Pentavalent Disabled Infectious Single Animal (DISA)/DIVA Vaccine Provides Protection in Sheep and Cattle against Different Serotypes of Bluetongue Virus

Bluetongue (BT) is a midge-borne OIE-notifiable disease of ruminants caused by the bluetongue virus (BTV). There are at least 29 BTV serotypes as determined by serum neutralization tests and genetic analyses of genome segment 2 encoding serotype immunodominant VP2 protein. Large parts of the world are endemic for multiple serotypes. The most effective control measure of BT is vaccination. Conventionally live-attenuated and inactivated BT vaccines are available but have their specific pros and cons and are not DIVA compatible. The prototype Disabled Infectious Single Animal (DISA)/DIVA vaccine based on knockout of NS3/NS3a protein of live-attenuated BTV, shortly named DISA8, fulfills all criteria for modern veterinary vaccines of sheep. Recently, DISA8 with an internal in-frame deletion of 72 amino acid codons in NS3/NS3a showed a similar ideal vaccine profile in cattle. Here, the DISA/DIVA vaccine platform was applied for other serotypes, and pentavalent DISA/DIVA vaccine for “European” serotypes 1, 2, 3, 4, 8 was studied in sheep and cattle. Protection was demonstrated for two serotypes, and neutralization Ab titers indicate protection against other included serotypes. The DISA/DIVA vaccine platform is flexible in use and generates monovalent and multivalent DISA vaccines to combat specific field situations with respect to Bluetongue.

Induction of Ascites in Mice

Smaller animals such as rats, mice, hamsters, and guinea pigs are usually poor choices for polyclonal antibody production because only small volumes of serum can be obtained. This problem can be reduced by inducing the formation of ascites in mice, which can provide up to 10 mL of ascites fluid from a single animal. Antibody titers in ascites fluids are almost as high as serum titers.

A Genetic Platform for Functionally Profiling Odorant Receptors Ex Vivo Using Olfactory Cilia.

The molecular basis for odor perception in humans remains a black box as odorant receptors (ORs) are notoriously difficult to study outside of their native environment. Efforts toward OR expression and functional profiling in heterologous systems have been met with limited success due to poor efficiency of cell surface expression and consequently reduced G-protein signal amplification. We previously reported a genetic strategy in mice to increase the number of sensory neurons expressing specific ORs, which transforms the 10 million neurons of the mouse nose into a bioreactor producing large quantities of fully functional OR protein. We now describe the isolation of cilia from these bioreactors for two human ORs. Cilia are known to contain all components of the olfactory signal transduction machinery and can be placed into an ex vivo well-plate assay to rapidly measure robust, reproducible odor-specific responses. Our OR1A1 and OR5AN1 isolated cilia reveal 10-100fold more sensitivity than existing assays. Tissue from a single animal produces up to 4,000 384-well assay wells, and isolated olfactory cilia can be stored frozen and thus preserved for long term usage. This pipeline offers a sensitive, highly scalable ex vivo odor screening platform that opens the door for decoding human olfaction.

De novo Assembly, Annotation, and Analysis of Transcriptome Data of the Ladakh Ground Skink Provide Genetic Information on High-Altitude Adaptation

The Himalayan Arc is recognized as a global biodiversity hotspot. Among its numerous cryptic and undiscovered organisms, this composite high-mountain ecosystem harbors many taxa with adaptations to life in high elevations. However, evolutionary patterns and genomic features have been relatively rarely studied in Himalayan vertebrates. Here, we provide the first well-annotated transcriptome of a Greater Himalayan reptile species, the Ladakh Ground skink Asymblepharus ladacensis (Squamata: Scincidae). Based on tissues from the brain, an embryonic disc, and pooled organ material, using pair-end Illumina NextSeq 500 RNAseq, we assembled ~77,000 transcripts, which were annotated using seven functional databases. We tested ~1600 genes, known to be under positive selection in anurans and reptiles adapted to high elevations, and potentially detected positive selection for 114 of these genes in Asymblepharus. Even though the strength of these results is limited due to the single-animal approach, our transcriptome resource may be valuable data for further studies on squamate reptile evolution in the Himalayas as a hotspot of biodiversity.

A Single Animal Species-Based Prediction of Human Clearance and First-in-Human Dose of Monoclonal Antibodies: Beyond Monkey

These days, there is a lot of emphasis on the prediction of human clearance (CL) from a single species for monoclonal antibodies (mabs). Many studies indicate that monkey is the most suitable species for the prediction of human clearance for mabs. However, it is not well established if rodents (mouse or rat) can also be used to predict human CL for mabs. The objectives of this study were to predict and compare human CL as well as first-in-human dose of mabs from mouse or rat, ormonkey. Four methods were used for the prediction of human CL of mabs. These methods were: use of four allometric exponents (0.75, 0.80, 0.85, and 0.90), a minimal physiologically based pharmacokinetics method (mPBPK), lymph flow rate, and liver blood flow rate. Based on the predicted CL, first-in-human dose of mabs was projected using either exponent 1.0 (linear scaling) or exponent 0.85, and human-equivalent dose (HED) from each of these species. The results of the study indicated that rat or mouse could provide a reasonably accurate prediction of human CL as well as first-in-human dose of mabs. When exponent 0.85 was used for CL prediction, there were 78%, 95%, and 92% observations within a 2-fold prediction error for mouse, rat, and monkey, respectively. Predicted human dose fell within the observed human dose range (administered to humans) for 10 out of 13 mabs for mouse, 11 out of 12 mabs for rat, and 12 out of 15 mabs for monkey. Overall, the clearance and first-in-human dose of mabs were predicted reasonably well by all three species (a single species). On average, monkey may be the best species for the prediction of human clearance and human dose but mouse or rat especially; rat can be a very useful species for conducting the aforementioned studies.

Evaluation of analgesic (in vivo) activity of Ariflex liniment in comparison with diclofenac gel by acetic acid induced writhing model

Background: Adverse effects of available medications for osteoarthritis (OA) and rheumatoid arthritis (RA) necessitate development of safer and effective alternative medicinal substitutes. The present study was conducted to evaluate analgesic activity of Ariflex liniment (conceptualized and developed by Ari Healthcare Pvt. Ltd.) in comparison with diclofenac gel by using acetic acid induced writhing model.Methods: Albino mice of either sex weighing 20-25 g were taken and divided into 3 groups with 5 animals in each group, i.e., group 1 (control group), group 2 (diclofenac gel) and group 3 (Ariflex liniment). After 1 hour of topical application of study drugs writhing was induced in mice using intra-peritonial injection of 1% acetic acid in volume of 0.1 ml/10 g body weight. Then the writhing episodes were recorded for 30 minutes and results were noted.Results: In the control group, the total number of writhes were 260±29.73 (mean±S. E. M.). The total number of writhes was 12.17±11.81 (mean ± S. E. M.) in diclofenac group. In Ariflex liniment group, not a single animal felt pain, hence there were no writhes recorded. When compared to control group, the difference in number of writhes was statistically significant. The analgesic activity of Ariflex liniment was found to be superior to that of diclofenac gel used as standard drug.Conclusions: It can be concluded that Ariflex liniment possesses analgesic activity.

A realistic locomotory model of Drosophila larva for behavioral simulations.

The Drosophila larva is extensively used as model species in experiments where behavior is recorded via tracking equipment and evaluated via population-level metrics. Although larva locomotion neuromechanics have been studied in detail, no comprehensive model has been proposed for realistic simulations of foraging experiments directly comparable to tracked recordings. Here we present a virtual larva for simulating autonomous behavior, fitting empirical observations of spatial and temporal kinematics. We propose a trilayer behavior-based control architecture for larva foraging, allowing to accommodate increasingly complex behaviors. At the basic level, forward crawling and lateral bending are generated via coupled, interfering oscillatory processes under the control of an intermittency module, alternating between crawling bouts and pauses. Next, navigation in olfactory environments is achieved via active sensing and top-down modulation of bending dynamics by concentration changes. Finally, adaptation at the highest level entails associative learning. We could accurately reproduce behavioral experiments on autonomous free exploration, chemotaxis, and odor preference testing. Inter-individual variability is preserved across virtual larva populations allowing for single animal and population studies. Our model is ideally suited to interface with neural circuit models of sensation, memory formation and retrieval, and spatial navigation.

The circadian clock gates Drosophila adult emergence by controlling the timecourse of metamorphosis

The daily rhythm of adult emergence of holometabolous insects is one of the first circadian rhythms to be studied. In these insects, the circadian clock imposes a daily pattern of emergence by allowing or stimulating eclosion during certain windows of time and inhibiting emergence during others, a process that has been described as “gating.” Although the circadian rhythm of insect emergence provided many of the key concepts of chronobiology, little progress has been made in understanding the bases of the gating process itself, although the term “gating” suggests that it is separate from the developmental process of metamorphosis. Here, we follow the progression through the final stages of Drosophila adult development with single-animal resolution and show that the circadian clock imposes a daily rhythmicity to the pattern of emergence by controlling when the insect initiates the final steps of metamorphosis itself. Circadian rhythmicity of emergence depends on the coupling between the central clock located in the brain and a peripheral clock located in the prothoracic gland (PG), an endocrine gland whose only known function is the production of the molting hormone, ecdysone. Here, we show that the clock exerts its action by regulating not the levels of ecdysone but that of its actions mediated by the ecdysone receptor. Our findings may also provide insights for understanding the mechanisms by which the daily rhythms of glucocorticoids are produced in mammals, which result from the coupling between the central clock in the suprachiasmatic nucleus and a peripheral clock located in the suprarenal gland.

Evaluation of the i-STAT Alinity v in a veterinary clinical setting

Many point-of-care (POC) analyzers are available for the measurement of electrolytes and acid-base status in animals. We assessed the precision of the i-STAT Alinity v, a recently introduced POC analyzer, and compared it to 2 commonly used and previously validated POC analyzers (i-STAT 1, Stat Profile pHOx Ultra). Precision was evaluated by performing multiple analyses of whole blood samples from healthy dogs, cats, and horses on multiple i-STAT Alinity v analyzers. For comparison between analyzers, whole blood samples from dogs and cats presented to the emergency room were run concurrently on all 3 POC instruments. Reported values were compared by species (dogs and cats only) using Pearson correlation, and all values from all species were analyzed together for the Bland–Altman analysis. Results suggested that the i-STAT Alinity v precision was very good, with median coefficients of variability <2.5% for all measured parameters (except the anion gap), with variable ranges of coefficients of variation. In addition, good-to-excellent correlation was observed between the i-STAT Alinity v and i-STAT 1, and between the i-STAT Alinity v and Stat Profile pHOx Ultra for all parameters in both cats and dogs, respectively. In this cohort, the i-STAT Alinity v had clinically acceptable bias compared to the currently marketed analyzers and can be used for monitoring measured analytes in cats and dogs, although serial measurements in a single animal should be performed on the same analyzer whenever possible.

Disentanglement of Cape fur seals (Arctocephalus pusillus pusillus) with reversible medetomidine-midazolam-butorphanol

Anaesthesia in pinnipeds is considered a much higher risk than in most terrestrial mammals because of their frequent proximity to water and physiological and anatomical adaptations related to diving, which also influence their anaesthesia management. Anaesthetising and immobilising entangled seals does not allow for selection of animals that are at a safe distance from the water’s edge. Medetomidine-midazolam-butorphanol (MMB) sedation was trialled on eight entangled Cape fur seals (CFS) (Arctocephalus pusillus pusillus) to determine if it was safe to use on animals that entered the water post-darting. The MMB was given at an estimated dose of 0.03 mg/kg, 0.2 mg/kg and 0.2 mg/kg, respectively, via remote darting. Sedation was reversed with intramuscular atipamezole (0.15 mg/kg) and naltrexone (0.4 mg/kg) to antagonise the effects of medetomidine and butorphanol, respectively. Moderate sedation was achieved in six animals. Six of the animals entered the water after being darted. There was a single mortality and a single animal that was too lightly sedated for capture. The preliminary results indicate that MMB produces suitable sedation for disentanglement of CFS. Additionally, MMB might be suitable for application to field-based biological research.