On the Biocompatibility and Teat Retention of In Situ Gelling Intramammary Formulations: Cattle Mastitis Prevention and Treatment.

Treatment and prevention of cattle mastitis remains a formidable challenge due to the anatomical and physiological constraints of the cow udder. In this study, we investigated polymeric excipients and solvents that can form, (when combined) novel, non-toxic and biocompatible in situ gelling formulations in the mammary gland of bovine cattle. We also report on a new approach to screen intramammary formulations using fresh excised cow teats. Fourteen hydrophilic polymers and six solvents were evaluated for in vitro cytotoxicity and biocompatibility towards cultured bovine mammary epithelial cells (MAC-T), microscopic and macroscopic examination upon contact with excised cow teats. No significant cytotoxicity (p > 0.05) was observed with polyethylene oxides, hydroxypropyl methylcellulose, carboxymethyl cellulose, sodium alginate and xanthan gum. Polycarbophil and carbopol polymers showed significantly higher cytotoxicity (p < 0.05). Concentration-dependent cytotoxicity was observed for glycerin, propylene glycol, polyethylene glycol 400, ethanol, N-methyl-2-pyrrolidone and 2-pyrrolidone, with the 2-pyrrolidone solvents showing higher cytotoxic effects (p < 0.05). In situ gelling formulations comprising hydroxypropyl methylcellulose or carboxymethyl cellulose and solvents in specific ratios were biocompatible at higher concentrations with MAC-T cells compared to alginates. All investigated formulations could undergo in situ sol-to-gel phase transformation, forming non-toxic gels with good biocompatibility in excised cow teats hence, showing potential for use as intramammary carriers for sustained drug delivery.

Negative effects of energy supplementation at peak lactation of sheep can be offset by the addition of Lactobacillus-fermented plant extracts.

Energy supplementation may reduce oxidative stress by correcting a negative energy balance, but in some contexts, it has been shown to increase oxidative stress, especially at peak lactation. The current experiment examined if a pelleted energy supplement with or without the addition of Lactobacillus-fermented seaweed or seaweed plus terrestrial plants extracts affected oxidative stress of ewes from late gestation through to weaning and ewe and lamb production from lambing to weaning. Treatments were either no supplement (CON-), a pelleted supplement only (CON+, 100 g/ewe per d), CON+ with seaweed extract only (SWO, 10 mL/ewe per day), or CON+ with seaweed plus an arrangement of terrestrial plant extract (SWP, 10 mL/ewe per d). Ewes (n = 160; mean initial BW = 72.3 ± 9.5 kg [mean ± SD]) were randomized to pastures (n = 4 pastures per treatment with 10 ewes each). After lambing, ewes with twins were reallocated to pastures (n = 3 pastures per treatment with 10 ewes each) according to lambing date. At 4 wk in milk, supplementation tended to reduce total antioxidant status (TAS; P = 0.10) and increased glutathione peroxidase (GPx) activity compared with nonsupplemented ewes (P = 0.04). The addition of seaweed and terrestrial plants extracts to the concentrate, that is, SWO and SWP, increased TAS and reduced GPx activity compared with CON+ (P < 0.01). Supplementation increased milk yield at weeks 4, 6, and 8 of lactation, and protein, lactose, and total milk solids yield at peak lactation (week 4; P < 0.05). The CON- ewes had greater somatic cell count than the supplemented ewes at weeks 4, 8, and 10 of lactation (P = 0.03). Our results suggest that energy supplementation, alone, increases oxidative stress of lactating ewes, which may relate to increased oxidative phosphorylation. Most importantly, these results indicate that in situations where energy supplementation is needed to increase animal performance, negative effects of energy supplementation around peak lactation can be offset by the addition of Lactobacillus-fermented plant extracts (SWO and SWP) to improve antioxidant status.

Viability assessment of Bifidobacterium longum ATCC 15707 on non-dairy foods using quantitative fluorescence microscopy.

This study demonstrates an effective technique for separating and purifying viable bacteria from samples that interfere with viability staining. The viability of Bifidobacterium longum ATCC 15707 was assessed using Percoll Buoyant Density Gradient Centrifugation (PBDC) to separate bacteria from complex non-dairy food matrices and Quantitative Fluorescence Microscopy (QFM) to determine individual cells using LIVE/DEAD BacLight bacterial viability staining. Water agar (3%) was used to retain cells of B. longum and offered a lower fluorescence background with BacLight viability staining, compared with fixation on polycarbonate (PC) black membrane. The effect of drying temperatures and non-dairy foods on viability of B. longum was assessed. B. longum coated on oat, peanut or raisin was separated by filtration, low- and high-speed centrifugation, flotation and sedimentation buoyant density centrifugation. Purified cells were subsequently deposited on water agar for rehydration followed by LIVE/DEAD BacLight viability staining and enumeration. Conventional plate counting was also conducted to compare viability results. Finally, this method was applied to assess cell membrane damages of B. longum incorporated onto non-dairy foods during 24 h drying. Furthermore, viability assessment of B. longum coated onto oat, peanut, or raisin was much lower by plate counting compared to viability staining. Drying appeared to have a greater impact when viability was assessed by plate counting compared to viability staining. IMPORTANCE: Enumeration of viable beneficial bacteria from function foods presents a significant bottleneck for product development and quality control. Interference with microscopic and/or fluorescent techniques by ingredients, time required to incubate plated microbes, and the transient nature of the colony forming unit make rapid assessment of viable bacteria difficult. Viability assessment of Bifidobacterium longum ATCC 15707 by Percoll Buoyant Density Gradient Centrifugation with LIVE/DEAD BacLight viability staining on water agar (3%) was in agreement with serial dilution enumeration. Without the need for incubation viability assessment by staining provided a more rapid means to assess the impact of drying on the viability of B. longum coated onto oat, peanut or raisin.

A 3D printed chitosan-pectin hydrogel wound dressing for lidocaine hydrochloride delivery.

A chitosan-pectin (CS-PEC) biopolymeric hydrogel wound dressing was investigated for lidocaine delivery. Here we demonstrate for the first time the feasibility of three-dimensional (3D) printed CS-PEC hydrogel incorporating the local anaesthetic drug lidocaine hydrochloride (LDC) as a potential wound dressing candidate. The hydrogels were prepared by physical crosslinking of CS and PEC polysaccharides. The scaffolds were printed using an extrusion-based 3D printer using a mechanical positive displacement dispensing system followed by lyophilisation. The 3D printed hydrogels showed good printability, dimensional integrity and self-adhesion to skin. The high swelling ratio and water absorption of 3D printed hydrogels indicated suitability for absorbing exudates and maintaining a moist wound healing environment. Fourier transform infrared (FTIR) spectroscopy results indicated that the CS-PEC hydrogel was formed by hydrogen bonds. Incorporation of LDC in the hydrogel did not interfere with its functional stability. In vitro drug release studies of LDC over 6 h fitted the Korsmeyer-Peppas model. This work demonstrates the possibility of a 3D printed hydrogel as a suitable candidate for wound dressings.

Development of a Long-Term Drug Delivery System with Levonorgestrel-Loaded Chitosan Microspheres Embedded in Poly(vinyl alcohol) Hydrogel.

This study reports on the fabrication of a controlled release system for the delivery of levonorgestrel (LNG) for long-term contraception. LNG was encapsulated in chemically cross-linked chitosan (CS) microspheres, and microspheres presented a spherical geometry with a good particle size distribution (polydispersity index (PDI) < 0.1). The LNG-CS microspheres were classified based on their particle size range, <63, 63-125, and 125-300 µm, where the 125-300 µm particles were selected to be incorporated into a physically cross-linked and annealed PVA hydrogel matrix to prolong the drug release. PVA concentrations and the annealing treatment influenced the swelling behavior of PVA hydrogels. Fourier transform infrared (FTIR) spectroscopy indicated that CS was successfully cross-linked through the formation of a Schiff base; the PVA hydrogel was formed through hydrogen bonding without reacting with LNG, which was only physically entrapped, thus maintaining its stability. Differential scanning calorimetry (DSC) showed that freeze-thaw and annealing processes increased the degree of crystallinity in the PVA hydrogel. In vitro drug release assessments for all formulations showed zero order without any burst release. Over a period of 100 days, 34, 27, and 21% of LNG was released from the CS-LNG microspheres in the size ranges < 63, 63-125, and 125-300 µm, respectively, while only 14, 11, and 9% of LNG was released when the CS-LNG microspheres were incorporated into 10, 15, and 20% PVA hydrogels, respectively. The drug release kinetics exhibited both diffusion- and swelling-controlled mechanisms following the Korsmeyer-Peppas model. This work presents a promising delivery system for long-term contraception with controlled zero-order release behaviors.

Non-dairy probiotic food products: An emerging group of functional foods.

The functional food sector has shown tremendous growth in recent years with the application of probiotic bacteria as "food additives". The utilization of probiotic bacteria in food presents many challenges related to their growth, survival, viability, stability and functionality in food processing, storage and consumption as well as changes of sensory characteristics of probiotic foods. Although dairy foods are currently the most common food carrier to deliver probiotics, an increasing number of non-dairy food matrices exhibit potential for delivery of probiotics. This review provides more recent insight into the emergence of non-dairy probiotics products, the interactions between probiotics and different food matrices and the challenges in developing such products. Some of the technical issues are also reviewed and discussed. These issues include the efficacy of probiotic bacteria in non-chilled, low pH or high water activity foods; the potential loss of bacterial viability, additionally unwanted fermentation and changes of the sensory characteristics of food products which may result in poor microbiological quality and low acceptability to consumers.

Computed tomography provides enhanced techniques for longitudinal monitoring of progressive intracranial volume loss associated with regional neurodegeneration in ovine neuronal ceroid lipofuscinoses.

INTRODUCTION: The neuronal ceroid lipofuscinoses (NCLs; Batten disease) are a group of fatal neurodegenerative lysosomal storage diseases of children caused by various mutations in a range of genes. Forms associated with mutations in two of these, CLN5 and CLN6, are being investigated in well-established sheep models. Brain atrophy leading to psychomotor degeneration is among the defining features, as is regional progressive ossification of the inner cranium. Ongoing viral-mediated gene therapy trials in these sheep are yielding encouraging results. In vivo assessment of brain atrophy is integral to the longitudinal monitoring of individual animals and provides robust data for translation to treatments for humans. METHODS: Computed tomography (CT)-based three-dimensional reconstruction of the intracranial volume (ICV) over time reflects the progression of cortical brain atrophy, verifying the use of ICV measurements as a surrogate measure for brain size in ovine NCL. RESULTS: ICVs of NCL-affected sheep increase for the first few months, but then decline progressively between 5 and 13 months in CLN5-/- sheep and 11-15 months in CLN6-/- sheep. Cerebral ventricular volumes are also increased in affected animals. To facilitate ICV measures, the radiodensities of ovine brain tissue and cerebrospinal fluid were identified. Ovine brain tissue exhibited a Hounsfield unit (HU) range of (24; 56) and cerebrospinal fluid a HU range of (-12; 23). CONCLUSIONS: Computed tomography scanning and reconstruction verify that brain atrophy ovine CLN5 NCL originates in the occipital lobes with subsequent propagation throughout the whole cortex and these regional differences are reflected in the ICV loss.

Development of customised 3D printed biodegradable projectile for administrating extended-release contraceptive to wildlife.

Customisation of sustained and controlled release of contraceptives plays a key role in veterinary applications. A biodegradable projectile containing different doses of contraceptive progesterone was prepared using fused deposition modelling 3D printing. Three concentrations of progesterone (2, 5 and 10% w/w) with polylactic acid was prepared as a 1.75 mm filament by hot melt extrusion. Solvent dissolution tests confirmed the successful incorporation of progesterone in the polymer while microscopic (SEM) studies indicated the drug was melted and thoroughly mixed with the polymer matrix and pore-formation after dissolution. A significant suppression of melting temperature of polymer from 166 to 145 °C was noted by thermal analysis (DSC) studies of the drug loaded systems. Interaction between the contraceptive drug and the polymer via hydrogen bonding was revealed from the spectroscopic (FTIR) studies. In vitro release behaviour was assessed over a five-month period, for 2% and 5% progesterone loaded projectiles release profiles fitted zero order whereas 10% loaded projectiles fitted the Higuchi model. Penetration assessment confirmed the drug loaded PLA projectiles provided sufficient specific kinetic energy required to penetrate thin and medium-thickness skins. This work demonstrates the feasibility of fused deposition modelling 3D printing as suitable process for manufacturing ballistic customised drug delivery devices.

Drug delivery to the lens for the management of cataracts.

Cataracts are one of the most prevalent diseases of the lens, affecting its transparency and are the leading cause of reversible blindness in the world. The clarity of the lens is essential for its normal physiological function of refracting light onto the retina. Currently there is no pharmaceutical treatment for prevention or cure of cataracts and surgery to replace the affected lens remains the gold standard in the management of cataracts. Pharmacological treatment for prevention of cataracts is hindered by many physiological barriers that must be overcome by a therapeutic agent to reach the avascular lens. Various therapeutic agents and formulation strategies are currently being investigated to prevent cataract formation as access to surgery is limited. This review provides a summary of recent research in the field of drug delivery to the lens for the management of cataracts including models used to study cataract treatments and discusses the future perspectives in the field.

Short communication: A study of Lactobacillus isolates' adherence to and influence on membrane integrity of human Caco-2 cells.

The selection criteria of ideal probiotic bacteria are complex and involve many factors. One key criterion is based on the ability of the probiotic bacteria to adhere to the epithelial lining of the gastrointestinal tract. The objective of this study was to evaluate and compare the adherence and influence on membrane integrity of 2 selected lactobacilli isolates-Lactobacillus rhamnosus MI13 (dairy food origin) and L. plantarum RC2 (bovine rumen origin)-to Caco-2 cells in the presence and absence of Escherichia coli. The adhesion and influence on membrane integrity properties of the 2 Lactobacillus isolates were compared with Escherichia coli, a human commensal bacterium. From the adhesion studies, we concluded that the bovine rumen isolate exhibited better adherence to Caco-2 cells than the dairy food isolate. In contrast, the dairy food isolate better protected the Caco-2 monolayer from damage induced by ethanol.

Effect of Non-Dairy Food Matrices on the Survival of Probiotic Bacteria during Storage.

The viability of probiotics in non-dairy food products during storage is required to meet content criteria for probiotic products. This study investigated whether non-dairy foods could be matrices for probiotics. Selected probiotic bacteria were coated on non-dairy foods under two storage conditions, and viabilities were assessed. The non-dairy foods were coated with 5-7 log cfu g-1 of Lactobacillus acidophilus ATCC4356T, Lactobacillus plantarum RC30, and Bifidobacterium longum ATCC15707T. The coated non-dairy foods were stored at 20 °C and 20% relative humidity (RH) or 30 °C and 50% RH. Viability of probiotic bacteria was determined after 0, 2, and 4 weeks of storage. B. longum showed the highest survival at week 4 of 6.5-6.7 log cfu g-1 on wheat bran and oat, compared with 3.7-3.9 log cfu g-1 of L. acidophilus and 4.2-4.8 log cfu g-1 of L. plantarum at 20 °C 20% RH. Under the storage conditions of 30 °C 50% RH, survival of 4.5 log cfu g-1 of B. longum was also found on oat and peanut. This was two and four times higher than the population of L. acidophilus and L. plantarum, respectively. The results suggest that probiotics can survive on non-dairy foods under ambient storage conditions. However, the storage conditions, food matrices, and probiotic strains should be carefully chosen to maximize probiotic bacteria survival.

Coated Solid Substrate Microbe Formulations: Pseudomonas spp. and Zeolite.

Formulation provides a means to stabilize for storage and delivery biocontrol and bioremediation agents based on microbes such as bacteria and fungi. Typically it is difficult to both stabilize and deliver fragile non-spore-forming bacteria. Fungal spores might intuitively appear to be easy to stabilize; however, their tendency to germinate in low moisture environments presents challenges for the formulation scientist. Here we present a light background regarding issues with formulating microbes and strategies to help overcome instability and delivery issues.

Comparison of Microbiological and Probiotic Characteristics of Lactobacilli Isolates from Dairy Food Products and Animal Rumen Contents.

Lactobacilli are employed in probiotic food preparations and as feed additives in poultry and livestock, due to health benefits associated with their consumption. The objective of this study was to evaluate and compare the probiotic potential of ten lactobacilli strains isolated from commercial dairy food products and animal rumen contents in New Zealand. Genetic identification of the isolates revealed that all belonged to the genus Lactobacillus, specifically the species L. reuteri, L. rhamnosus and L. plantarum. All isolates did not show any haemolytic behaviour. Isolates of dairy origin showed better tolerance to low pH stress. On the other hand, rumen isolates exhibited a higher tolerance to presence of bile salts. All isolates exhibited resistance to aminoglycoside antibiotics, however most were sensitive to ampicillin. Isolates of rumen origin demonstrated a higher inhibitory effect on Listeria monocytogenes, Enterobacter aerogenes and Salmonella menston. Bacterial adherence of all isolates increased with a decrease in pH. This screening study on lactobacilli isolates has assessed and identified potential probiotic candidates for further evaluation.

Design and characterisation of a polyethylene oxide matrix with the potential use as a teat insert for prevention/treatment of bovine mastitis.

This manuscript reports (for the first time) on antibiotic-free polymeric inserts for the prevention and/or treatment of bovine mastitis. Polyethylene oxide (PEO)-based inserts were prepared using different concentrations of various hydrophilic polymers and water-soluble and water-insoluble drug-release-modifying excipients. A simple and scalable melt-extrusion method was employed to prepare the inserts. The prepared inserts were characterised for their dimension, rheological and mechanical properties. The in vitro release of a model bacteriostatic drug (salicylic acid) from the prepared inserts was studied to demonstrate the effectiveness and reproducibility of the melt-extrusion manufacturing method. Further, the in vitro stability of the inserts was evaluated using gel permeation chromatography (GPC) to monitor any change in molecular weight under real-time and accelerated storage conditions. The investigated inserts were stable at accelerated storage conditions over a period of 6 months. PEO inserts have the potential to serve a dual purpose, act as a physical barrier against pathogens invading the teat canal of cows and possibly control the release of a drug.

Characterization and evaluation of ß-glucan formulations as injectable implants for protein and peptide delivery.

CONTEXT: Injectable implants are biodegradable, syringeable formulations that are injected as liquids, but form a gel inside the body due to a change in pH, ions or temperature. OBJECTIVE: To investigate the effect of polymer concentration, pH, ions and temperature on the gel formation of ß-glucan, a natural cell-wall polysaccharide derived from barley, with particular emphasis on two-phase system formation after addition of dextran or PEG. MATERIALS AND METHODS: Oscillation viscometry was used to evaluate the gel character by measuring flow index (N), storage (G') and loss (G") moduli. Two-phase systems were further characterized for hardness and syringeability using a texture analyzer. Finally, in vitro release characteristics were determined using Franz diffusion cells. RESULTS: Oscillation viscometry revealed that only addition of dextran or PEG resulted in distinct gel formation. This was seen by a decrease in N after polymer addition. Moreover, hardness (in g) of the gels increased significantly (p < 0.001) from 3.65 ± 0.43 to 34.30 ± 8.90 (dextran) and 805.80 ± 5.30 (PEG) 24 h after polymer addition. In vitro release profiles showed significantly (p < 0.05) reduced AUC(0-8 h), k and percentage of drug released from two-phase systems compared to ß-glucan dispersions, with the PEG system resulting in the lowest amount released over 8 h (15.1 ± 1.6%). DISCUSSION: The unfavorable mixing enthalpy and higher water affinity of PEG resulted in the formation of a dense ß-glucan gel. CONCLUSION: 1.5% (w/w) ß-glucan combined with PEG at a ratio of 1:3 seemed to be the most promising injectable formulation with respect to fastest gel formation, increased hardness and sustained release.

Control, communication and monitoring of intravaginal drug delivery in dairy cows.

We present the design of an electronically controlled drug delivery system. The intravaginally located device is a low-invasive platform that can measure and react inside the cow vagina while providing external control and monitoring ability. The electronics manufactured from off the shelf components occupies 16 mL of a Theratron syringe. A microcontroller reads and logs sensor data and controls a gascell. The generated gas pressure propels the syringe piston and releases the formulation. A two way radio link allows communication between other devices or a base station. Proof of principle experiments confirm variable-rate, arbitrary profile drug delivery qualified by internal sensors. A total volume of 30 mL was dispensed over a 7-day-period with a volume error of +/- 1 mL or +/- 7% for larger volumes. Delivery was controlled or overridden via the wireless link, and proximity to other devices was detected and recorded. The results suggest that temperature and activity sensing or social grouping determined via proximity can be used to detect oestrus and trigger appropriate responses.

Development of an injection molded poly(epsilon-caprolactone) intravaginal insert for the delivery of progesterone to cattle.

This paper reports experiments conducted to research, develop and clinically evaluate an injection molded intravaginal insert manufactured from the biodegradable polyester poly(epsilon-caprolactone). The study demonstrated that it is possible to engineer poly(epsilon-caprolactone) into a shape that is well retained, and can be used as a platform for the controlled delivery of progesterone via the vagina of cows. Field evaluation showed that the poly(epsilon-caprolactone) intravaginal inserts containing 10% (w/w) progesterone were at least as effective clinically as the commercially available CIDR intravaginal insert.

Reengineering of a commercially available bovine intravaginal insert (CIDR insert) containing progesterone.

The purpose of this study was to reengineer a commercially available intravaginal insert containing 1.9 g progesterone (CIDR intravaginal insert) for a 7-day insertion period in cattle. The reengineering process resulted in a reduced initial drug load (1.38 g) and a reduction in the residual drug load following insertion, while at the same time maintaining the biological performance of the insert. The in vitro and in vivo pharmaceutical properties of the commercially available CIDR intravaginal insert were characterized initially to gain a thorough understanding of the factors that affected progesterone release from the insert. The effect of changing a selection of formulation and physical variables of the insert was also investigated (including surface area, drug load, addition of pore forming materials, silicone shore hardness and drug particle size). The knowledge gained from these studies was used to define the characteristics of the reengineered insert which was then manufactured and shown to be bioequivalent and clinically equivalent to the commercially available insert.