Sustained release of inactivated H1N1 virus from degradable microparticles for extended vaccination.

Influenza vaccines administered as intramuscularly injected inactivated viruses or intranasally administered live-attenuated viruses usually provide short-term protection against influenza infections. Biodegradable particles that provide sustained release of the antigen has been studied as an approach to extend vaccine protection. Here, we investigate sustained release of ultraviolet killed influenza virus (A/PR/8/34) (kPR8) loaded into poly(D,L-lactic-co-glycolic acid) (PLGA) microparticles. Particles were prepared using the double emulsion method, and polymer molecular weight (MW), polymer hydrophobicity, polymer concentration in the organic phase, and the amount of killed virus were varied to obtain a range of particles. Formulations included PLGA 50:50 (2-6, 7-17 kDa), PLGA 75:25 (4-15 kDa), and 50/50 PLGA 75:25 (4-15 kDa)/PCL (14 kDa). Additionally, NaOH was co-encapsulated in some cases to enhance particle degradation. The structure of the particles was explored by size measurements and electron microscopy. The kPR8 release profiles were measured using hemagglutinin ELISA. The concentration of the polymer (PLGA) in the organic phase and polymer MW significantly influenced virus loading, while polymer MW and co-encapsulation of NaOH modulated the release profiles. Mice receiving a single intramuscular injection of NaOH microparticle-encapsulated kPR8 were partially protected against a lethal influenza challenge 32 weeks post immunization. Microparticle (MP) vaccination induced a gradual increase in PR8-specific IgGs dominated by IgG1 in contrast to the rapid IgG2a-biased response elicited by soluble kPR8 immunization. Our results indicate that vaccine-NaOH co-loaded PLGA particles show potential as a single dose vaccination strategy for extended protection against influenza virus infection.

Sustained release system from PLGA particles co-encapsulated with inactivated influenza virus with natural killer T cell agonist α-galactosylceramide.

Vaccines against influenza and many other infectious diseases require multiple boosters in addition to the primary dose to improve efficacy, but this approach is not ideal for compliance. The multiple doses could potentially be replaced by sustained or pulsatile release of antigens encapsulated in degradable microparticles (MPs). The efficacy of a vaccine is improved by adding an adjuvant, which can be co-delivered from the particles to enhance immunogenicity. Here, we developed degradable poly-lactic-co-glycolic acid (PLGA) (7-17 kDa) MPs capable of sustained release of ultraviolet killed influenza virus (A/PR/8/34) (kPR8) vaccine and the natural killer T (NKT) cell agonist alpha-galactosylceramide (α-GalCer) and tested their effectiveness at providing long-term protection against influenza virus infection in mice. Multiple formulations were developed for encapsulating the virus and adjuvant separately, and in combination. The MPs exhibited sustained release of both the virus and the adjuvant lasting more than a month. Co-encapsulation significantly increased the encapsulation efficiency (EE) of the vaccine but reduced the release duration. On the other hand, co-encapsulation led to a reduction in EE for the α-GalCer and a change in release profile to a higher initial burst followed by a linear release compared to a low initial burst and slower linear release. The α-GalCer also had considerably longer release duration compared to the vaccine. Mice injected with particle formulations co-encapsulating kPR8 and α-GalCer were protected from a lethal influenza virus infection 30 weeks after vaccination. This study demonstrates that PLGA MP based vaccines are promising for providing effective vaccination and possibly for replacing multiple doses with a single injection.

Comparison of oseltamivir and α-galactosylceramide for reducing disease and transmission in pigs infected with 2009 H1N1 pandemic influenza virus.

Influenza virus infections are a major cause of respiratory disease in humans. Neuraminidase inhibitors (NAIs) are the primary antiviral medication used to treat ongoing influenza infections. However, NAIs are not always effective for controlling virus shedding and lung inflammation. Other concerns are the emergence of NAI-resistant virus strains and the risk of side effects, which are occasionally severe. Consequently, additional anti-influenza therapies to replace or combine with NAIs are desirable. Here, we compared the efficacy of the NAI oseltamivir with the invariant natural killer T (iNKT) cell superagonist, α-galactosylceramide (α-GalCer), which induces innate immune responses that inhibit influenza virus replication in mouse models. We show that oseltamivir reduced lung lesions and lowered virus titers in the upper respiratory tract of pigs infected with A/California/04/2009 (CA04) pandemic H1N1pdm09. It also reduced virus transmission to influenza-naïve contact pigs. In contrast, α-GalCer had no impact on virus replication, lung disease, or virus transmission, even when used in combination with oseltamivir. This is significant as iNKT-cell therapy has been studied as an approach for treating humans with influenza.

Characterization of a Novel Reassortant Epizootic Hemorrhagic Disease Virus Serotype 6 Strain Isolated from Diseased White-Tailed Deer (Odocoileus virginianus) on a Florida Farm.

We report an outbreak of a novel reassortant epizootic hemorrhagic disease virus serotype 6 (EHDV-6) in white-tailed deer (WTD) on a Florida farm in 2019. At necropsy, most animals exhibited hemorrhagic lesions in the lung and heart, and congestion in the lung, liver, and spleen. Histopathology revealed multi-organ hemorrhage and congestion, and renal tubular necrosis. Tissues were screened by RT-qPCR and all animals tested positive for EHDV. Tissues were processed for virus isolation and next-generation sequencing was performed on cDNA libraries generated from the RNA extracts of cultures displaying cytopathic effects. Six isolates yielded nearly identical complete genome sequences of a novel U.S. EHDV-6 strain. Genetic and phylogenetic analyses revealed the novel strain to be most closely related to a reassortant EHDV-6 strain isolated from cattle in Trinidad and both strains received segment 4 from an Australian EHDV-2 strain. The novel U.S. EHDV-6 strain is unique in that it acquired segment 8 from an Australian EHDV-8 strain. An RNAscope® in situ hybridization assay was developed against the novel U.S. EHDV-6 strain and labeling was detected within lesions of the heart, kidney, liver, and lung. These data support the novel U.S. reassortant EHDV-6 strain as the cause of disease in the farmed WTD.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Central nervous system diseases of cattle in Georgia, 2001-2017.

In a review of 159 archived bovine autopsy cases submitted to the Georgia Veterinary Diagnostic Laboratory System from 2001 to 2017, we evaluated histologic lesions and other laboratory test results in cases diagnosed with central nervous system (CNS) disease to determine the most common disorders and the rate of definitive morphologic and etiologic diagnosis within the population. The most common breed was Aberdeen Angus or Angus (31%), and female animals comprised 60% of the overall submissions. Gross anatomic changes were described in 33% of cases that had histologic lesions. Suppurative meningoencephalitis was the most common diagnosis (28% of cases overall) and was frequently associated with bacterial sepsis. Lymphocytic meningoencephalitis without an identified etiology was diagnosed in 17% of cases. Polioencephalomalacia was the most commonly identified noninfectious diagnosis (17%). Hydrocephalus was the most common CNS comorbidity and potential congenital defect. Identification of specific agents was sporadic, and infectious disease testing was largely dependent on fluorescent antibody testing, almost all of which were negative. Our findings indicate the common differential diagnoses for cattle with neurologic signs in the southeastern United States, as well the need for a well-defined, methodical diagnostic approach, including autopsy, histology, and appropriate additional laboratory testing.

Evaluation of Targeted Next-Generation Sequencing for Detection of Bovine Pathogens in Clinical Samples.

The laboratory diagnosis of infectious diseases, especially those caused by mixed infections, is challenging. Routinely, it requires submission of multiple samples to separate laboratories. Advances in next-generation sequencing (NGS) have provided the opportunity for development of a comprehensive method to identify infectious agents. This study describes the use of target-specific primers for PCR-mediated amplification with the NGS technology in which pathogen genomic regions of interest are enriched and selectively sequenced from clinical samples. In the study, 198 primers were designed to target 43 common bovine and small-ruminant bacterial, fungal, viral, and parasitic pathogens, and a bioinformatics tool was specifically constructed for the detection of targeted pathogens. The primers were confirmed to detect the intended pathogens by testing reference strains and isolates. The method was then validated using 60 clinical samples (including tissues, feces, and milk) that were also tested with other routine diagnostic techniques. The detection limits of the targeted NGS method were evaluated using 10 representative pathogens that were also tested by quantitative PCR (qPCR), and the NGS method was able to detect the organisms from samples with qPCR threshold cycle (CT ) values in the 30s. The method was successful for the detection of multiple pathogens in the clinical samples, including some additional pathogens missed by the routine techniques because the specific tests needed for the particular organisms were not performed. The results demonstrate the feasibility of the approach and indicate that it is possible to incorporate NGS as a diagnostic tool in a cost-effective manner into a veterinary diagnostic laboratory.

Knowingly but naively: The overpowering influence of innocence on interrogation rights decision-making.

Most suspects waive the guaranteed protections that interrogation rights afford them against police intimidation. One factor thought to motivate suspects' inclination to waive their rights stems from the acquiescence bias whereby suspects mindlessly comply with interrogators' requests. However, research bearing on the phenomenology of innocence has demonstrated the power of innocents' mindset, which could motivate some innocent suspects to waive their rights knowingly (instead of mindlessly complying). To test these ideas, participants (N = 178) were (a) rightfully (guilty) or wrongfully (innocent) accused of wrongdoing during an experimental session, (b) administered 1 of 2 forms that by signing either waived or invoked their rights to a student advocate, and (c) given questions to assess their degree of knowing during the decision-making process (i.e., extent to which individuals were cognizant of their decisions). Results demonstrated that unknowing innocent and guilty individuals tended to passively comply, engaging in a pre-interrogation acquiescence bias by signing waive and invoke forms at similar rates. But, as participants became more cognizant of their decisions, they acquiesced at lower rates and their change from acquiescence differed depending on their status. As innocents became more cognizant, they signed the waiver form at higher rates than the invoke form, thereby demonstrating that innocence can motivate some suspects to knowingly forgo their rights. Conversely, as guilty individuals became more cognizant, they signed the invoke form at higher rates than the waiver form. These findings have implications for reforming pre-interrogation protocols, protecting suspects' civil liberties, and preventing innocents from offering false self-incriminating evidence. (PsycINFO Database Record

Septicemia and meningoencephalitis caused by Listeria monocytogenes in two neonatal llamas.

Listeriosis is a disease of humans and domestic mammals (mainly ruminants) with variable manifestations, primarily encephalitis, septicemia, and abortion. Although Listeria monocytogenes readily causes illness in ruminants, the prevalence among domestic South American camelids (llamas and alpacas) is low and has not been documented in their wild counterparts, the vicuna and guanaco. We describe herein the clinical signs, autopsy findings, and histopathology of septicemia and suppurative meningoencephalitis caused by L. monocytogenes in 2 neonatal llamas ( Llama glama) from the same herd. L. monocytogenes was isolated in pure culture and identified by real-time PCR on fresh and paraffin-embedded tissue samples of the brain from both crias. This presentation of septicemic listeriosis with meningoencephalitis in 2 animals from the same group is unusual, especially among llamas.

Evaluation of Fourteen Aurone Derivatives as Potential Anti-Cancer Agents.

BACKGROUND: Aurones are a sub-set of the flavone family that possess a number of biological activities, including anti-cancer, anti-inflammatory, anti-microbial, anti-parasitic and anti-viral. Due to their high availability, simple synthesis, and generally low toxicity, aurones could be attractive candidates for safer cancer drugs. This study aims to evaluate the anti-proliferation and anti-metastasis activity of a series of synthesized aurone derivatives. METHODS: A series of aurone derivatives with simple unsubstituted coumaranone (benzofuranone) fragments and a range of alkylidene fragments have been prepared and tested for anti-proliferation activity against human cancer cell lines A549 (lung), BT20 (breast) and MCF7 (breast), and antimetastasis activity against A549. RESULTS: Several of these compounds displayed significant levels of activity and high levels of selectivity for the inhibition of the growth of cancerous cell lines versus the corresponding normal cell lines. This growth inhibition was found to be associated with the induction of apoptosis in cancer cells. Moreover, several aurone derivatives showed remarkable inhibition on the motility of lung cancer cells A549 at a concentration as low as 6.25 µM. Analysis of the structure-activity relationship revealed that the aurone derivatives based upon five-membered heteroaromatic rings exhibited the most significant anti-cancer activity. CONCLUSION: Aurone derivatives devoid of the unusual oxygenation found in the coumaranone fragment are potential leads for new anti-cancer agents.

First report of vascular invasion of demodex mites with thrombi and dissemination to visceral lymph nodes in a dog.

An adult-onset generalized canine demodecosis is a life threatening form of demodecosis often seen in immunocompromised dogs. A generalized, adult-onset canine demodecosis with an unusual vascular invasion, thrombi and dissemination to peripheral, and visceral lymph nodes was diagnosed in an adult, female, American Bulldog of unknown age. Organized thrombi with intralesional mites were observed in multiple lymphatic vessels and in a section of blood vessel. Vascular invasion of mites with dissemination of mites into visceral lymph nodes, is an unusual findings not previously recorded in canine generalized demodecosis. Dissemination of mites to visceral lymph nodes through vascular channels may be part of the pathogenesis of chronic, untreated, and disseminated, adult-onset generalized canine demodecosis.

Helping and hurting others: Person and situation effects on aggressive and prosocial behavior as assessed by the Tangram task.

The Tangram Help/Hurt Task is a laboratory-based measure designed to simultaneously assess helpful and hurtful behavior. Across five studies we provide evidence that further establishes the convergent and discriminant validity of the Tangram Help/Hurt Task. Cross-sectional and meta-analytic evidence finds consistently significant associations between helpful and hurtful scores on the Tangram Task and prosocial and aggressive personality traits. Experimental evidence reveals that situational primes known to induce aggressive and prosocial behavior significantly influence helpful and hurtful scores on the Tangram Help/Hurt Task. Additionally, motivation items in all studies indicate that tangram choices are indeed associated with intent of helping and hurting. We discuss the advantages and limitations of the Tangram Help/Hurt Task relative to established measures of helpful and hurtful behavior. Aggr. Behav. 43:133-146, 2017. © 2016 Wiley Periodicals, Inc.

Overcoming Innocents' Naiveté: Pre-interrogation Decision-making Among Innocent Suspects.

Suspects, especially innocent ones, are highly susceptible to waiving their interrogation rights. This research tested the ability of two strategies to overcome innocent suspects' willingness to waive their rights. One strategy was based on the social influence of scarcity (i.e., not constraining the pre-interrogation time limit). The other strategy focused on disrupting individuals' cognitive fluency during the decision-making process (i.e., violating their induced expectation of offering a waiver). Disrupting innocent individuals' cognitive fluency increased their willingness to invoke their rights and, notably, was not qualified by interactions with any other factors. However, scarcity did not influence individuals' pre-interrogation decision-making. Results also further established the association between innocent individuals' naïve mindset and their willingness to waive their rights - specifically, innocents' willingness to waive their rights increased with the strength of their just world beliefs. The theoretical and applied implications of these findings are discussed. The importance and benefit of reforming pre-interrogation protocols using fair and feasible strategies that would disrupt suspects' cognitive fluency are emphasized. Copyright © 2016 John Wiley & Sons, Ltd.

A fast slewing cryostat for small sample in vacuo electrical/optical testing in the range 30 K-500 K.

A cold finger vacuum cryostat is described in which a sapphire heat conducting element is used to achieve very fast slew rates for a small sample stage over a wide temperature range with optimal matching to a closed cycle helium refrigerator head. When the set temperature is reached it is maintained with very high stability (±5 mK). The target applications are associated with semiconductor electrical material characterisation measurements which require temperature scanning, e.g., thermally stimulated current, deep level transient spectroscopy, and applications which require annealing followed by rapid transition to lower temperature for measurement.

Outbreak of equine piroplasmosis in Florida.

CASE DESCRIPTION: A 7-year-old Quarter Horse gelding was hospitalized in Ocala, Fla, because of lethargy, fever, anorexia, and swelling of distal aspects of the limbs. A tentative diagnosis of equine piroplasmosis (EP) was made on the basis of examination of a blood smear. The case was reported to the Florida State Veterinarian, and infection with Babesia equi was confirmed. The subsequent investigation included quarantine and testing of potentially exposed horses for B equi and Babesia caballi infections, tick surveillance, and owner-agent interviews. CLINICAL FINDINGS: 210 horses on 25 premises were tested for infection with EP pathogens. Twenty B equi-infected horses on 7 premises were identified; no horses tested positive for B caballi. Seven horses, including the index case, had clinical findings consistent with EP Dermacentor variabilis was considered the only potential tick vector for B equi collected, and all D variabilis specimens tested negative for Babesia organisms via PCR assay. Results of the epidemiological investigation suggested that B equi was spread by use of shared needles and possibly blood transfusions. All horses that tested positive were involved in nonsanctioned Quarter Horse racing, and management practices were thought to pose substantial risk of transmission of blood-borne pathogens. TREATMENT AND OUTCOME: Final outcome of B equi-infected horses was euthanasia, death from undetermined causes, or shipment to a US federal research facility. CLINICAL RELEVANCE: This investigation highlights the importance of collaboration between private veterinary practitioners, state veterinary diagnostic laboratories, and regulatory officials in the recognition, containment, and eradication of foreign animal disease.

The splicing factor PSF is part of a large complex that assembles in the absence of pre-mRNA and contains all five snRNPs.

PSF (PTB-associated splicing factor) is a large nuclear protein that has been implicated in numerous processes including transcription and RNA splicing. It has been shown to directly associate with U5 snRNA and has also been found within numerous purified splicing complexes. Here, we show that when HeLa nuclear extracts are adjusted to splicing conditions, PSF is found as part of a large complex that contains all five snRNPs and most known splicing factors. Formation of the complex does not require addition of exogenous pre-mRNA substrate and occurs at 4 degrees C but is salt sensitive. Sedimentation experiments and identification of individual components by mass spectrometry revealed association with multiple nuclear factors, most of which overlap with spliceosome components.