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Anthrax is a notorious disease of public health importance caused by Bacillus anthracis. The causative agent can also be used as a biological weapon. Spores of these bacteria can sustain extreme environmental conditions and remain viable in soil for decades. Domestic and wild ruminants are highly susceptible to this pathogen, which usually presents as a peracute to acute disease. In humans, cutaneous anthrax is frequent but pulmonary and enteric anthrax are more serious. Humans, animals, and the environment are all involved, making anthrax a perfect target for a One Health approach. The environment plays a key role in disease transmission. At a time when the One Health concept is not mere slogans, collaborative efforts of medical professionals, veterinarians, and environmental scientists will be valuable for the prevention and control of this disease. In this review, we discussed the transmission dynamics of anthrax in the environment, animals, and humans, as well as One Health strategies to control and prevent anthrax.

ABSTRACTAdult females of reproductive age develop greater antibody responses to inactivated influenza vaccines (IIV) than males. How sex, age, and sex steroid concentrations impact B cells and durability of IIV-induced immunity and protection over 4 months post-vaccination (mpv) was analyzed. Vaccinated adult females had greater germinal center B cell and plasmablast frequencies in lymphoid tissues, higher neutralizing antibody responses 1–4 mpv, and better protection against live H1N1 challenge than adult males. Aged mice, regardless of sex, had reduced B cell frequencies, less durable antibody responses, and inferior protection after challenge than adult mice, which correlated with diminished estradiol among aged females. To confirm that greater IIV-induced immunity was caused by sex hormones, four core genotype (FCG) mice were used, in which the testes-determining gene, Sry, was deleted from chromosome Y (ChrY) and transferred to Chr3 to separate gonadal sex (i.e., ovaries or testes) from sex chromosome complement (i.e., XX or XY complement). Vaccinated, gonadal female FCG mice (XXF and XYF) had greater numbers of B cells, higher antiviral antibody titers, and reduced pulmonary virus titers following live H1N1 challenge than gonadal FCG males (XYM and XXM). To establish that lower estradiol concentrations cause diminished immunity, adult and aged females received either a placebo or estradiol replacement therapy prior to IIV. Estradiol replacement significantly increased IIV-induced antibody responses and reduced morbidity after the H1N1 challenge among aged females. These data highlight that estradiol is a targetable mechanism mediating greater humoral immunity following vaccination among adult females.IMPORTANCEFemales of reproductive ages develop greater antibody responses to influenza vaccines than males. We hypothesized that female-biased immunity and protection against influenza were mediated by estradiol signaling in B cells. Using diverse mouse models ranging from advanced-age mice to transgenic mice that separate sex steroids from sex chromosome complement, those mice with greater concentrations of estradiol consistently had greater numbers of antibody-producing B cells in lymphoid tissue, higher antiviral antibody titers, and greater protection against live influenza virus challenge. Treatment of aged female mice with estradiol enhanced vaccine-induced immunity and protection against disease, suggesting that estradiol signaling in B cells is critical for improved vaccine outcomes in females.

ABSTRACTPregnant patients are at greater risk of hospitalization with severe COVID-19 than non-pregnant people. This was a retrospective observational cohort study of remnant clinical specimens from patients who visited acute care hospitals within the Johns Hopkins Health System in the Baltimore, MD–Washington DC, area between October 2020 and May 2022. Participants included confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected pregnant people and matched non-pregnant people (the matching criteria included age, race/ethnicity, area deprivation index, insurance status, and vaccination status to ensure matched demographics). The primary dependent measures were clinical COVID-19 outcomes, infectious virus recovery, viral RNA levels, and mucosal anti-spike (S) IgG titers from upper respiratory tract samples. A total of 452 individuals (117 pregnant and 335 non-pregnant) were included in the study, with both vaccinated and unvaccinated individuals represented. Pregnant patients were at increased risk of hospitalization (odds ratio [OR] = 4.2; confidence interval [CI] = 2.0–8.6), intensive care unit admittance (OR = 4.5; CI = 1.2–14.2), and being placed on supplemental oxygen therapy (OR = 3.1; CI = 1.3–6.9). Individuals infected during their third trimester had higher mucosal anti-S IgG titers and lower viral RNA levels (P < 0.05) than those infected during their first or second trimesters. Pregnant individuals experiencing breakthrough infections due to the Omicron variant had reduced anti-S IgG compared to non-pregnant patients (P < 0.05). The observed increased severity of COVID-19 and reduced mucosal antibody responses particularly among pregnant participants infected with the Omicron variant suggest that maintaining high levels of SARS-CoV-2 immunity through booster vaccines may be important for the protection of this at-risk population.IMPORTANCEIn this retrospective observational cohort study, we analyzed remnant clinical samples from non-pregnant and pregnant individuals with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections who visited the Johns Hopkins Hospital System between October 2020 and May 2022. Disease severity, including intensive care unit admission, was greater among pregnant than non-pregnant patients. Vaccination reduced recovery of infectious virus and viral RNA levels in non-pregnant patients, but not in pregnant patients. In pregnant patients, increased nasopharyngeal viral RNA levels and recovery of infectious virus were associated with reduced mucosal IgG antibody responses, especially among women in their first trimester of pregnancy or experiencing breakthrough infections from Omicron variants. Taken together, this study provides insights into how pregnant patients are at greater risk of severe COVID-19. The novelty of this study is that it focuses on the relationship between the mucosal antibody response and its association with virus load and disease outcomes in pregnant people, whereas previous studies have focused on serological immunity. Vaccination status, gestational age, and SARS-CoV-2 omicron variant impact mucosal antibody responses and recovery of infectious virus from pregnant patients.

In this study, we evaluated sex differences during infection with mouse-adapted H1N1 and H3N2 influenza A viruses (IAVs) in the C57BL/6J mouse model and compared the cytokine and antibody responses between plasma and serum samples during IAV infection and vaccination. Lethal doses for both H1N1 and H3N2 IAVs were lower for adult females and they suffered with greater morbidity than adult males when infected with sublethal doses. In influenza virus-infected mice, cytokine responses differed between plasma and serum samples. After inactivated influenza virus vaccination and drift variant challenge, adult female mice had greater antibody responses and were better protected. In influenza-vaccinated and challenged mice, binding antibodies were unaffected between paired plasma or serum samples. However, functional antibody assays, including hemagglutination inhibition, microneutralization, and antibody-dependent cellular cytotoxicity assays, were affected by the use of plasma and serum sample types. Our results indicate that careful consideration is required while selecting plasma versus serum samples to measure cytokine and antibody responses during IAV infection and vaccination.

In this study, chopping board meat samples collected from meat shops of Bharatpur Metropolitan City, Nepal, were screened for the presence of Escherichia coli (E. coli), with a special emphasis on the identification of extended-spectrum β-lactamase (ESBL)-producing strains. Representatives from the meat shops were also interviewed to understand the sanitary status and hygienic practices. E. coli bacteria were detected in one third (33/99) of the meat samples, while none of the samples had ESBL-producing strains. While 60.6% (60/99) of the meat shop personnel wore protective clothing, 15.15% (15/99) used gloves, and only 5.05% (5/99) had separate equipment for cleaning the viscera of animals. This study highlights the need for the regular screening of meat samples to identify pathogenic bacteria such as E. coli and for improvements in the sanitary status and hygienic practices of retail meat shops in Bharatpur Metropolitan City, Nepal.

Low pathogenic avian influenza (LPAI) of subtype H9 outbreaks have been frequently occurring in major commercial hubs of Nepal including Chitwan, a central lowland area, causing substantial economic losses to the farmers. However, the risk factors associated with these outbreaks have been poorly understood, and hence, this case-control study was conducted in Chitwan, Nawalpur, and Makawanpur districts of Nepal from October 2019 to March 2020. A total of 102 farms were selected in which 51 were case farms, and 51 were controls. Case farms were avian influenza (AI)-subtype-H9-confirmed farms through polymerase chain reaction (PCR) assays on poultry samples. Control farms included farms that were AI-negative in the antigen test brought to the National Avian Disease Investigation Laboratory, Chitwan, for diagnosis during the study period. Each farm was visited to collect information using a semi-structured questionnaire. A total of 25 variables representing farm characteristics and biosecurity measures were considered as potential risk factors. The final multivariable model showed that distance of less than 0.5 km from the main road (OR = 4.04, 95% CI = 1.20–13.56, p = 0.023), distance of less than 1 km from a nearest infected farm (OR = 76.42, 95% CI = 7.17–814.06, p = 0.0003), and wild birds coming around the farm (OR = 6.12, 95% CI = 1.99–18.79, p = 0.0015) were risk factors for avian influenza type H9, whereas using apron or separate cloth inside the shed (OR = 0.109, 95% CI = 0.020–0.577, p = 0.0092) was shown to reduce the risk of farms being positive for AI subtype H9. These findings suggest that due consideration should be given to site selection while establishing the farms and the importance of implementing appropriate biosecurity measures, such as using separate cloth inside the shed and preventing the entry of wild birds inside the farm to reduce the potential risk of introduction of avian influenza type H9 to their poultry farms.

African swine fever (ASF) is a highly contagious viral infection of domestic and wild pigs with high mortality. First reported in East Africa in the early 1900s, ASF was largely controlled in domestic pigs in many countries. However, in recent years ASF outbreaks have been reported in several countries in Europe and Asia. The occurrence of ASF in China, the largest pork producer in the world, in 2018 and in India, the country that surrounds and shares open borders with Nepal, has increased the risk of ASF transmission to Nepal. Lately, the pork industry has been growing in Nepal, overcoming traditional religious and cultural biases against it. However, the emergence of viral infections such as ASF could severely affect the industry’s growth and sustainability. Because there are no effective vaccines available to prevent ASF, the government should focus on preventing entry of the virus through strict quarantine measures at the borders, controls on illegal trade, and effective management practices, including biosecurity measures.

ABSTRACT In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and “to develop, validate, improve, and implement serological testing and associated technologies” (https://www.cancer.gov/research/key-initiatives/covid-19/coronavirus-research-initiatives/serological-sciences-network). SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology standard reference material and first WHO international standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. SeroNet institutions reported development of a total of 27 enzyme-linked immunosorbent assay (ELISA) methods, 13 multiplex assays, and 9 neutralization assays and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. In conclusion, SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons. IMPORTANCE SeroNet institutions have developed or implemented 61 diverse COVID-19 serological assays and are collaboratively working to harmonize these assays using reference materials to establish standardized reporting units. This will facilitate clinical interpretation of serology results and cross-comparison of research data.

ABSTRACT Inactivated influenza vaccines induce greater antibody responses in females than males among both humans and mice. To test the breadth of protection, we used recombinant mouse-adapted A/California/2009 (maA/Cal/09) H1N1 viruses containing mutations at one (1M), two (2M), or three (3M) antigenic sites, in addition to a virus containing the 1M mutation and a substitution of the Ca2 antigenic site (Sub) with one derived from an H5 hemagglutinin (HA) to challenge mice of both sexes. Following maA/Cal/09 vaccination, females produced greater virus-specific, class-switched total IgG and IgG2c antibodies against the vaccine and all mutant viruses, and antibodies from females recognized a greater number of unique, linear HA epitopes than did antibodies from males. While females had greater neutralizing antibody titers against the vaccine virus, both sexes showed a lower neutralization capacity against mutant viruses. After virus challenge, vaccinated females had lower pulmonary virus titers and reduced morbidity than males for the 1M and 2M viruses, but not the Sub virus. Females generated greater numbers of germinal center (GC) B cells containing superior somatic hypermutation (SHM) frequencies than vaccinated males. Deletion of activation-induced cytidine deaminase (Aicda) eliminated female-biased immunity and protection against the 2M virus. Harnessing methods to improve GC B cell responses and frequencies of SHM, especially in males, should be considered in the development of universal influenza vaccines. IMPORTANCE Adult females develop greater antibody responses to influenza vaccines than males. We hypothesized that female-biased immunity and protection would be dependent on the extent of virus diversity as well as molecular mechanisms in B cells which constrain the breadth of epitope recognition. We developed a panel of mouse-adapted (ma) A/Cal/09 viruses that had mutations in the immunodominant hemagglutinin. Following vaccination against maA/Cal/09, females were better able to neutralize maA/Cal/09 than males, but neutralization of mutant maA/Cal/09 viruses was equally poor in both sexes, despite vaccinated females being better protected against these viruses. Vaccinated females benefited from the greater production of class-switched, somatically hypermutated antibodies generated in germinal center B cells, which increased recognition of more diverse maA/Cal/09 hemagglutinin antigen epitopes. Female-biased protection against influenza infection and disease after vaccination is driven by differential mechanisms in males versus females and should be considered in the design of novel vaccine platforms.

ABSTRACT The impact of obesity on the human microbiota, immune maturation, and influenza virus infection has not been yet established in natural host animal models of influenza. In this study, gnotobiotic (Gn) pigs were colonized with human fecal microbiota (HFM) of obese (oHFM) or healthy lean (hHFM) children and infected at different periods (2-, 3-, and 5-weeks post-transplantation) using a zoonotic influenza virus strain. The infected oHFM pigs were characterized by lower levels of Firmicutes (Lactococcus, Lactobacillus, Turicibacter, and Streptococcus) and Actinobacteria (Bifidobacterium), which was associated with higher levels of Proteobacteria (Klebsiella), Bacteroidetes, and Verrucomicrobia (Akkermansia) compared with the infected hHFM group (P

Abstract Several vaccines have been introduced to combat the coronavirus infectious disease‐2019 (COVID‐19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Current SARS‐CoV‐2 vaccines include mRNA‐containing lipid nanoparticles or adenoviral vectors that encode the SARS‐CoV‐2 Spike (S) protein of SARS‐CoV‐2, inactivated virus, or protein subunits. Despite growing success in worldwide vaccination efforts, additional capabilities may be needed in the future to address issues such as stability and storage requirements, need for vaccine boosters, desirability of different routes of administration, and emergence of SARS‐CoV‐2 variants such as the Delta variant. Here, we present a novel, well‐characterized SARS‐CoV‐2 vaccine candidate based on extracellular vesicles (EVs) of Salmonella typhimurium that are decorated with the mammalian cell culture‐derived Spike receptor‐binding domain (RBD). RBD‐conjugated outer membrane vesicles (RBD‐OMVs) were used to immunize the golden Syrian hamster (Mesocricetus auratus) model of COVID‐19. Intranasal immunization resulted in high titres of blood anti‐RBD IgG as well as detectable mucosal responses. Neutralizing antibody activity against wild‐type and Delta variants was evident in all vaccinated subjects. Upon challenge with live virus, hamsters immunized with RBD‐OMV, but not animals immunized with unconjugated OMVs or a vehicle control, avoided body mass loss, had lower virus titres in bronchoalveolar lavage fluid, and experienced less severe lung pathology. Our results emphasize the value and versatility of OMV‐based vaccine approaches.

Abstract COVID-19 caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originated in Wuhan (Hubei province, China) during late 2019. It has spread across the globe affecting nearly 21 million people with a toll of 0.75 million deaths and restricting the movement of most of the world population during the past 6 months. COVID-19 became the leading health, economic, and humanitarian challenge of the twenty-first century. In addition to the considerable COVID-19 cases, hospitalizations, and deaths in humans, several cases of SARS-CoV-2 infections in animal hosts (dog, cat, tiger, lion, and mink) have been reported. Thus, the concern of pet owners is increasing. Moreover, the dynamics of the disease requires further explanation, mainly concerning the transmission of the virus from humans to animals and vice versa. Therefore, this study aimed to gather information about the reported cases of COVID-19 transmission in animals through a literary review of works published in scientific journals and perform genomic and phylogenetic analyses of SARS-CoV-2 isolated from animal hosts. Although many instances of transmission of the SARS-CoV-2 have been reported, caution and further studies are necessary to avoid the occurrence of maltreatment in animals, and to achieve a better understanding of the dynamics of the disease in the environment, humans, and animals. Future research in the animal–human interface can help formulate and implement preventive measures to combat the further transmission of COVID-19.

Coronaviruses are a group of enveloped, single-stranded, positive-sense RNA viruses which are broadly classified into Alpha, Beta, Gamma, and Delta coronaviruses genera based on the viral genome. Coronavirus was not thought to be a significant problem in humans until the outbreak of Severe Acute Respiratory Syndrome (SARS) in 2002 but infection in animals including pigs, cats, dogs and poultry has been problematic for long. The outbreak of Coronavirus Disease 2019 (COVID-19) in December 2019 in Wuhan, China drew special attention towards this virus once again. The intermediate host of this novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is yet to be determined, but it has a very close genomic relationship with the bat coronavirus (BatCoV), RaTG13 strain, and the pangolin coronaviruses. As veterinary medicine has long term experience dealing with coronaviruses, this could be helpful in better understanding to detect the origin of SARS-CoV-2 and drive human medicine towards the development of vaccines and antiviral drugs through the collaborative and transdisciplinary approach of One Health.

Habitat suitability is crucial to ensure the long-term persistence of species and can be identified based on relationships between species occurrences and underlying abiotic and biotic factors. We identified potential nesting habitat for the Egyptian vulture (Neophron percnopterus) in Nepal using ecological niche modeling with climatic variables. We estimated the currently suitable nesting habitat for Egyptian vulture in Nepal at 38,204 km2. We found a high probability of suitable nesting habitat on east-facing aspects, and the probability of a suitable nesting habitat was greater in more mountainous areas, particularly in central and western regions of Nepal. Precipitation was a major factor for predicting probability of the presence of nest sites for Egyptian vultures. After identifying potentially suitable habitat, we identified environmental factors affecting landscape-level suitable nesting habitat for Egyptian vultures using generalized linear models. For Egyptian vultures, sites near forests and human settlements were most suitable for nesting, roosting, and foraging, especially in central and western Nepal. Based on potentially suitable nesting habitat and previous work on Egyptian vulture foraging and roosting habitat, we recommend protecting forests near water sources and open areas for their long-term conservation.

Swine influenza is a highly contagious respiratory disease of pigs caused by influenza A viruses (IAV-S). IAV-S causes significant economic losses to the swine industry and poses challenges to public health given its zoonotic potential. Thus effective IAV-S vaccines are needed and highly desirable and would benefit both animal and human health. Here, we developed two recombinant orf viruses, expressing the hemagglutinin (HA) gene (OV-HA) or the HA and the nucleoprotein (NP) genes of IAV-S (OV-HA-NP). The immunogenicity and protective efficacy of these two recombinant viruses were evaluated in pigs. Both OV-HA and OV-HA-NP recombinants elicited robust virus neutralizing antibody response in pigs, with higher levels of neutralizing antibodies (NA) being detected in OV-HA-NP-immunized animals pre-challenge infection. Although both recombinant viruses elicited IAV-S-specific T-cell responses, the frequency of IAV-S-specific proliferating CD8+ T cells upon re-stimulation was higher in OV-HA-NP-immunized animals than in the OV-HA group. Importantly, IgG1/IgG2 isotype ELISAs revealed that immunization with OV-HA induced Th2-biased immune responses, whereas immunization with OV-HA-NP virus resulted in a Th1-biased immune response. While pigs immunized with either OV-HA or OV-HA-NP were protected when compared to non-immunized controls, immunization with OV-HA-NP resulted in incremental protection against challenge infection as evidenced by a reduced secondary antibody response (NA and HI antibodies) following IAV-S challenge and reduced virus shedding in nasal secretions (lower viral RNA loads and frequency of animals shedding viral RNA and infectious virus), when compared to animals in the OV-HA group. Interestingly, broader cross neutralization activity was also observed in serum of OV-HA-NP-immunized animals against a panel of contemporary IAV-S isolates representing the major genetic clades circulating in swine. This study demonstrates the potential of ORFV-based vector for control of swine influenza virus in swine.

ABSTRACT In the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), more severe outcomes are reported in males than in females, including hospitalizations and deaths. Animal models can provide an opportunity to mechanistically interrogate causes of sex differences in the pathogenesis of SARS-CoV-2. Adult male and female golden Syrian hamsters (8 to 10 weeks of age) were inoculated intranasally with 105 50% tissue culture infective dose (TCID50) of SARS-CoV-2/USA-WA1/2020 and euthanized at several time points during the acute (i.e., virus actively replicating) and recovery (i.e., after the infectious virus has been cleared) phases of infection. There was no mortality, but infected male hamsters experienced greater morbidity, losing a greater percentage of body mass, developed more extensive pneumonia as noted on chest computed tomography, and recovered more slowly than females. Treatment of male hamsters with estradiol did not alter pulmonary damage. Virus titers in respiratory tissues, including nasal turbinates, trachea, and lungs, and pulmonary cytokine concentrations, including interferon-β (IFN-β) and tumor necrosis factor-α (TNF-α), were comparable between the sexes. However, during the recovery phase of infection, females mounted 2-fold greater IgM, IgG, and IgA responses against the receptor-binding domain of the spike protein (S-RBD) in both plasma and respiratory tissues. Female hamsters also had significantly greater IgG antibodies against whole-inactivated SARS-CoV-2 and mutant S-RBDs as well as virus-neutralizing antibodies in plasma. The development of an animal model to study COVID-19 sex differences will allow for a greater mechanistic understanding of the SARS-CoV-2-associated sex differences seen in the human population. IMPORTANCE Men experience more severe outcomes from coronavirus disease 2019 (COVID-19) than women. Golden Syrian hamsters were used to explore sex differences in the pathogenesis of a human isolate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). After inoculation, male hamsters experienced greater sickness, developed more severe lung pathology, and recovered more slowly than females. Sex differences in disease could not be reversed by estradiol treatment in males and were not explained by either virus replication kinetics or the concentrations of inflammatory cytokines in the lungs. During the recovery period, antiviral antibody responses in the respiratory tract and plasma, including to newly emerging SARS-CoV-2 variants, were greater in female than in male hamsters. Greater lung pathology during the acute phase combined with lower antiviral antibody responses during the recovery phase of infection in males than in females illustrate the utility of golden Syrian hamsters as a model to explore sex differences in the pathogenesis of SARS-CoV-2 and vaccine-induced immunity and protection.

Unprecedented and unforeseen highly infectious Coronavirus Disease 2019 (COVID-19) has become a significant public health concern for most of the countries worldwide, including Nepal, and it is spreading rapidly. Undoubtedly, every nation has taken maximum initiative measures to break the transmission chain of the virus. This review presents a retrospective analysis of the COVID-19 pandemic in Nepal, analyzing the actions taken by the Government of Nepal (GoN) to inform future decisions. Data used in this article were extracted from relevant reports and websites of the Ministry of Health and Population (MoHP) of Nepal and the WHO. As of January 22, 2021, the highest numbers of cases were reported in the megacity of the hilly region, Kathmandu district (population = 1,744,240), and Bagmati province. The cured and death rates of the disease among the tested population are ~98.00 and ~0.74%, respectively. Higher numbers of infected cases were observed in the age group 21–30, with an overall male to female death ratio of 2.33. With suggestions and recommendations from high-level coordination committees and experts, GoN has enacted several measures: promoting universal personal protection, physical distancing, localized lockdowns, travel restrictions, isolation, and selective quarantine. In addition, GoN formulated and distributed several guidelines/protocols for managing COVID-19 patients and vaccination programs. Despite robust preventive efforts by GoN, pandemic scenario in Nepal is, yet, to be controlled completely. This review could be helpful for the current and future effective outbreak preparedness, responses, and management of the pandemic situations and prepare necessary strategies, especially in countries with similar socio-cultural and economic status.

Introduction: Laryngoscopy and intubation are stressful as they lead to a rise in heart rate and blood pressure. Though transient, it may be detrimental to the cardiac and neurosurgical patients. There is a need to explore the possibility of obtunding the pressor response to laryngoscopy and intubation with the use of McCoy blade laryngoscope. We aimed to find out the hemodynamic response to laryngoscopy and intubation using McCoy laryngoscope in adult patients undergoing general anesthesia. Methods: The descriptive cross-sectional study was conducted in 37 American Society of Anesthesiologists' Physical Status I/IIpatients, with normal airway from December 2019- May 2020 in a tertiary care hospital. Ethical approval was obtained from Institutional Research Committee (reference number.: MEMG/IRC/290/GA). Convenience sampling method was used. The mean systolic and diastolic blood pressures were measured at baseline, one, three and five minutes after laryngoscopy and intubation. Data were analyzed using the Statistical Package for the Social Sciences Version 21.0. Results: In the first minute after laryngoscopy and intubation, the rise in mean blood pressure was noted in 14 (37.83%) cases. The peak rise in mean blood pressure was 3%, note done minute after laryngoscopy and intubation. Conclusions: We noted better attenuation of pressor response to laryngoscopy and intubation using McCoy blade laryngoscope in adult patients undergoing general anesthesia.

Abstract Virus infections possess persistent health challenges in swine industry leading to severe economic losses worldwide. The economic burden caused by virus infections such as Porcine Reproductive and Respiratory Syndrome Virus, Swine influenza virus, Porcine Epidemic Diarrhea Virus, Porcine Circovirus 2, Foot and Mouth Disease Virus and many others are associated with severe morbidity, mortality, loss of production, trade restrictions and investments in control and prevention practices. Pigs can also have a role in zoonotic transmission of some viral infections to humans. Inactivated and modified-live virus vaccines are available against porcine viral infections with variable efficacy under field conditions. Thus, improvements over existing vaccines are necessary to: (1) Increase the breadth of protection against evolving viral strains and subtypes; (2) Control of emerging and re-emerging viruses; (3) Eradicate viruses localized in different geographic areas; and (4) Differentiate infected from vaccinated animals to improve disease control programs. Nanoparticles (NPs) generated from virus-like particles, biodegradable and biocompatible polymers and liposomes offer many advantages as vaccine delivery platform due to their unique physicochemical properties. NPs help in efficient antigen internalization and processing by antigen presenting cells and activate them to elicit innate and adaptive immunity. Some of the NPs-based vaccines could be delivered through both parenteral and mucosal routes to trigger efficient mucosal and systemic immune responses and could be used to target specific immune cells such as mucosal microfold (M) cells and dendritic cells (DCs). In conclusion, NPs-based vaccines can serve as novel candidate vaccines against several porcine viral infections with the potential to enhance the broader protective efficacy under field conditions. This review highlights the recent developments in NPs-based vaccines against porcine viral pathogens and how the NPs-based vaccine delivery system induces innate and adaptive immune responses resulting in varied level of protective efficacy.

Parenteral administration of killed/inactivated swine influenza A virus (SwIAV) vaccine in weaned piglets provides variable levels of immunity due to the presence of preexisting virus specific maternal derived antibodies (MDA). To overcome the effect of MDA on SwIAV vaccine in piglets, we developed an intranasal deliverable killed SwIAV antigen (KAg) encapsulated chitosan nanoparticles called chitosan-based NPs encapsulating KAg (CS NPs-KAg) vaccine. Further, to target the candidate vaccine to dendritic cells and macrophages which express mannose receptor, we conjugated mannose to chitosan (mCS) and formulated KAg encapsulated mCS nanoparticles called mannosylated chitosan-based NPs encapsulating KAg (mCS NPs-KAg) vaccine. In MDA-positive piglets, prime-boost intranasal inoculation of mCS NPs-KAg vaccine elicited enhanced homologous (H1N2-OH10), heterologous (H1N1-OH7), and heterosubtypic (H3N2-OH4) influenza virus-specific secretory IgA (sIgA) antibody response in nasal passage compared to CS NPs-KAg vaccinates. In vaccinated upon challenged with a heterologous SwIAV H1N1, both mCS NPs-KAg and CS NPs-KAg vaccinates augmented H1N2-OH10, H1N1-OH7, and H3N2-OH4 virus-specific sIgA antibody responses in nasal swab, lung lysate, and bronchoalveolar lavage (BAL) fluid; and IgG antibody levels in lung lysate and BAL fluid samples. Whereas, the multivalent commercial inactivated SwIAV vaccine delivered intramuscularly increased serum IgG antibody response. In mCS NPs-KAg and CS NPs-KAg vaccinates increased H1N2-OH10 but not H1N1-OH7 and H3N2-OH4-specific serum hemagglutination inhibition titers were observed. Additionally, mCS NPs-KAg vaccine increased specific recall lymphocyte proliferation and cytokines IL-4, IL-10, and IFNγ gene expression compared to CS NPs-KAg and commercial SwIAV vaccinates in tracheobronchial lymph nodes. Consistent with the immune response both mCS NPs-KAg and CS NPs-KAg vaccinates cleared the challenge H1N1-OH7 virus load in upper and lower respiratory tract more efficiently when compared to commercial vaccine. The virus clearance was associated with reduced gross lung lesions. Overall, mCS NP-KAg vaccine intranasal immunization in MDA-positive pigs induced a robust cross-reactive immunity and offered protection against influenza virus.

We designed the killed swine influenza A virus (SwIAV) H1N2 antigen (KAg) with polyriboinosinic:polyribocytidylic acid [(Poly(I:C)] adsorbed corn-derived Nano-11 particle based nanovaccine called Nano-11-KAg+Poly(I:C), and evaluated its immune correlates in maternally derived antibody (MDA)-positive pigs against a heterologous H1N1 SwIAV infection. Immunologically, in tracheobronchial lymph nodes (TBLN) detected enhanced H1N2-specific cytotoxic T-lymphocytes (CTLs) in Nano-11-KAg+Poly(I:C) vaccinates, and in commercial vaccinates detected CTLs with mainly IL-17A+ and early effector phenotypes specific to both H1N2 and H1N1 SwAIV. In commercial vaccinates, activated H1N2- and H1N1-specific IFNγ+&TNFα+, IL-17A+ and central memory T-helper/Memory cells, and in Nano-11-KAg+Poly(I:C) vaccinates H1N2-specific central memory, IFNγ+ and IFNγ+&TNFα+, and H1N1-specific IL-17A+ T-helper/Memory cells were observed. Systemically, Nano-11-KAg+Poly(I:C) vaccine augmented H1N2-specific IFNγ+ CTLs and H1N1-specific IFNγ+ T-helper/Memory cells, and commercial vaccine boosted H1N2- specific early effector CTLs and H1N1-specific IFNγ+&TNFα+ CTLs, as well as H1N2- and H1N1-specific T-helper/Memory cells with central memory, IFNγ+&TNFα+, and IL-17A+ phenotypes. Remarkably, commercial vaccine induced an increase in H1N1-specific T-helper cells in TBLN and naive T-helper cells in both TBLN and peripheral blood mononuclear cells (PBMCs), while H1N1- and H1N2-specific only T-helper cells were augmented in Nano-11-KAg+Poly(I:C) vaccinates in both TBLN and PBMCs. Furthermore, the Nano-11-KAg+Poly(I:C) vaccine stimulated robust cross-reactive IgG and secretory IgA (SIgA) responses in lungs, while the commercial vaccine elicited high levels of serum and lung IgG and serum hemagglutination inhibition (HI) titers. In conclusion, despite vast genetic difference (77% in HA gene identity) between the vaccine H1N2 and H1N1 challenge viruses in Nano-11-KAg+Poly(I:C) vaccinates, compared to over 95% identity between H1N1 of commercial vaccine and challenge viruses, the virus load and macroscopic lesions in the lungs of both types of vaccinates were comparable, but the Nano-11-KAg+Poly(I:C) vaccine cleared the virus from the nasal passage better. These data suggested the important role played by Nano-11 and Poly(I:C) in the induction of polyfunctional, cross-protective cell-mediated immunity against SwIAV in MDA-positive pigs.

Rabies is a global problem, but the burden is higher in less developed or developing countries of Asia and Africa. In Nepal, rabies is reported to kill around 500 animals and up to 32 human beings in recent years, with possible under-reporting of cases and deaths. As a prophylactic measure, around 30,000 livestock and 300,000 humans get vaccinated each year in Nepal. This review summarizes the past, present and future perspectives of rabies control and prevention in Nepal. The global strategic plan of World Health Organization (WHO) aims to bring human deaths from dog-transmitted rabies to zero by 2030. To achieve this goal of ‘Zero by 30’, the concerned governmental and non-governmental agencies in Nepal should work together using the One Health concept.

Coronavirus disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), was first reported in late 2019 from Wuhan, China. Considering COVID-19's alarming levels of spread and severity, the World Health Organization (WHO) declared a global pandemic on March 11, 2020. The first case of COVID-19 in Nepal was reported on January 23, 2020. The Government of Nepal implemented different public health measures to contain COVID-19, including border closures and a countrywide lockdown. We collected the daily data provided by the Ministry of Health and Population (MoHP) of the Government of Nepal and illustrated the early epidemiological characteristics of COVID-19 in Nepal. By May 31, 2020, 1,572 cases and eight deaths were reported in Nepal associated with COVID-19. The estimate of prevalence for COVID-19 among tested populations was 2.25% (95% CI: 2.15–2.37%) and case-fatality rate was 0.5%. The majority of the cases were young males (n = 1,454, 92%), with overall average age being 30.5 years (ranging from 2 months to 81 years) and were mostly asymptomatic. There were only five cases from three districts until the end of March, but cases surged from April and spread to 57 out of 77 districts of Nepal by the end of May 2020 despite the continuous lockdown. Most of these cases are from the southern plains of Nepal, bordering India. As the effect of COVID-19 is expected to persist longer, the Government of Nepal should make appropriate strategies for loosening lockdowns in a phase-wise manner while maintaining social distancing and personal hygiene and increasing its testing, tracking, and medical capacity.

Circulating androgens can modulate immune cell activity, but the impact of androgens on viral pathogenesis remains unclear. Previous data demonstrate that testosterone reduces the severity of influenza A virus (IAV) infection in male mice by mitigating pulmonary inflammation rather than by affecting viral replication. To examine the immune responses mediated by testosterone to mitigate IAV-induced inflammation, adult male mice remained gonadally intact or were gonadectomized and treated with either placebo or androgen-filled (i.e., testosterone or dihydrotestosterone) capsules prior to sublethal IAV infection. Like intact males, treatment of gonadectomized males with androgens improved the outcome of IAV infection, which was not mediated by changes in the control of virus replication or pulmonary cytokine activity. Instead, androgens accelerated pulmonary leukocyte contraction to limit inflammation. To identify which immune cells were contracting in response to androgens, the composition of pulmonary cellular infiltrates was analyzed and revealed that androgens specifically accelerated the contraction of total pulmonary inflammatory monocytes during peak disease, as well as CD8+ T cells, IAV-specific CD8+ T numbers, cytokine production and degranulation by IAV-specific CD8+ T cells, and the influx of eosinophils into the lungs following clearance of IAV. Neither depletion of eosinophils nor adoptive transfer of CD8+ T cells could reverse the ability of testosterone to protect males against IAV suggesting these were secondary immunologic effects. The effects of testosterone on the contraction of immune cell numbers and activity were blocked by co-administration of the androgen receptor antagonist flutamide and mimicked by treatment with dihydrotestosterone, which was also able to reduce the severity of IAV in female mice. These data suggest that androgen receptor signaling creates a local pulmonary environment that promotes downregulation of detrimental inflammatory immune responses to protect against prolonged influenza disease.

The current novel coronavirus disease 2019 (COVID-19) pandemic is revealing profound differences between men and women in disease outcomes worldwide. In the United States, there has been inconsistent reporting and analyses of male-female differences in COVID-19 cases, hospitalizations, and deaths. We seek to raise awareness about the male-biased severe outcomes from COVID-19, highlighting the mechanistic differences including in the expression and activity of angiotensin-converting enzyme 2 (ACE2) as well as in antiviral immunity. We also highlight how sex differences in comorbidities, which can be associated with both age and race, impact male-biased outcomes from COVID-19.

Vaccination is the most effective preventive measure of COVID-19 available at present, but its success depends on the global accessibility of vaccines and the willingness of people to be vaccinated. As the vaccination rollouts are increasing worldwide, it is important to assess public perception and willingness towards vaccination, so that the aim of mass vaccination will be successful. This study aimed to understand public perception towards COVID-19 vaccines and their willingness to get vaccinated in Nepal. This cross-sectional online survey was conducted among 1196 residents of Nepal in August 2021; most of the participants of this online survey were young adults (18–47 years) with university-level education. A total of 64.5% (771/1196) of the participants perceived COVID-19 vaccines to be safe and risk-free, while 68.6% (820/1196) agreed that vaccination would be efficient in the fight against this pandemic. Most of the participants (841/1196, 70.3%) disagreed that people are getting COVID-19 vaccines easily in Nepal, while they agree with the prioritization of older adults and healthcare workers for vaccination. A total of 61.1% (731/1196) of the participants had received at least one dose of the vaccine. Among the unvaccinated, 93.3% (434/465) were willing to get vaccinated when their turn came. The higher confidence of younger adults in vaccines and the vaccination process is encouraging, as that can help educate others who are hesitant or are not positive towards the idea of receiving vaccines. Dissemination of correct and current information, acquisition of enough doses of vaccines, and equitable distribution of vaccines will be required to achieve successful completion of the COVID-19 vaccination campaign in Nepal.

The gut microbiome plays an important role in the immune system development, maintenance of normal health status, and in disease progression. In this study, we comparatively examined the fecal microbiomes of Amish (rural) and non-Amish (urban) infants and investigated how they could affect the mucosal immune maturation in germ-free piglets that were inoculated with the two types of infant fecal microbiota (IFM). Differences in microbiome diversity and structure were noted between the two types of fecal microbiotas. The fecal microbiota of the non-Amish (urban) infants had a greater relative abundance of Actinobacteria and Bacteroidetes phyla, while that of the Amish (rural) counterparts was dominated by Firmicutes. Amish infants had greater species richness compared with the non-Amish infants' microbiota. The fecal microbiotas of the Amish and the non-Amish infants were successfully transplanted into germ-free piglets, and the diversity and structure of the microbiota in the transplanted piglets remained similar at phylum level but not at the genus level. Principal coordinates analysis (PCoA) based on Weighted-UniFrac distance revealed distinct microbiota structure in the intestines of the transplanted piglets. Shotgun metagenomic analysis also revealed clear differences in functional diversity of fecal microbiome between Amish and non-Amish donors as well as microbiota transplanted piglets. Specific functional features were enriched in either of the microbiota transplanted piglet groups directly corresponding to the predominance of certain bacterial populations in their gut environment. Some of the colonized bacterial genera were correlated with the frequency of important lymphoid and myeloid immune cells in the ileal submucosa and mesenteric lymph nodes (MLN), both important for mucosal immune maturation. Overall, this study demonstrated that transplantation of diverse IFM into germ-free piglets largely recapitulates the differences in gut microbiota structure between rural (Amish) and urban (non-Amish) infants. Thus, fecal microbiota transplantation to germ-free piglets could be a useful large animal model system for elucidating the impact of gut microbiota on the mucosal immune system development. Future studies can focus on determining the additional advantages of the pig model over the rodent model.

The livestock and poultry sectors are an integral part of Nepalese economy and lifestyle. Livestock and poultry populations have continuously been increasing in the last decade in Nepal and are likely to follow that trend as the interests in this field is growing. Infectious diseases such as Foot and Mouth Disease (FMD), Peste des Petits Ruminants (PPR), hemorrhagic septicemia (HS), black quarter (BQ), swine fever, avian influenza, and Newcastle disease (ND) constitute one of the major health challenges to the Nepalese livestock and poultry industry. Vaccinations are an efficient means of preventing the occurrence and spread of several diseases in animals and birds. Considering this fact, the government of Nepal began the production of veterinary vaccines in the 1960s. Nepal is self-reliant in producing several vaccines for cattle and buffaloes, sheep and goats, pigs, and poultry. Despite these efforts, the demand for vaccines is not met, especially in the commercial poultry sector, as Nepal spends billions of rupees in vaccine imports each year. There is a need of strengthening laboratory facilities for the isolation and characterization of field strains of pathogens and capacity building for the production of different types of vaccines using the latest technologies to be self-reliant in veterinary vaccine production in the future in Nepal.

Intranasal vaccination elicits secretory IgA (SIgA) antibodies in the airways, which is required for cross-protection against influenza. To enhance the breadth of immunity induced by a killed swine influenza virus antigen (KAg) or conserved T cell and B cell peptides, we adsorbed the antigens together with the TLR3 agonist poly(I:C) electrostatically onto cationic alpha-D-glucan nanoparticles (Nano-11) resulting in Nano-11-KAg-poly(I:C) and Nano-11-peptides-poly(I:C) vaccines. In vitro, increased TNF-α and IL-1ß cytokine mRNA expression was observed in Nano-11-KAg-poly(I:C)-treated porcine monocyte-derived dendritic cells. Nano-11-KAg-poly(I:C), but not Nano-11-peptides-poly(I:C), delivered intranasally in pigs induced high levels of cross-reactive virus-specific SIgA antibodies secretion in the nasal passage and lungs compared to a multivalent commercial influenza virus vaccine administered intramuscularly. The commercial and Nano-11-KAg-poly(I:C) vaccinations increased the frequency of IFNγ secreting T cells. The poly(I:C) adjuvanted Nano-11-based vaccines increased various cytokine mRNA expressions in lymph nodes compared to the commercial vaccine. In addition, Nano-11-KAg-poly(I:C) vaccine elicited high levels of virus neutralizing antibodies in bronchoalveolar lavage fluid. Microscopic lung lesions and challenge virus load were partially reduced in poly(I:C) adjuvanted Nano-11 and commercial influenza vaccinates. In conclusion, compared to our earlier study with Nano-11-KAg vaccine, addition of poly(I:C) to the formulation improved cross-protective antibody and cytokine response.

Annually, swine influenza A virus (SwIAV) causes severe economic loss to swine industry. Currently used inactivated SwIAV vaccines administered by intramuscular injection provide homologous protection, but limited heterologous protection against constantly evolving field viruses, attributable to the induction of inadequate levels of mucosal IgA and cellular immune responses in the respiratory tract. A novel vaccine delivery platform using mucoadhesive chitosan nanoparticles (CNPs) administered through intranasal (IN) route has the potential to elicit strong mucosal and systemic immune responses in pigs. In this study, we evaluated the immune responses and cross-protective efficacy of IN chitosan encapsulated inactivated SwIAV vaccine in pigs. Killed SwIAV H1N2 (δ-lineage) antigens (KAg) were encapsulated in chitosan polymer-based nanoparticles (CNPs-KAg). The candidate vaccine was administered twice IN as mist to nursery pigs. Vaccinates and controls were then challenged with a zoonotic and virulent heterologous SwIAV H1N1 (γ-lineage). Pigs vaccinated with CNPs-KAg exhibited an enhanced IgG serum antibody and mucosal secretory IgA antibody responses in nasal swabs, bronchoalveolar lavage (BAL) fluids, and lung lysates that were reactive against homologous (H1N2), heterologous (H1N1), and heterosubtypic (H3N2) influenza A virus strains. Prior to challenge, an increased frequency of cytotoxic T lymphocytes, antigen-specific lymphocyte proliferation, and recall IFN-γ secretion by restimulated peripheral blood mononuclear cells in CNPs-KAg compared to control KAg vaccinates were observed. In CNPs-KAg vaccinated pigs challenged with heterologous virus reduced severity of macroscopic and microscopic influenza-associated pulmonary lesions were observed. Importantly, the infectious SwIAV titers in nasal swabs [days post-challenge (DPC) 4] and BAL fluid (DPC 6) were significantly (p

Japanese encephalitis (JE) is a mosquito borne zoonotic disease caused by JE virus (JEV). JE has been endemic in Terai region, the lowland plains of Nepal bordering India, since 1978. However, in recent years cases of JE has been continuously reported from high altitude zones of hills and mountains. Irrigated rice farming system, expanded pig husbandry practices, inadequate vaccine coverage, low level of public awareness and climate change favoring mosquito breeding in higher altitudes might be the probable risk factors for emergence and re-emergence of JE in Nepal. Repeated outbreak in endemic areas and geographical expansion to newer areas have created huge challenge for JE prevention and control. At present, JE is one of the major public health concern of Nepal. Expanding vaccine coverage, improving agricultural practices, generating public awareness, supporting for use of mosquito avoiding practices and regional collaboration at border against JE can be helpful in getting better control over it in future.

The current inactivated influenza vaccines provide satisfactory protection against homologous viruses but limited cross-protection against antigenically divergent strains. Consequently, there is a need to develop more broadly protective vaccines. The highly conserved extracellular domain of the matrix protein 2 (M2e) has shown promising results as one of the components of a universal influenza vaccine in different animal models. As an approach to overcome the limited, strain specific, protective efficacy of inactivated influenza vaccine (IIV), a combination of recombinant M2e expressed on the surface of norovirus P particle (M2eP) and IIV was tested in chickens. Co-immunization of birds with both vaccines did not affect the production of M2e-specific IgG antibodies compared to the group vaccinated with M2eP alone. However, the co-immunized birds developed significantly higher pre-challenge hemagglutination inhibition antibody titers against the homologous IIV antigen and heterologous challenge virus. These combined vaccine groups also had cross reactive antibody responses against different viruses (H5, H6, and H7 subtypes) compared to the IIV alone vaccinated group. Upon intranasal challenge with homologous and heterologous viruses, the combined vaccine groups showed greater reduction in viral shedding in tracheal swabs compared to those groups receiving IIV alone. Moreover, M2eP antisera from vaccinated birds were able to bind to the native M2 expressed on the surface of whole virus particles and infected cells, and inhibit virus replication in vitro. Our results support the potential benefit of supplementing IIV with M2eP, to expand the vaccine cross protective efficacy.

Pigs are believed to be one of the important sources of emerging human and swine influenza viruses (SwIV). Influenza virus conserved peptides have the potential to elicit cross-protective immune response, but without the help of potent adjuvant and delivery system they are poorly immunogenic. Biodegradable polylactic-co-glycolic acid (PLGA) nanoparticle (PLGA-NP) based vaccine delivery system enhances cross-presentation of antigens by the professional antigen presenting cells. In this study, Norovirus P particle containing SwIV M2e (extracellular domain of the matrix protein 2) chimera and highly conserved two each of H1N1 peptides of pandemic 2009 and classical human influenza viruses were entrapped in PLGA-NPs. Influenza antibody-free pigs were vaccinated with PLGA-NPs peptides cocktail vaccine twice with or without an adjuvant, Mycobacterium vaccae whole cell lysate, intranasally as mist. Vaccinated pigs were challenged with a virulent heterologous zoonotic SwIV H1N1, and one week later euthanized and the lung samples were analyzed for the specific immune response and viral load. Clinically, pigs vaccinated with PLGA-NP peptides vaccine had no fever and flu symptoms, and the replicating challenged SwIV was undetectable in the bronchoalveolar lavage fluid. Immunologically, PLGA-NP peptides vaccination (without adjuvant) significantly increased the frequency of antigen-specific IFNγ secreting CD4 and CD8 T cells response in the lung lymphocytes, despite not boosting the antibody response both at pre- and post-challenge. In summary, our data indicated that nanoparticle-mediated delivery of conserved H1N1 influenza peptides induced the virus specific T cell response in the lungs and reduced the challenged heterologous virus load in the airways of pigs.

Japanese encephalitis (JE) is a mosquito-borne zoonotic disease that has pigs as the major amplifying hosts. It is the most important cause of viral encephalitis in people in Nepal and is spreading in its geographic distribution in that country. Pig farming is increasing in Nepal due to reducing cultural biases against pigs and government programs to support pig farming for poverty alleviation. Major strategies for JE prevention and control include education, vector control, and immunization of people and pigs. This study used a survey of 400 pig farmers in 4 areas of Nepal with different JE and pig farming histories to explore regional variations in farmer awareness and actions towards JE, the association of awareness and actions with farm and farmer variables, and the implications of these associations for public health education. Exposure to JE risk factors was common across pig farms and pig farming districts but there were significant district level differences in knowledge and practices related to on-farm JE risk reduction. Social factors such as literacy, gender, and cultural practices were associated with farmer attitudes, knowledge and practices for JE control. JE vaccine uptake was almost non-existent and mosquito control steps were inconsistently applied across all 4 districts. Income was not a determining factor of the differences, but all farmers were very poor. The low uptake of vaccine and lack of infrastructure or financial capacity to house pigs indoors or away from people suggest that farmer personal protection should be a priority target for education in Nepal. This study re-enforces the need to attack root causes of people's personal disease prevention behaviours and take into account local variation in needs and capacities when designing health or agriculture education programs.