Animal Biowarfare Research: Historical Perspective and Potential Future Attacks

A biological attack on livestock or poultry could result in the loss of valuable animals, costs related to the containment of outbreaks and the disposal of carcasses, lost trade and other economic effects involving suppliers, transporters, distributors and restaurants; however, it is not possible to secure all livestock, and livestock are much less well guarded than human targets. Thus, the vulnerability of the livestock industry to the introduction of biological agents varies for the following reasons: (i) the majority of lethal and contagious biological agents are environmentally resilient, endemic in foreign countries and harmless to humans, making it easier for terrorists to acquire, handle and deploy these pathogens, (ii) with animals concentrated in fewer production facilities and frequently transported between these facilities, a single pathogen introduction could cause widespread infection and (iii) the extent of human travel around the globe makes it difficult to exclude exotic animal diseases as possible biological agents. Historically, many governments have developed and planned to use biological agents for direct attacks on livestock or poultry. In the past, developed nations have actively developed biological weapons to target animals. The potential spectrum of bioterrorism ranges from isolated acts against individuals by individuals to tactical and strategic military attacks and state‐sponsored international terrorism intended to cause mass casualties in animals, humans or both. This review provides an overview of the past development and use of biological weapons and describes potential future attacks.     

Exogenous DNA Uptake of Boar Spermatozoa by a Magnetic Nanoparticle Vector System

The sperm‐mediated gene transfer method is applicable to transgenesis in many species that use spermatozoa for reproduction recently, which has been shown various results. In the current study, we show that transgenic porcine embryos can be efficiently produced by employing a simple transfection method that uses magnetic nanoparticles (MNPs). The complexes formed between plasmid DNA and MNPs were bounded on ejaculated boar spermatozoa at a higher efficiency compared to methods using DNA alone or lipofection. Using confocal microscopy, rhodamine fluorophore‐labelled MNPs were detected on external surfaces of the spermatozoa membrane, which were bounded on zona pellucida of in vitro maturated oocyte during in vitro fertilization. Electron microscopy revealed that clusters of MNPs were detected in inside of plasma membrane and nucleus of the spermatozoa head. Additionally, we found that magnetofected boar spermatozoa could be fertilized with oocytes in vitro and that the resulting gene of green fluorescent protein was detected in fertilized eggs by genomic PCR analysis. Taken together, these results suggest that MNPs can be used to efficiently introduce a transgene into embryo via spermatozoa.     

Cytokine response in Balb/c mice infected with Francisella tularensis LVS and the Pohang isolate

We investigated the immune response induced by the Francisella (F.) tularensis live vaccine strain (LVS) and the Pohang isolate. After the Balb/c mice were infected intradermally (i.d) with 2 × 10⁴ cfu of F. tularensis LVS and Pohang, respectively, their blood and organs were collected at different times; 0, 3, 6, 24, 72, 96, 120 and 168 h after infection. Using these samples, RT-PCR and ELISA analysis were carried out for the comparative study of the cytokines, including TNF-α, INF-γ, IL-2, IL-4, IL-10 and IL-12. In the Pohang-infected mice at 120 h, the liver showed a 53 times higher level of TNF-α and a 42 times higher level of IFN-γ than the respective levels at the early time points after infection. The levels of TNF-α and IFN-γ induced by LVS were 5 times lower than those induced by the Pohang isolate. Also, the organs from the Pohang-infected mice showed higher levels of TNF-α, IFN-γ, IL-10 and IL-12 than the levels in the LVS-infected mice. The blood from the Pohang-infected mice at 120 h revealed about a 40 times increased level of IFN-γ, and IL-10 was also increased by 4 times at 96 h compared to an early infection time point, while IL-4 was not induced during the whole infection period. These results suggest that F. tularensis may induce a Th1-mediated immune response to in vivo infection and the Pohang isolate has a higher capacity than the LVS to induce an acute immune response in Blab/c mice.     

Molecular analysis of endogenous avian leukosis/sarcoma virus genomes in Korean chicken embryos

Since the status of endogenous avian leucosis/sarcoma virus (ALSV) infections in Korean broiler chickens is unclear, this study examined embryonated eggs obtained from broiler farms and Korean native chicken breeds in Korea using PCR with the primer sets specific for endogenous ALSVs. The PCR assays detected the genomes of EAV, ev, ev/J and ART-CH belonging to the endogenous ALSV from all embryos tested. Phylogenetically, the Korean EAV genomes were more closely related to the prototype EAV-0 than to the other prototype, E51. The Korean ART-CH elements clustered together but were distinct from the prototype ART-CH clones, 5 and 14. Although there was comparatively little divergence in the nucleotide and amino acid sequences of the Korean ev and ev/J genomes compared with the other known ev and ev/J genomes, the Korean genomes had phylogenetically distinct branches. From these results, endogenous genomes are quite prevalent in Korean broiler chickens. In addition, the endogenous genomes circulating in Korean broiler chickens are genetically different from the other known endogenous genomes. These results are expected to provide useful information for the control and establishment of a surveillance system for endogenous ALSVs in Korea.     

Flow cytometric evaluation of disseminated intravascular coagulation in a canine endotoxemia model

Sepsis is associated with substantial morbidity and mortality in dogs. Alterations in hemostasis by systemic inflammation play an important role in the pathophysiology of sepsis. To evaluate the functional hemostatic changes in sepsis, we evaluated coagulation profiles and flow cytometric measurement of P-selectin (CD62P) expression on platelets, as well as platelet-leukocyte aggregation from a lipopolysaccharide (LPS)-induced endotoxemia model in dogs (n = 7). A sublethal dose of LPS [1 mg/kg body weight (BW)] induced thrombocytopenia and increased activated partial thromboplastin time (aPTT), prothrombin time (PT), and D-dimer concentrations. Flow cytometry analysis showed a significant increase in P-selectin expression on platelets between 1 and 24 h of a total 48 h of the experiment. In addition, platelet-leukocyte aggregation was significantly increased in the early stage of endotoxemia (at 1 and < 6 h for platelet-monocyte aggregation and at 3 h for platelet-neutrophil aggregation). Our results suggest that CD62P expression on platelets and platelet-leukocyte aggregation, as measured by flow cytometry, can be useful biomarkers of disseminated intravascular coagulation (DIC) in canine sepsis. These functional changes contribute to our understanding of the pathophysiology of hemostasis in endotoxemia.     

In vitro neuronal and osteogenic differentiation of mesenchymal stem cells from human umbilical cord blood

Mesenchymal stem cells (MSCs) have the capabilities for self-renewal and differentiation into cells with the phenotypes of bone, cartilage, neurons and fat cells. These features of MSCs have attracted the attention of investigators for using MSCs for cell-based therapies to treat several human diseases. Because bone marrow-derived cells, which are a main source of MSCs, are not always acceptable due to a significant drop in their cell number and proliferative/differentiation capacity with age, human umbilical cord blood (UCB) cells are good substitutes for BMCs due to the immaturity of newborn cells. Although the isolation of hematopoietic stem cells from UCB has been well established, the isolation and characterization of MSCs from UCB still need to be established and evaluated. In this study, we isolated and characterized MSCs. UCB-derived mononuclear cells, which gave rise to adherent cells, exhibited either an osteoclast or a mesenchymal-like phenotype. The attached cells with mesenchymal phenotypes displayed fibroblast-like morphologies, and they expressed mesenchym-related antigens (SH2 and vimentin) and periodic acid Schiff activity. Also, UCB-derived MSCs were able to transdifferentiate into bone and 2 types of neuronal cells, in vitro. Therefore, it is suggested that the MSCs from UCB might be a good alternative to bone marrow cells for transplantation or cell therapy.