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Domestic rabbits (Oryctolagus cuniculus) are the fourth most common species admitted to the British Columbia Society for the Prevention of Cruelty to Animals (BC SPCA) shelter system. However, shelter data analysis has largely focused on cats and dogs and little is known about the population dynamics of rabbits in shelters. We analyzed five years of rabbit records (n = 1567) at the BC SPCA to identify trends in intake and predictors of length of stay (LOS) of rabbits. The majority of rabbits were surrendered by their owners (40.2%), with most rabbits being surrendered for human-related reasons (96.9%). Overall, rabbit intakes decreased over the study period. When analyzing by month of intake, rabbit intakes were found to be the highest in May. Most rabbits in our data were adults (46.7%), non-brachycephalic (66.7%), erect-eared (82.5%), short-furred (76.2%), and subsequently adopted (80.3%). The median LOS of rabbits was 29 days, highlighting the pressing need to improve their time to adoption. A linear model was constructed to identify predictors of LOS of adopted rabbits (n = 1203) and revealed that intake year, intake month, source of intake, age, cephalic type, and breed size significantly predicted time to adoption for rabbits (F(37, 1165) = 7.95, p < 2.2e-16, adjusted R2 = 0.18). These findings help characterize shelter population dynamics for rabbits, shed light on the challenges associated with unwanted rabbits, and offer a foundation for animal shelters to design programs and marketing strategies tailored to reduce LOS of rabbits with particular characteristics. Shelter rabbits represent an understudied population and our study highlights the importance of further research in companion rabbits. [ABSTRACT FROM AUTHOR]

Candidate gene is a powerful approach to study gene-trait association and offers valuable information for genetic improvement using marker-assisted selection. The current work aimed to study the polymorphisms of four single nucleotide polymorphism (SNPs) at located growth hormone (GH), insulin-like growth factor-II (IGF-II), fat mass and obesity-associated (FTO), and insulin receptor substrate-1 (IRS-1) genes, and their association with the carcass, and meat quality traits in rabbits. The SNPs were genotyped using RFLP-PCR in New Zealand White and local Baladi rabbits. The results revealed that the heterozygous genotype was the most frequent in all cases, except for the FTO SNP in LB rabbits. There was a significant effect for GH genotypes on meat lightness after slaughter and hind-part weight. While, IGF-II mutation significantly affected slaughter, hot carcass, commercial carcass, and hind-part weights. The FTO SNP was associated with cooking loss and intramuscular fat weight, and the IRS-1 SNP was significantly associated with drip loss and intramuscular fat. Specific-breed effects were obtained for IGF-II SNP on cooking loss, and for the intramuscular fat. Although the results suggested that these mutations are useful candidate genes for selection, more research for detecting more variants associated with carcass and meat quality traits in rabbits are recommended. [ABSTRACT FROM AUTHOR]

Chlamydia psittaci is a primary zoonotic pathogen with a broad host range causing severe respiratory and reproductive system infection in animals and humans. To reduce the global burden of C. psittaci-associated diseases on animal welfare and health and to control the pathogen spread in husbandry, effective vaccines based on promising vaccine candidate(s) are required. Recently, the caprine C. psittaci AMK-16 strain (AMK-16) demonstrated a high level of protection (up to 80–100%) in outbred mice and pregnant rabbits immunized with these formaldehyde-inactivated bacteria against experimental chlamydial wild-type infection. This study investigated the molecular characteristics of AMK-16 by whole-genome sequencing followed by molecular typing, phylogenetic analysis and detection of main immunodominant protein(s) eliciting the immune response in mouse model. Similarly to other C. psittaci, AMK-16 harbored an extrachromosomal plasmid. The whole-genome phylogenetic analysis proved that AMK-16 strain belonging to ST28 clustered with only C. psittaci but not with Chlamydia abortus strains. However, AMK-16 possessed the insert which resulted from the recombination event as the additional single chromosome region of a 23,100 bp size with higher homology to C. abortus (98.38–99.94%) rather than to C. psittaci (92.06–92.55%). At least six of 16 CDSs were absent in AMK-16 plasticity zone and 41 CDSs in other loci compared with the reference C. psittaci 6BC strain. Two SNPs identified in the AMK-16 ompA sequence resulted in MOMP polymorphism followed by the formation of a novel genotype/subtype including three other C. psittaci strains else. AMK-16 MOMP provided marked specific cellular and humoral immune response in 100% of mice immunized with the inactivated AMK-16 bacteria. Both DnaK and GrpE encoded by the recombination region genes were less immunoreactive, inducing only a negligible T-cell murine immune response, while homologous antibodies could be detected in 50% and 30% of immunized mice, respectively. Thus, AMK-16 could be a promising vaccine candidate for the development of a killed whole cell vaccine against chlamydiosis in livestock. [ABSTRACT FROM AUTHOR]

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic cause of adult T-cell leukemia/lymphoma (ATL) and encodes a viral oncoprotein (Hbz) that is consistently expressed in asymptomatic carriers and ATL patients, suggesting its importance in the development and maintenance of HTLV-1 leukemic cells. Our previous work found Hbz protein is dispensable for virus-mediated T-cell immortalization but enhances viral persistence. We and others have also shown that hbz mRNA promotes T-cell proliferation. In our current studies, we evaluated the role of hbz mRNA on HTLV-1-mediated immortalization in vitro as well as in vivo persistence and disease development. We generated mutant proviral clones to examine the individual contributions of hbz mRNA, hbz mRNA secondary structure (stem-loop), and Hbz protein. Wild-type (WT) and all mutant viruses produced virions and immortalized T-cells in vitro. Viral persistence and disease development were also evaluated in vivo by infection of a rabbit model and humanized immune system (HIS) mice, respectively. Proviral load and sense and antisense viral gene expression were significantly lower in rabbits infected with mutant viruses lacking Hbz protein compared to WT or virus with an altered hbz mRNA stem-loop (M3 mutant). HIS mice infected with Hbz protein-deficient viruses showed significantly increased survival times compared to animals infected with WT or M3 mutant virus. Altered hbz mRNA secondary structure, or loss of hbz mRNA or protein, has no significant effect on T-cell immortalization induced by HTLV-1 in vitro; however, the Hbz protein plays a critical role in establishing viral persistence and leukemogenesis in vivo. Author summary: Human T-cell leukemia virus type 1 (HTLV-1) is the cause of an aggressive malignancy called adult T-cell leukemia/lymphoma. HTLV-1 encodes a viral oncoprotein (Hbz) that is consistently expressed in patients, suggesting its importance in the development and maintenance of leukemic cells. Remarkably, the Hbz gene is bimodal, with functions at both the mRNA and protein levels. In our study, we generated mutant proviral clones to examine the individual contributions of hbz mRNA, hbz mRNA secondary structure (stem-loop), and Hbz protein. Viral persistence and disease development were evaluated in vivo by infection of a rabbit model and humanized immune system (HIS) mice, respectively. Proviral load and viral gene expression were significantly lower in rabbits infected with mutant viruses lacking Hbz protein compared to WT or virus with an altered hbz mRNA stem-loop (M3 mutant). HIS mice infected with Hbz protein-deficient viruses showed significantly increased survival times compared to animals infected with WT or M3 mutant virus. Our data demonstrate that, despite the structure-dependent function that hbz mRNA exerts to stimulate proliferation of cell lines, alteration of a single 5' stem-loop structure of the RNA alone has no effect on the ability of HTLV-1 to persist and induce leukemogenesis in vivo. [ABSTRACT FROM AUTHOR]

Acid sphingomyelinase (ASM) promotes atherogenesis and acute cardiovascular events. We previously demonstrated ASM inhibitor desipramine attenuated oxidized-LDL-induced macrophage apoptosis in vitro. Here, we aim to determine whether ASM-mediated apoptosis in plaque improves stability in vivo. In this study, rabbits with abdominal aorta balloon injury and a 12-week high-cholesterol diet (HCD) were used to simulate an atherosclerotic plaque model. Atherosclerotic rabbits received oral administration of saline (Control group), atorvastatin (Ator group), or desipramine (DES group). ASM activity and ceramide level were measured by ultra-performance liquid chromatography (UPLC). Plaque morphology was assessed by histochemistry and immunohistochemistry. Apoptosis was evaluated by SPECT/CT imaging of 99mTc-duramycin uptake and TUNEL. We found that increasing ASM activity and ceramide level in atherosclerotic rabbits was abated by additional atorvastatin and desipramine treatment. Meanwhile, the DES and Ator groups were similar in plaque stability, with smaller plaque size, areas of macrophages, higher smooth muscle cell content, and decreased apoptosis and matrix metalloproteinase (MMP) activities relative to the Control group. 99mTc-duramycin uptake of rabbit aorta was significantly higher in Control than in the Normal group, while it was reduced by desipramine and atorvastatin administration. Moreover, the uptake of 99mTc-duramycin positively correlated with apoptotic cell number, macrophage infiltration, and plaque instability. The present study demonstrated that desipramine exerted plaque-stabilizing effects partially by suppressing apoptosis and MMP activity in a rabbit model. And 99mTc-duramycin SPECT/CT imaging allowed noninvasively monitoring of atherosclerotic disease and evaluation of anti-atherosclerotic therapy. [ABSTRACT FROM AUTHOR]

Background: Liver tumor remains an important cause of cancer-related death. Nanosecond pulsed electric fields (nsPEFs) are advantageous in the treatment of melanoma and pancreatic cancer, but their therapeutic application on liver tumors need to be further studied. Methods: Hep3B cells were treated with nsPEFs. The biological behaviors of cells were detected by Cell Counting Kit-8, 5-ethynyl-20-deoxyuridine, and transmission electron microscopy (TEM) assays. In vivo, rabbit VX2 liver tumor models were ablated by ultrasound-guided nsPEFs and radiofrequency ablation (RFA). Contrast-enhanced ultrasound (CEUS) was used to evaluate the ablation effect. HE staining and Masson staining were used to evaluate the tissue morphology after ablation. Immunohistochemistry was performed to determine the expression of Ki67, proliferating cell nuclear antigen, and α-smooth muscle actin at different time points after ablation. Results: The cell viability of Hep3B cells was continuously lower than that of the control group within 3 days after pulse treatment. The proliferation of Hep3B cells was significantly affected by nsPEFs. TEM showed that Hep3B cells underwent significant morphological changes after pulse treatment. In vivo, CEUS imaging showed that nsPEFs could completely ablate model rabbit VX2 liver tumors. After nsPEFs ablation, the area of tumor fibrosis and the expression of Ki67, proliferating cell nuclear antigen, and α-smooth muscle actin were decreased. However, after RFA, rabbit VX2 liver tumor tissue showed complete necrosis, but the expression of PCNA and α-smooth muscle actin did not decrease compared to the tumor group. Conclusions: nsPEFs can induce Hep3B cells apoptosis and ablate rabbit VX2 liver tumors in a non-thermal manner versus RFA. The ultrasound contrast agent can monitor immediate effect of nsPEF ablation. This study provides a basis for the clinical study of nsPEFs ablation of liver cancer. [ABSTRACT FROM AUTHOR]

Background: The rupture of vulnerable atherosclerotic plaque is the main cause of acute ischemic vascular events, and is characterized by pathological degradation of matrix collagen in the fibrous cap. In a previous study, we reported that 5-aminolevulinic acid-mediated sonodynamic therapy suppressed collagen degradation in rabbit plaque. However, the underlying molecular mechanism has yet to be fully elucidated. Methods: We applied sinoporphyrin sodium-mediated sonodynamic therapy (DVDMS-SDT) to balloon-denuded rabbit and apolipoprotein E-deficient (ApoE−/−) mouse models to observe collagen content in plaque. Cultured human THP-1 and mouse peritoneal macrophage-derived foam cells were used for in vitro mechanistic studies. Results: We observed that DVDMS-SDT decreased plaque area and increased the percentages of collagen and smooth muscle cells and reduced the percentage of macrophages in rabbit and ApoE-/- mouse advanced plaques. In vitro, DVDMS-SDT modulated the caspase 3-pigment epithelium-derived factor/hypoxia-inducible factor-1α (PEDF/HIF-1α)-matrix metalloprotease-2/9 (MMP-2/MMP-9) signaling in macrophage foam cells. Conclusions: Our findings show that DVDMS-SDT effectively inhibits matrix collagen degradation in advanced atherosclerotic plaque by modulating caspase 3-PEDF/HIF-1α-MMP-2/MMP-9 signaling in macrophage foam cells and therefore represents a suitable and promising clinical regimen to stabilize vulnerable plaques. [ABSTRACT FROM AUTHOR]

Rabbit Haemorrhagic Disease Virus (RHDV), which is a calicivirus, is used as a biocontrol agent to suppress European wild rabbit populations in Australia. The transmission of RHDV can be influenced by social interactions of rabbits; however, there is a paucity of this knowledge about juvenile rabbits and the roles they may play in the transmission of RHDV. We aimed to quantify the social interactions of juvenile (< 900 g) and adult (> 1200 g) rabbits in a locally abundant population in the Central Tablelands of New South Wales, Australia. Twenty-six juvenile and 16 adult rabbits were fitted with VHF proximity loggers to monitor intra- and inter-group pairings. Use of multiple warrens by these rabbits was investigated using VHF base stations at nine warrens and on foot with a hand-held Yagi antenna. Juvenile rabbits were strongly interconnected with both juveniles and adults within and outside their warren of capture, and almost all juveniles were well-connected to other individuals within their own social group. Inter-group pairings were infrequent and fleeting between adults. Both juvenile and adult rabbits used multiple warrens. However, visits to warrens outside their warren of capture, particularly those within 50 m, were more common and longer in duration in juveniles than in adults. The high connectivity of juveniles within and between warrens in close proximity increases potential pathogen exchange between warrens. Therefore, juvenile rabbits could be of greater importance in lagovirus transmission than adult rabbits. The strength of juvenile rabbit inter- and intra-group pairings, and their tendency to use multiple warrens, highlight their potential to act as 'superspreaders' of both infection and immunity for lagoviruses and other pathogens with similar lifecycles. Confirmation of this potential is required through examination of disease progress and rabbit age-related immune responses during outbreaks. [ABSTRACT FROM AUTHOR]

Kyasanur Forest disease virus (KFDV) and the closely related Alkhurma hemorrhagic disease virus (AHFV) are emerging flaviviruses that cause severe viral hemorrhagic fevers in humans. Increasing geographical expansion and case numbers, particularly of KFDV in southwest India, class these viruses as a public health threat. Viral pathogenesis is not well understood and additional vaccines and antivirals are needed to effectively counter the impact of these viruses. However, current animal models of KFDV pathogenesis do not accurately reproduce viral tissue tropism or clinical outcomes observed in humans. Here, we show that pigtailed macaques (Macaca nemestrina) infected with KFDV or AHFV develop viremia that peaks 2 to 4 days following inoculation. Over the course of infection, animals developed lymphocytopenia, thrombocytopenia, and elevated liver enzymes. Infected animals exhibited hallmark signs of human disease characterized by a flushed appearance, piloerection, dehydration, loss of appetite, weakness, and hemorrhagic signs including epistaxis. Virus was commonly present in the gastrointestinal tract, consistent with human disease caused by KFDV and AHFV where gastrointestinal symptoms (hemorrhage, vomiting, diarrhea) are common. Importantly, RNAseq of whole blood revealed that KFDV downregulated gene expression of key clotting factors that was not observed during AHFV infection, consistent with increased severity of KFDV disease observed in this model. This work characterizes a nonhuman primate model for KFDV and AHFV that closely resembles human disease for further utilization in understanding host immunity and development of antiviral countermeasures. Author summary: Kyasanur Forest disease virus (KFDV) and Alkhurma hemorrhagic disease virus (AHFV) are tick-borne flaviviruses that cause viral hemorrhagic fevers in India and the Arabian Peninsula, respectively. Bonnet macaques and black-faced langurs are susceptible to KFDV infection, but these animals do not experience hemorrhagic signs as seen in human cases with KFDV. This work characterizes for the first time experimental infection of KFDV and AHFV in pigtailed macaques (PTMs). Infected PTMs can develop moderate to severe disease that models multiple features of human disease, including some hemorrhagic signs. Together these data describe the PTM model for KFDV and AHFV as a valuable tool for future work to study viral pathogenesis and for assessing the efficacy of vaccines and antivirals. [ABSTRACT FROM AUTHOR]

Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development. Author summary: Syphilis is still an endemic disease in many low- and middle-income countries, and it has been resurgent in high-income nations for almost two decades. In endemic areas, syphilis causes significant morbidity and mortality, particularly when its causative agent, the spirochete Treponema pallidum subsp. pallidum (T. pallidum) is transmitted to the fetus during pregnancy. A better understanding of T. pallidum biology and syphilis pathogenesis would help devise better control strategies for this infection. One of the limitations associated with working with T. pallidum was our inability to genetically alter this pathogen to evaluate the function of genes encoding virulence factors or create attenuated strains that could be informative for vaccine development when studied using the rabbit model of the disease. Here, we report a transformation protocol that allowed us to replace a specific region of the T. pallidum genome containing a pseudogene (i.e., a non-functional gene) with a stably integrated kanamycin resistance gene. To our knowledge, this is the first-ever report of a method to achieve a genetically modified T. pallidum strain. [ABSTRACT FROM AUTHOR]

The rabbit retinal vein occlusion (RVO) model is an experimental system that mimics retinal ischemic diseases in humans. The rabbit RVO model is widely used to assess the therapeutic efficacy of various experimental surgical procedures. In the present study, we measured temporal retinal expression of Vegfa, which is known as an ischemic response gene, in rabbit RVO. This analysis revealed that the retinal Vegfa transcriptional response began 7 days after generation of RVO, rather than immediately after induction of ischemia. Next, in order to analyze ischemia-induced changes in gene expression profiles, we performed microarray analysis of day 7 RVO retina versus control retina. The angiogenic regulators Dcn and Mmp1 and pro-inflammatory factors Mmp12 and Cxcl13 were significantly upregulated in RVO retinas. Further, we suggest that epigenetic regulation via the REST/cofactor-complex could contribute to RVO pathology. Among human homologous genes in rabbits, genes associated with hypoxia, angiogenesis, and inflammation were significantly upregulated in RVO retinas. Components of the Tumor necrosis factor-alpha (TNFα) and Nuclear factor-kappa B (NF-κB) pathways, which play regulatory roles in angiogenesis and inflammation, were significantly upregulated in RVO, and the expression levels of downstream factors, such as the transcription factor AP-1 and chemokines, were increased. Further, connectivity map analyses suggested that inhibitors of the NF-κB pathway are potential therapeutic agents for retinal ischemic disease. The present study revealed new insights into the pathology of retinal ischemia using the rabbit RVO model, which accurately recapitulates human disease. [ABSTRACT FROM AUTHOR]

Norovirus is the major cause of epidemic nonbacterial gastroenteritis worldwide. Lack of structural information on infection and replication mechanisms hampers the development of effective vaccines and remedies. Here, using cryo-electron microscopy, we show that the capsid structure of murine noroviruses changes in response to aqueous conditions. By twisting the flexible hinge connecting two domains, the protruding (P) domain reversibly rises off the shell (S) domain in solutions of higher pH, but rests on the S domain in solutions of lower pH. Metal ions help to stabilize the resting conformation in this process. Furthermore, in the resting conformation, the cellular receptor CD300lf is readily accessible, and thus infection efficiency is significantly enhanced. Two similar P domain conformations were also found simultaneously in the human norovirus GII.3 capsid, although the mechanism of the conformational change is not yet clear. These results provide new insights into the mechanisms of non-enveloped norovirus transmission that invades host cells, replicates, and sometimes escapes the hosts immune system, through dramatic environmental changes in the gastrointestinal tract. Author summary: The capsid structure of caliciviruses has been reported to be classified into two different types, according to the species and genotype. One is the rising type of P domain conformation as shown in human norovirus GII.10 and rabbit hemorrhagic disease virus (RHDV), where the P domain rises from the S domain surface. The other is the resting type of P domain conformation as shown in human norovirus GI.1, sapovirus and San Miguel sea lion virus (SMSV), where the P domain rests upon the S domain. Here, we demonstrate that the P domain of the murine noroviruses changes reversibly between the rising and resting P domain conformation types in response to aqueous conditions. We also found the similar two P domain conformations in human norovirus GII.3 VLPs at the same time. Our findings provide new insights into the mechanisms of viral infection of caliciviruses. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) is a viral pathogen transmitted by the fecal-oral route and is a major cause of waterborne acute hepatitis in many developing countries. In addition to infecting humans, HEV has been identified in swine, wild boars, rabbits and other mammals; with swine and wild boars being main reservoirs for zoonotic transmission of HEV. There are four major HEV genotypes known to infect humans; genotypes 1 (HEV-1) and 2 (HEV-2) are restricted to humans, and genotypes 3 (HEV-3) and 4 (HEV-4) are zoonotic. Herein, three human HEV strains originating in France were sequenced and near full-length genomes were characterized. Phylogenetic analysis showed that two strains were genotype 3 and closely grouped (a 100% bootstrap value) with subtype 3i reference strains. In percent nucleotide identities, these two strains were 94% identical to each other, 90–93% identical to subtype 3i strains, 82–86% identical to other HEV-3, and 77–79% identical to rabbit HEV strains excluding the two divergent strains KJ013414 and KJ013415 (74%); these two strains were less than 77% identical to strains of HEV genotypes 1, 2 and 4. The third strain was found distinct from any known HEV strains in the database, and located between the clusters of HEV-3 and rabbit HEV strains. This unique strain was 74–75% identical to HEV-1, 73% to HEV-2, 81–82% to HEV-3, 77–79% to rabbit HEV again excluding the two divergent strains KJ013414 and KJ013415 (74%), and 74–75% to HEV-4, suggesting a novel unclassified strain associated with HEV-3 and rabbit HEV. SimPlot and BootScan analyses revealed a putative recombination of HEV-3 and rabbit HEV sequences at four breakpoints. Phylogenetic trees of the five fragments of the genome confirmed the presence of two HEV-3 derived and three unclassified sequences. Analyses of the amino acid sequences of the three open reading frames (ORF1-3) encoded proteins of these three novel strains showed that some amino acid residues specific to rabbit HEV strains were found solely in this unclassified strain but not in the two newly identified genotype 3i strains. The results obtained by SimPlots, BootScans, phylogenetic analyses, and amino acid sequence comparisons in this study all together appear to suggest that this novel unclassified strain is likely carrying a mosaic genome derived from HEV-3 and rabbit HEV sequences, and is thus designated as a putative genotype 3/rabbit HEV recombinant. [ABSTRACT FROM AUTHOR]

Introduction: Increasing use of factor Xa (FXa) inhibitors necessitates effective reversal agents to manage bleeding. Andexanet alfa, a novel modified recombinant human FXa, rapidly reverses the anticoagulation effects of direct and indirect FXa inhibitors. Objective: To evaluate the ability of andexanet to reverse anticoagulation in vitro and reduce bleeding in rabbits administered edoxaban. Materials and methods: In vitro studies characterized the interaction of andexanet with edoxaban and its ability to reverse edoxaban-mediated anti-FXa activity. In a rabbit model of surgically induced, acute hemorrhage, animals received edoxaban vehicle+andexanet vehicle (control), edoxaban (1 mg/kg)+andexanet vehicle, edoxaban+andexanet (75 mg, 5-minute infusion, 20 minutes after edoxaban), or edoxaban vehicle+andexanet prior to injury. Results: Andexanet bound edoxaban with high affinity similar to FXa. Andexanet rapidly and dose-dependently reversed the effects of edoxaban on FXa activity and coagulation pharmacodynamic parameters in vitro. In edoxaban-anticoagulated rabbits, andexanet reduced anti-FXa activity by 82% (from 548±87 to 100±41 ng/ml; P<0.0001), mean unbound edoxaban plasma concentration by ~80% (from 100±10 to 21±6 ng/ml; P<0.0001), and blood loss by 80% vs. vehicle (adjusted for control, 2.6 vs. 12.9 g; P = 0.003). The reduction in blood loss correlated with the decrease in anti-FXa activity (r = 0.6993, P<0.0001) and unbound edoxaban (r = 0.5951, P = 0.0035). Conclusion: These data demonstrate that andexanet rapidly reversed the anticoagulant effects of edoxaban, suggesting it could be clinically valuable for the management of acute and surgery-related bleeding. Correlation of blood loss with anti-FXa activity supports the use of anti-FXa activity as a biomarker for assessing anticoagulation reversal in clinical trials. [ABSTRACT FROM AUTHOR]

Encephalitozoon cuniculi is a microsporidian species which can induce subclinical to serious disease in mammals including rabbits, a definitive natural host. The pathophysiology of infection has not been comprehensively elucidated. In this exploratory study, we utilized two mass spectrometry approaches: first, the analysis of the humoral response by profiling the microsporidian antigens as revealed by Western blot screening, and second, implementing the iTRAQ®-labeling protocol to focus on the changes within the host proteome during infection. Seven E. cuniculi proteins were identified at one-dimensional gel regions where specific seropositive reaction was observed by Western blot, including polar tube protein 3, polar tube protein 2, and for the first time reported: heat shock related 70kDa protein, polysaccharide deacetylase domain-containing protein, zinc finger protein, spore wall and anchoring disk complex protein EnP1, and translation elongation factor 1 alpha. In addition, there was a significant increase of nine host proteins in blood samples from E. cuniculi-diseased rabbits in comparison with non-diseased control subjects undergoing various inflammatory processes. This included serum paraoxonase, alpha-1-antiproteinase F precursor and alpha-1-antiproteinase S-1 which have presumptive catalytic activity likely related to infection control, and cystatin fetuin-B-type, an enzyme regulator that has been poorly studied to date. Notably, 11 proteins were found to be statistically increased in rabbits with neurological versus renal clinical presentation of E. cuniculi infection. Overall, this novel analysis based on mass spectrometry has provided new insights on the inflammatory and humoral responses during E. cuniculi infection in rabbits. [ABSTRACT FROM AUTHOR]

Increasing evidence demonstrates that hepatitis E virus (HEV) can be transmitted across species. According to previous reports, swine HEV has two genotypes, genotype 3 and 4, and both can infect humans by the fecal-oral route. Thus, it is crucial for the control of HEV zoonotic transmission to evaluate the dynamics of viral shedding and distribution in different tissues during cross-species infection by HEV. In this study, rabbits were infected with genotype 4 swine HEV by the intraperitoneal route. The results showed that HEV RNA not only shed in the feces but also in the saliva of some rabbits during infection with swine HEV. Viremia appeared late after infection, and anti-HEV IgG was not obvious until the appearance of high viremia levels. After the rabbits were euthanized, a histopathological examination showed that the livers developed overt hepatitis accompanied by an elevation of alanine aminotransferase (ALT) and aspartate transaminase (AST). Furthermore, HEV RNA was detected in various tissues, especially in the salivary glands and tonsils. Subsequently, negative-stranded HEV RNA was practiced in tissues with positive HEV RNA, which demonstrated that HEV replicated in the tissues. Next, we harvested additional tissues from the liver, salivary gland, tonsil, spleen, thymus gland, lymph node and intestine, which are known as replication sites of swine HEV. Additionally, we also observed the HEV antigen distributed in the organs above through immunohistochemical staining. These results demonstrate that rabbits could be used as an animal model for researching cross-species infection of genotype 4 HEV. It is also noteworthy that HEV can shed in the saliva and presents the risk of droplet transmission. These new data provide valuable information for understanding cross-species infection by HEV. [ABSTRACT FROM AUTHOR]

In addition to being an important human pathogen, Staphylococcus aureus is able to cause a variety of infections in numerous other host species. While the S. aureus strains causing infection in several of these hosts have been well characterised, this is not the case for companion rabbits (Oryctolagus cuniculus), where little data are available on S. aureus strains from this host. To address this deficiency we have performed antimicrobial susceptibility testing and genome sequencing on a collection of S. aureus isolates from companion rabbits. The findings show a diverse S. aureus population is able to cause infection in this host, and while antimicrobial resistance was uncommon, the isolates possess a range of known and putative virulence factors consistent with a diverse clinical presentation in companion rabbits including severe abscesses. We additionally show that companion rabbit isolates carry polymorphisms within dltB as described as underlying host-adaption of S. aureus to farmed rabbits. The availability of S. aureus genome sequences from companion rabbits provides an important aid to understanding the pathogenesis of disease in this host and in the clinical management and surveillance of these infections. [ABSTRACT FROM AUTHOR]

Many studies aimed at investigating bone repair have been conducted through animal models in recent years. However, limitations do exist in these models due to varying regeneration potential among different animal species. Even using the same animal, big differences exist in the size of critical size defects (CSD) involving the same region. This study aimed to investigate the standardization of radial bone defect models in rabbits and further establish more reliable CSD data. A total of 40 6-month-old New Zealand white rabbits of clean grade totaling 80 radial bones were prepared for bone defect models, according to the principle of randomization. Five different sizes (1.0, 1.2, 1.4, 1.7 and 2.0 cm) of complete periosteal defects were introduced under anesthesia. At 12 weeks postoperatively, with the gradual increase in defect size, the grades of bone growth were significantly decreased in all 5 groups. X-ray, CT scans and H&E staining of the 1.4, 1.7, and 2.0-cm groups showed lower grades of bone growth than that of the 1.0 and 1.2-cm groups respectively (P < 0.05). Using rabbit radial defect model involving 6-month-old healthy New Zealand white rabbits, this study indicates that in order to be critical sized, defects must be greater than 1.4 cm. [ABSTRACT FROM AUTHOR]

The article deals with mechanism of genetic determinism of pathogenecity in Rabbit hemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV). Topics discussed include the types of RHDV strains, the evolution of the viruses from pre-existing nonpathogenic viruses and species jump, and documentation of RHDV strains in Iberian hares. The origins of European leporids with lagoviruses are also discussed.

Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D) and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel), elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 μg. [ABSTRACT FROM AUTHOR]

Rabbits (Oryctolagus cuniculus) are a popular companion animal, food animal, and animal model of human disease. Abnormal red cell shapes (poikilocytes) have been observed in rabbits, but their significance is unknown. The objective of this study was to investigate the prevalence and type of poikilocytosis in pet rabbits and its association with physiologic factors, clinical disease, and laboratory abnormalities. We retrospectively analyzed blood smears from 482 rabbits presented to the University of California-Davis Veterinary Medical Teaching Hospital from 1990 to 2010. Number and type of poikilocytes per 2000 red blood cells (RBCs) were counted and expressed as a percentage. Acanthocytes (>3% of RBCs) were found in 150/482 (31%) rabbits and echinocytes (>3% of RBCs) were found in 127/482 (27%) of rabbits, both healthy and diseased. Thirty-three of 482 (7%) rabbits had >30% acanthocytes and echinocytes combined. Mild to moderate (>0.5% of RBCs) fragmented red cells (schistocytes, microcytes, keratocytes, spherocytes) were found in 25/403 (6%) diseased and 0/79 (0%) healthy rabbits (P = 0.0240). Fragmentation and acanthocytosis were more severe in rabbits with inflammatory disease and malignant neoplasia compared with healthy rabbits (P<0.01). The % fragmented cells correlated with % polychromasia, RDW, and heterophil, monocyte, globulins, and fibrinogen concentrations (P<0.05). Echinocytosis was significantly associated with renal failure, azotemia, and acid-base/electrolyte abnormalities (P<0.05). Serum cholesterol concentration correlated significantly with % acanthocytes (P<0.0001), % echinocytes (P = 0.0069), and % fragmented cells (P = 0.0109), but correlations were weak (Spearman ρ <0.02). These findings provide important insights into underlying pathophysiologic mechanisms that appear to affect the prevalence and type of naturally-occurring poikilocytosis in rabbits. Our findings support the need to carefully document poikilocytes in research investigations and in clinical diagnosis and to determine their diagnostic and prognostic value. [ABSTRACT FROM AUTHOR]

Infection of the central nervous system is considered a complication of Anthrax and was reported in humans and non-human primates. Previously we have reported that Bacillus anthracis possesses a toxin-independent virulent trait that, like the toxins, is regulated by the major virulence regulator, AtxA, in the presence of pXO2. This toxin-independent lethal trait is exhibited in rabbits and Guinea pigs following significant bacteremia and organ dissemination. Various findings, including meningitis seen in humans and primates, suggested that the CNS is a possible target for this AtxA-mediated activity. In order to penetrate into the brain tissue, the bacteria have to overcome the barriers isolating the CNS from the blood stream. Taking a systematic genetic approach, we compared intracranial (IC) inoculation and IV/SC inoculation for the outcome of the infection in rabbits/GP, respectively. The outstanding difference between the two models is exhibited by the encapsulated strain VollumΔpXO1, which is lethal when injected IC, but asymptomatic when inoculated IV/SC. The findings demonstrate that there is an apparent bottleneck in the ability of mutants to penetrate into the brain. Any mutant carrying either pXO1 or pXO2 will kill the host upon IC injection, but only those carrying AtxA either on pXO1 or in the chromosome in the background of pXO2 can penetrate into the brain following peripheral inoculation. The findings were corroborated by histological examination by H&E staining and immunofluorescence of rabbits' brains following IV and IC inoculations. These findings may have major implications on future research both on B. anthracis pathogenicity and on vaccine development. [ABSTRACT FROM AUTHOR]

For the treatment of chronic ocular diseases such as glaucoma, continuous instillations of eye drops are needed. However, frequent administrations of hypotensive topical formulations can produce adverse ocular surface effects due to the active substance or other components of the formulation, such as preservatives or other excipients. Thus the development of unpreserved formulations that are well tolerated after frequent instillations is an important challenge to improve ophthalmic chronic topical therapies. Furthermore, several components can improve the properties of the formulation in terms of efficacy. In order to achieve the mentioned objectives, we have developed formulations of liposomes (150–200 nm) containing components similar to those in the tear film and loaded with the hypotensive melatonin analog 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT, 100 µM). These formulations were combined with mucoadhesive (sodium hyaluronate or carboxymethylcellulose) or amphiphilic block thermosensitive (poloxamer) polymers to prolong the hypotensive efficacy of the drug. In rabbit eyes, the decrease of intraocular pressure with 5-MCA-NAT-loaded liposomes that were dispersed with 0.2% sodium hyaluronate, 39.1±2.2%, was remarkably higher compared to other liposomes formulated without or with other bioadhesive polymers, and the effect lasted more than 8 hours. According to the results obtained in the present work, these technological strategies could provide an improved modality for delivering therapeutic agents in patients with glaucoma. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) constitutes a significant health burden worldwide, with an estimated approximately 33% of the world’s population exposed to the pathogen. The recent licensed HEV 239 vaccine in China showed excellent protective efficacy against HEV of genotypes 1 and 4 in the general population and pregnant women. Because hepatitis E is a zoonosis, it is also necessary to ascertain whether this vaccine can serve to manage animal sources of human HEV infection. To test the efficacy of the HEV 239 vaccine in protecting animal reservoirs of HEV against HEV infection, twelve specific-pathogen-free (SPF) rabbits were divided randomly into two groups of 6 animals and inoculated intramuscularly with HEV 239 and placebo (PBS). All animals were challenged intravenously with swine HEV of genotype 4 or rabbit HEV seven weeks after the initial immunization. The course of infection was monitored for 10 weeks by serum ALT levels, duration of viremia and fecal virus excretion and HEV antibody responses. All rabbits immunized with HEV 239 developed high titers of anti-HEV and no signs of HEV infection were observed throughout the experiment, while rabbits inoculated with PBS developed viral hepatitis following challenge, with liver enzyme elevations, viremia, and fecal virus shedding. Our data indicated that the HEV 239 vaccine is highly immunogenic for rabbits and that it can completely protect rabbits against homologous and heterologous HEV infections. These findings could facilitate the prevention of food-borne sporadic HEV infection in both developing and industrialized countries. [ABSTRACT FROM AUTHOR]

It is generally accepted that high osmotic pressure (HOP) of lacrimal fluid is the core mechanism causing ocular inflammation and injury. However, the association between HOP and the regulation of cell inflammatory response and apoptotic pathways remains unclear. In the present study, we used HOP to interfere with in vitro cultured rabbit corneal epithelial cells, and found that HOP increased the generation of reactive oxygen species (ROS) in rabbit corneal epithelial cells, and increased ROS in turn induced the activation of JNK inflammatory signaling pathway, which further promoted the expression of pro-inflammatory factor NF-κβ and induced the generation of inflammatory factor IL-1β and TNF-α. In addition, HOP-induced ROS in rabbit corneal epithelial cells regulated the CD95/CD95L-mediated cell apoptotic signaling pathway by activating JNK inflammatory signaling pathway. These findings may serve as new theoretical basis and a new way of thinking about the treatment of ocular diseases, especially dry eye. [ABSTRACT FROM AUTHOR]

Objective: This study aims to investigate the potentially protective effect of neuroglobin (Ngb) gene-modified bone marrow mesenchymal stem cells (BMSCs) on traumatic spinal cord injury (SCI) in rabbits. Methods: A lentiviral vector containing an Ngb gene was constructed and used to deliver Ngb to BMSCs. Ngb gene-modified BMSCs were then injected at the SCI sites 24 hours after SCI. The motor functions of the rabbits were evaluated by the Basso–Beattie–Bresnahan rating scale. Fluorescence microscopy, quantitative real-time PCRs, Western blots, malondialdehyde (MDA) tests, and terminal deoxynucleotidyltransferase-mediated UTP end labeling assays were also performed. Results: Ngb expression in the Ngb-BMSC group increased significantly. A more significant functional improvement was observed in the Ngb-BMSC group compared with those in the other groups. Traumatic SCI seemingly led to an increase in MDA level and number of apoptotic cells, which can be prevented by Ngb-BMSC treatment. Conclusion: This study demonstrates that Ngb gene-modified BMSCs can strengthen the therapeutic benefits of BMSCs in reducing secondary damage and improving the neurological outcome after traumatic SCI. Therefore, the combined strategy of BMSC transplantation and Ngb gene therapy can be used to treat traumatic SCI. [ABSTRACT FROM AUTHOR]

Background: Cryptosporidium and Giardia are the two important zoonotic pathogens causing diarrhea of humans and animals worldwide. Considering the human cryptosporidiosis outbreak and sporadic cases caused by C. cuniculus, the important public health significance of G. duodenalis and little obtained information regarding rabbit infected with Cryptosporidium and Giardia in China, the aim of this study is to determine the prevalence and molecularly characterize Cryptosporidium and Giardia in rabbits in Heilongjiang Province, China. Methodology/Principal Findings: 378 fecal samples were obtained from rabbits in Heilongjiang Province. Cryptosporidium oocysts and Giardia cysts were detected using Sheather's sugar flotation technique and Lugol's iodine stain method, respectively. The infection rates of Cryptosporidium and Giardia were 2.38% (9/378) and 7.41% (28/378), respectively. Genotyping of Cryptosporidium spp. was done by DNA sequencing of the small subunit rRNA (SSU rRNA) gene and all the nine isolates were identified as Cryptosporidium cuniculus. The nine isolates were further subtyped using the 60-kDa glycoprotein (gp60) gene and two subtypes were detected, including VbA32 (n = 3) and a new subtype VbA21 (n = 6). G. duodenalis genotypes and subtypes were identified by sequence analysis of the triosephosphate isomerase (TPI) gene. The assemblage B (belonging to eight different subtypes B-I to B-VIII) was found in 28 G. duodenalis-positive samples. Conclusions/Significance: The rabbits have been infected with Cryptosporidium and Giardia in Heilongjiang Province. The results show that the rabbits pose a threat to human health in the studied areas. Genotypes and subgenotypes of C. cuniculus and G. duodenalis in this study might present the endemic genetic characterization of population structure of the two parasites. [ABSTRACT FROM AUTHOR]

Background: Cathelicidins and defensins are endogenous antimicrobial peptides (AMPs) that are downregulated in the mucosal epithelia of the large intestine in shigellosis. Oral treatment of Shigella infected rabbits with sodium butyrate (NaB) reduces clinical severity and counteracts the downregulation of cathelicidin (CAP-18) in the large intestinal epithelia. Aims: To develop novel regimen for treating infectious diseases by inducing innate immunity, we selected sodium 4- phenylbutyrate (PB), a registered drug for a metabolic disorder as a potential therapeutic candidate in a rabbit model of shigellosis. Since acute respiratory infections often cause secondary complications during shigellosis, the systemic effect of PB and NaB on CAP-18 expression in respiratory epithelia was also evaluated. Methods: The readouts were clinical outcomes, CAP-18 expression in mucosa of colon, rectum, lung and trachea (immunohistochemistry and real-time PCR) and release of the CAP-18 peptide/protein in stool (Western blot). Principal findings: Significant downregulation of CAP-18 expression in the epithelia of rectum and colon, the site of Shigella infection was confirmed. Interestingly, reduced expression of CAP-18 was also noticed in the epithelia of lung and trachea, indicating a systemic effect of the infection. This suggests a causative link to acute respiratory infections during shigellosis. Oral treatment with PB resulted in reduced clinical illness and upregulation of CAP-18 in the epithelium of rectum. Both PB and NaB counteracted the downregulation of CAP-18 in lung epithelium. The drug effect is suggested to be systemic as intravenous administration of NaB could also upregulate CAP-18 in the epithelia of lung, rectum and colon. Conclusion: Our results suggest that PB has treatment potential in human shigellosis. Enhancement of CAP-18 in the mucosal epithelia of the respiratory tract by PB or NaB is a novel discovery. This could mediate protection from secondary respiratory infections that frequently are the lethal causes in dysentery. [ABSTRACT FROM AUTHOR]

Background: Enterococcus faecalis is a significant cause of infective endocarditis, an infection of the heart endothelium leading to vegetation formation (microbes, fibrin, platelets, and host cells attached to underlying endothelial tissue). Our previous research determined that enterococcal aggregation substance (AS) is an important virulence factor in causation of endocarditis, although endocarditis may occur in the absence of AS production. Production of AS by E. faecalis causes the organism to form aggregates through AS binding to enterococcal binding substance. In this study, we assessed the ability of IgGs and IgG Fabs against AS to provide protection against AS+ E. faecalis endocarditis. Methodology/Principal Findings: When challenged with AS+ E. faecalis, 10 rabbits actively immunized against AS+ E. faecalis developed more significant vegetations than 9 animals immunized against AS- E. faecalis, and 9/10 succumbed compared to 2/9 (p,0.005), suggesting enhanced aggregation by IgG contributes significantly to disease. IgG antibodies against AS also enhanced enterococcal aggregation as tested in vitro. In contrast, Fab fragments of IgG from rabbits immunized against purified AS, when passively administered to rabbits (6/group) immediately before challenge with AS+ E. faecalis, reduced total vegetation (endocarditis lesion) microbial counts (7.9×106 versus 2.0×105, p = 0.02) and size (40 mg versus 10, p = 0.05). In vitro, the Fabs prevented enterococcal aggregation. Conclusions/Significance: The data confirm the role of AS in infective endocarditis formation and suggest that use of Fabs against AS will provide partial protection from AS+ E. faecalis illness. [ABSTRACT FROM AUTHOR]