolavirus bundibugyoense, where the virus is lethal, revealed molecular similarities in gene expression. This may suggest that certain viral processes may be conserved across species. These results highlight the potential role of pigs in ebolavirus spillover dynamics and underscore the importance of understanding the role of livestock in the emergence of these pathogens to guide prevention and mitigation strategies.

Marburg virus disease (MVD) is a zoonotic hemorrhagic disease with an estimated case fatality rate of up to 88%. Ghana recorded its first human MVD outbreak in June 2022 and although the outbreak was quickly brought under control, the transmission dynamics of the disease remained unclear. We assessed the presence of Marburg virus (MARV) antibodies in livestock and dogs and identified associated risk factors that increased the risk of these animals being exposed to MARV in five regions of Ghana. Sera collected from 3113 livestock and dogs in 2 climatic seasons (rainy and dry seasons) were tested for MARV antibodies using an indirect ELISA test. The samples were further tested using dot blotting to substantiate the presence of antibodies against MARV glycoprotein (GP). Overall, MARV antibodies were detected in 20.6% of the animals. The species-specific prevalence was 28.7% in cattle, 21.8% in sheep, 19.5% in goats, 15.3% in dogs and 11.2% in pigs. The seropositivity was higher in the rainy season [RR 1.5; 95% CI 1.3–1.8] and in older animals [RR = 2.6; 95% CI 1.9–3.4]. The findings underscore the importance of regular surveillance using the one health approach and future studies into the role of livestock and dogs as potential intermediaries in the circulation of MARV.

The Ebolavirus genus contains several of the deadliest zoonotic viruses known. One of these, Bundibugyo virus (BDBV), has been the causative agent of two outbreaks of human disease that have resulted in 211 known cases with a case fatality rate of 33.6%. Although bats are routinely implicated as the possible reservoir species for the ebolaviruses, the source of infection for index cases in almost all outbreaks is unknown with only limited epidemiological evidence directly linking human cases to bats. This lack of evidence leaves open the possibility that maintenance of one or more of these viruses could involve multiple host species or more complex spillover dynamics. Domestic pigs have been found naturally infected with Reston virus (RESTV) and are experimentally susceptible to infection with Ebola virus (EBOV), two other members of the Ebolavirus genus. Infection of pigs resulted in shedding of infectious virus with subsequent transmission to naïve animals being documented, including transmission to humans for RESTV and to nonhuman primates for EBOV. The susceptibility and subsequent viral shedding and pathogenesis of domestic pigs to other ebolaviruses and the potential role this species may play in virus ecology, spillover dynamics, and human public health risk is unknown. For these reasons, we conducted a series of studies aimed at determining the susceptibility of domestic pigs to BDBV thereby demonstrating that pigs are not only susceptible to experimental infection but that the development of productive infection, tissue dissemination, and shedding of infectious virus can also occur while animals remain clinically normal. The role of pigs as a possible interim or amplifying host for ebolaviruses is a concern for both human public health and food security.

Monkeypox virus (MPXV) is a re-emerging zoonotic poxvirus responsible for producing skin lesions in humans. Endemic in sub-Saharan Africa, the 2022 outbreak with a clade IIb strain has resulted in ongoing sustained transmission of the virus worldwide. MPXV has a relatively wide host range, with infections reported in rodent and non-human primate species. However, the susceptibility of many domestic livestock species remains unknown. Here, we report on a susceptibility/transmission study in domestic pigs that were experimentally inoculated with a 2022 MPXV clade IIb isolate or served as sentinel contact control animals. Several principal-infected and sentinel contact control pigs developed minor lesions near the lips and nose starting at 12 through 18 days post-challenge (DPC). No virus was isolated and no viral DNA was detected from the lesions; however, MPXV antigen was detected by IHC in tissue from a pustule of a principal infected pig. Viral DNA and infectious virus were detected in nasal and oral swabs up to 14 DPC, with peak titers observed at 7 DPC. Viral DNA was also detected in nasal tissues or skin collected from two principal-infected animals at 7 DPC post-mortem. Furthermore, all principal-infected and sentinel control animals enrolled in the study seroconverted. In conclusion, we provide the first evidence that domestic pigs are susceptible to experimental MPXV infection and can transmit the virus to contact animals.

Domestic pigs are a critical component of the food supply and one of the most commonly raised production animals. Pork consumption has driven the intensification of pig production expanding into environments conducive to increased emergence and spread of infectious diseases, including the spillover of pathogens into human populations. One of these emerging viruses, Reston virus (RESTV), is an enigma among the Orthoebolavirus genus in that its lack of human pathogenicity is in stark contrast to the high virulence associated with most other ebolaviruses. RESTV is, however, associated with outbreaks of highly lethal hemorrhagic disease in non-human primates (NHP), as well as poorly understood clinical manifestations of mixed virulence and lethality in naturally and experimentally infected domestic pigs. Our results show it is possible for RESTV derived from an NHP to infect domestic pigs resulting in a spectrum of disease, from asymptomatic to severe respiratory distress. Further, we report on the first experimental transmission of RESTV between infected pigs and a co-housed, naïve animal, as well as the first report of the successful use of group oral fluids for the detection of RESTV RNA and virus-specific IgA antibodies.

Classical swine fever (CSF) is a highly contagious transboundary viral disease of domestic and wild pigs. Despite mass vaccination and continuous eradication programs, CSF remains endemic in Asia, some countries in Europe, the Caribbean and South America. Since June 2013, Northern Colombia has reported 137 CSF outbreaks, mostly in backyard production systems with low vaccination coverage. The purpose of this study was to characterize the virus responsible for the outbreak. Phylogenetic analysis based on the full-length E2 sequence shows that the virus is closely related to CSF virus (CSFV) genotype 2.6 strains circulating in Southeast Asia. The pathotyping experiment suggests that the virus responsible is a moderately virulent strain. The 190 nucleotide stretch of the E2 hypervariable region of these isolates also shows high similarity to the CSFV isolates from Colombia in 2005 and 2006, suggesting a common origin for the CSF outbreaks caused by genotype 2.6 strains. The emergence of genotype 2.6 in Colombia suggests a potential transboundary spread of CSFV from Asia to the Americas, complicating the ongoing CSF eradication efforts in the Americas, and emphasizes the need for continuous surveillance in the region.

The global human population is growing at a rapid rate leading to the need for continued expansion of food animal production to meet the world's increasing nutritional requirements. As a consequence of this increased production demand, the use of high volume, animal dense systems have expanded providing high quality protein at reduced costs. Backyard animal production has also expanded. This increased food animal production has facilitated the rapid spread, mutation, and adaptation of pathogens to new hosts. This scenario continues to drive the emergence and reemergence of diseases in livestock species increasing the urgency for development and availability of vaccines for transboundary animal diseases (TADs). Even though vaccines are widely recognized as being an essential tool for control of TADs, there are many scientific, economic, political, and logistical challenges to having vaccine available to control an outbreak. This article will focus on examples of the challenges associated with having vaccines available for emergency response, as well as the characteristics of 'ideal' TAD vaccines, the need for complementary diagnostic assays, and hurdles involved in bringing efficacious veterinary TAD vaccines to market including regulatory constraints and considerations for stockpiling vaccines for emergency use in non-endemic countries. Examples will also highlight the complicated interplay between animal health and human health and demonstrate the lasting benefits that can be gained from an efficacious vaccine.

Rabbit haemorrhagic disease virus (RHDV) is associated with high morbidity and mortality in the European rabbit (Oryctolagus cuniculus). In 2010, a genetically distinct RHDV named RHDV2 emerged in Europe and spread to many other regions, including North America in 2016. Prior to this study it was unknown if eastern cottontails (ECT(s); Sylvilagus floridanus), one of the most common wild lagomorphs in the United States, were susceptible to RHDV2. In this study, 10 wild-caught ECTs and 10 New Zealand white rabbits (NZWR(s); O. cuniculus) were each inoculated orally with either RHDV (RHDVa/GI.1a; n = 5 per species) or RHDV2 (a recombinant GI.1bP-GI.2; n = 5 per species) and monitored for the development of disease. Three of the five ECTs that were infected with RHDV2 developed disease consistent with RHD and died at 4 and 6 days post-inoculation (DPI). The RHDV major capsid protein/antigen (VP60) was detected in the livers of three ECTs infected with RHDV2, but none was detected in the ECTs infected with RHDV. Additionally, RHD viral RNA was detected in the liver, spleen, intestine and blood of ECTs infected with RHDV2, but not in the ECTs infected with RHDV. RHD viral RNA was detected in urine, oral swabs and rectal swabs in at least two of five ECTs infected with RHDV2. One ECT inoculated with RHDV2 seroconverted and developed a high antibody titre by the end of the experimental period (21 DPI). ECTs inoculated with the classic RHDV did not seroconvert. In comparison, NZWRs inoculated with RHDV2 exhibited high mortality (five of five) at 2 DPI and four of five NZWRs inoculated with RHDV either died or were euthanized at 2 DPI indicating both of these viruses were highly pathogenic to this species. This experiment indicates that ECTs are susceptible to RHDV2 and can shed viral RNA, thereby suggesting this species could be involved in the epidemiology of this virus.

Livestock research is paramount to understanding the risks associated with unintentional and intentional introductions of emerging, reemerging, and transboundary animal diseases, including their relationship to both the security of the economy and the nation's food supply. Research involving large animal species conducted in maximum containment Biosafety Level (BSL)-3Ag and BSL-4 facilities include Health and Human Services and United States Department of Agriculture Select Agents that can have severe consequences on both animal agricultural industry and public health. This themed issue of ILAR spans the spectrum of concerns related to this special niche within the animal research community with an emphasis on a review of available research, current trends, and novel approaches relevant to those conducting large animal research with high-risk agents and those charged with regulating those facilities and programs. Articles are authored by those embedded in the high- and maximum-containment community, directly involved with the work, detailing the unique challenges associated with BSL-3 and BSL-4 livestock research.

Maximum-containment laboratories are a unique and essential component of the bioeconomy of the United States. These facilities play a critical role in the national infrastructure, supporting research on a select set of especially dangerous pathogens, as well as novel, emerging diseases. Understanding the ecology, biology, and pathology at the human-animal interface of zoonotic spillover events is fundamental to efficient control and elimination of disease. The use of animals as human surrogate models or as target-host models in research is an integral part of unraveling the interrelated components involved in these dynamic systems. These models can prove vitally important in determining both viral- and host-factors associated with virus transmission, providing invaluable information that can be developed into better risk mitigation strategies. In this article, we focus on the use of livestock in maximum-containment, biosafety level-4 agriculture (BSL-4Ag) research involving zoonotic, risk group 4 pathogens and we provide an overview of historical associated research and contributions. Livestock are most commonly used as target-host models in high-consequence, maximum-containment research and are routinely used to establish data to assist in risk assessments. This article highlights the importance of animal use, insights gained, and how this type of research is essential for protecting animal health, food security, and the agriculture economy, as well as human public health in the face of emerging zoonotic pathogens. The utilization of animal models in high-consequence pathogen research and continued expansion to include available species of agricultural importance is essential to deciphering the ecology of emerging and re-emerging infectious diseases, as well as for emergency response and mitigation preparedness.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent that causes coronavirus disease, has been shown to infect several species. The role of domestic livestock and associated risks for humans in close contact with food production animals remains unknown for many species. Determining the susceptibility of pigs to SARS-CoV-2 is critical to a One Health approach to manage potential risk for zoonotic transmission. We found that pigs are susceptible to SARS-CoV-2 after oronasal inoculation. Among 16 animals, we detected viral RNA in group oral fluids and in nasal wash from 2 pigs, but live virus was isolated from only 1 pig. Antibodies also were detected in only 2 animals at 11 and 13 days postinoculation but were detected in oral fluid samples at 6 days postinoculation, indicating antibody secretion. These data highlight the need for additional livestock assessment to determine the potential role of domestic animals in the SARS-CoV-2 pandemic.

SARS-CoV-2, the agent responsible for COVID-19 has been shown to infect a number of species. The role of domestic livestock and the risk associated for humans in close contact remains unknown for many production animals. Determination of the susceptibility of pigs to SARS-CoV-2 is critical towards a One Health approach to manage the potential risk of zoonotic transmission. Here, pigs undergoing experimental inoculation are susceptible to SARS-CoV-2 at low levels. Viral RNA was detected in group oral fluids and nasal wash from at least two animals while live virus was isolated from a pig. Further, antibodies could be detected in two animals at 11 and 13 days post infection, while oral fluid samples at 6 days post inoculation indicated the presence of secreted antibodies. These data highlight the need for additional livestock assessment to better determine the potential role domestic animals may contribute towards the SARS-CoV-2 pandemic. ### Competing Interest Statement The authors have declared no competing interest.

Development and maintenance of laboratory tick colonies provides reliable access to a variety of tick species at multiple life stages. Advances in techniques for the membrane feeding of ticks reduce the number of laboratory animals needed for colony maintenance. In the present study, modifications to the existing protocol for in vitro feeding of the argasid species Ornithodoros tartakovskyi were made. Adult O. tartakovskyi ticks of both sexes were allowed to feed to engorgement using a novel membrane feeding apparatus in a six-well plate format with well-inserts of laboratory-grade, wax sealing film. Of the 193 ticks placed on the membrane, 89% (n = 172) fed until engorgement and subsequently detached. The modified feeding method described will aid in future laboratory tick-based research because it allows for increased containment, ease of sorting, successful in vitro feeding, easy replacement of blood meals and a reduction in the total volume of blood meal required.

Many Institutional Biosafety Committees have expanded their role beyond their original mission, described in the National Institutes of Health Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. The guidelines are derived from the 1975 Asilomar Conference on Recombinant DNA Molecules that convened to examine and address societal concerns surrounding the (then) newly emerging recombinant DNA technology. This chapter will focus on emerging biological agents and cutting-edge technologies that present challenges to evaluating and assessing the biohazard risks associated with research protocols. Highly specialized and/or cross-disciplinary knowledge may be required for committee members to be able to evaluate benefit versus risk for research proposals addressing emerging infectious agents or technologies. The importance of designing a process that will account for the full range of requirements to fulfill the societal expectations of safety, efficiency, and scientific progress is highlighted.

Veterinary rabies vaccines are essential for safeguarding the public from exposure to rabies virus, as vaccination of domestic animals provides a barrier between humans and wildlife reservoirs. Ensuring rabies vaccines are potent and effective is paramount in preventing human exposure to rabies virus. The National Institutes of Health (NIH) test, a mouse vaccination-challenge assay, is the most widely used and internationally recognized assay for potency testing of inactivated rabies vaccines, and it is currently considered the method of choice. In the NIH test, vaccinated mice are challenged by the intracranial (IC) route. The response to the IC challenge can be variable, which often results in invalid tests. In addition, the IC challenge-exposure raises animal welfare concerns. The objective of this study was to evaluate the intranasal route of challenge as a modification to the NIH test to reduce animal pain and suffering until harmonized requirements for in vitro testing of rabies vaccines are developed. Results confirm the intranasal route is an effective route of rabies challenge in mice. However, a valid challenge requires the use of a more concentrated inoculum, in comparison to the intracranial method.

Potency testing of most human and veterinary rabies vaccines requires vaccination of mice followed by a challenge test using an intracerebral injection of live rabies virus. NICEATM, ICCVAM, and their international partners organized a workshop to review the availability and validation status of alternative methods that might reduce, refine, or replace the use of animals for rabies vaccine potency testing, and to identify research and development efforts to further advance alternative methods. Workshop participants agreed that general anesthesia should be used for intracerebral virus injections and that humane endpoints should be used routinely as the basis for euthanizing animals when conducting the mouse rabies challenge test. Workshop participants recommended as a near-term priority replacement of the mouse challenge with a test validated to ensure potency, such as the mouse antibody serum neutralization test for adjuvanted veterinary rabies vaccines for which an international collaborative study was recently completed. The workshop recommended that an in vitro antigen quantification test should be a high priority for product-specific validation of human and non-adjuvanted veterinary rabies vaccines. Finally, workshop participants recommended greater international cooperation to expedite development, validation, regulatory acceptance, and implementation of alternative test methods for rabies vaccine potency testing.

Rabies is an incurable zoonotic disease caused by rabies virus, a member of the rhabdovirus family. It is transmitted through the bite of an infected animal. Control methods, including oral rabies vaccination (ORV) programs, have led to a reduction in the spread and prevalence of the disease in wildlife. This study evaluated the stability of RABORAL, a recombinant vaccinia virus vaccine that is used in oral rabies vaccination programs. The vaccine was studied in various field microenvironments in order to describe its viability and facilitate effective baiting strategies. Field microenvironments influenced the stability of this vaccine in this study. This study emphasizes the importance of understanding how vaccines perform under varying field conditions in order to plan effective baiting strategies.

A high prevalence of fecal Salmonella shedding in a collection of healthy exotic felids precipitated a change to two new commercially available feline diets. One year after initiation of the new diets, 18 fecal samples from individual felines, their exhibits, and representative samples of the diets were cultured for Salmonella spp. Only one culture grew a Salmonella sp. Salmonella uganda was cultured from the feces of one snow leopard (Felis uncia). Feeding a diet with minimal to no Salmonella contamination lowered Salmonella shedding rates in this collection of captive exotic felids.

The Ebolavirus genus contains several of the deadliest zoonotic viruses known. Though bats are routinely implicated as the possible reservoir host for the ebolaviruses, the involvement of other species in ecology of these viruses is unclear. In 2008, domestic pigs were shown to be naturally infected with Reston virus (RESTV), the only ebolavirus known to be non-pathogenic in humans. Subsequent experimental infections have demonstrated that pigs are also susceptible to highly lethal, human-pathogenic Ebola virus, raising further concerns about food safety, pathogenicity, and the role pigs may play in ebolavirus ecology and spillover dynamics. Whether infection of domestic pigs can support the eventual emergence of a human-pathogenic RESTV is unclear, necessitating further investigation into the pig-ebolavirus relationship. Here, we describe the successful experimental infection of domestic pigs with a non-human primate-derived isolate of RESTV resulting in severe pulmonary pathology with systemic dissemination of virus, shedding of infectious virus, and transmission to and subsequent infection of a co-housed, naïve contact animal. Our data show that pig-adaptation of the virus was not required for infection and that natural infection of pigs could arise directly from non-human primates, or vice versa. We also provide the first report describing the susceptibility of domestic pigs to Bundibugyo virus (BDBV), another highly pathogenic ebolavirus responsible for outbreaks of lethal disease in humans. We show that pigs are not only susceptible to experimental infection with BDBV, but that the development of productive infection, tissue dissemination, and shedding of infectious virus can occur while animals remain clinically asymptomatic. The results of these studies further support the hypothesis that pigs may serve as intermediate or amplifying hosts in ebolavirus ecology, leading to concerns for both human public health and food security.