Immunogenic fusion proteins induce neutralizing SARS-CoV-2 antibodies in the serum and milk of sheep.

Antigen-specific polyclonal immunoglobulins derived from the serum, colostrum, or milk of immunized ruminant animals have potential as scalable therapeutics for the control of viral diseases including COVID-19. Here we show that the immunization of sheep with fusions of the SARS-CoV-2 receptor binding domain (RBD) to ovine IgG2a Fc domains promotes significantly higher levels of antigen-specific antibodies compared to native RBD or full-length spike antigens. This antibody population contained elevated levels of neutralizing antibodies that suppressed binding between the RBD and hACE2 receptors in vitro. A second immune-stimulating fusion candidate, Granulocyte-macrophage colony-stimulating factor (GM-CSF), induced high neutralizing responses in select animals but narrowly missed achieving significance. We further demonstrated that the antibodies induced by these fusion antigens were transferred into colostrum/milk and possessed cross-neutralizing activity against diverse SARS-CoV-2 variants. Our findings highlight a new pathway for recombinant antigen design in ruminant animals with applications in immune milk production and animal health.

Short-term postprandial changes in select serum biochemistry analytes in healthy adult cats.

OBJECTIVES: The aim of this study was to evaluate the magnitude, duration and significance of postprandial changes to select serum biochemistry analytes in healthy adult cats in the 12 h period after a meal. METHODS: Nine adult research cats fed commercial food were included. Blood samples were taken after a 12 h fast (hour 0), cats were offered and consumed a meal, and postprandial samples were obtained over a 12 h period starting 2 h after the baseline blood draw (hours 2, 4, 6, 8, 10 and 12). Serum samples were run on a Roche Cobas C501 chemistry analyzer to obtain concentrations of blood urea nitrogen (BUN), creatinine, phosphorus, total calcium, bicarbonate, cholesterol, magnesium, sodium, potassium and chloride. Serum concentrations of each analyte at hours 2, 4, 6, 8, 10 and 12 were compared with concentrations prior to feeding. RESULTS: Serum concentration for at least one postprandial time point was different from baseline fasted concentration for BUN (hour 2, P = 0.006; hour 4, P <0.0001; hour 6, P = 0.002; hour 8, P = 0.026), phosphorus (hour 2, P = 0.019), bicarbonate (hours 2, 4, 6, 8 and 10; all P <0.01), glucose (hour 12, P = 0.014), magnesium (hour 10, P = 0.029) and chloride (hour 2, P = 0.026; hour 4, P = 0.044; hour 12, P = 0.019). No significant difference was seen at any postprandial sampling point compared with baseline for serum creatinine, total calcium, cholesterol, sodium or potassium concentrations. CONCLUSIONS AND RELEVANCE: Short-term postprandial serum concentrations of BUN, phosphorus, bicarbonate and chloride differed at multiple time points within a 12 h period compared with the fasted state at baseline, with most values remaining within the reference intervals. Veterinarians should be aware of these alterations, though they are unlikely to be mistaken for pathological disease states in healthy adult cats.

The impacts of bovine milk, soy beverage, or almond beverage on the growing rat microbiome.

Background: Milk, the first food of mammals, helps to establish a baseline gut microbiota. In humans, milk and milk products are consumed beyond infancy, providing comprehensive nutritional value. Non-dairy beverages, produced from plant, are increasingly popular as alternatives to dairy milk. The nutritive value of some plant-based products continues to be debated, whilst investigations into impacts on the microbiome are rare. The aim of this study was to compare the impact of bovine milk, soy and almond beverages on the rat gut microbiome. We previously showed soy and milk supplemented rats had similar bone density whereas the almond supplemented group had compromised bone health. There is an established link between bone health and the microbiota, leading us to hypothesise that the microbiota of groups supplemented with soy and milk would be somewhat similar, whilst almond supplementation would be different. Methods: Three-week-old male Sprague Dawley rats were randomly assigned to five groups (n = 10/group) and fed ad libitum for four weeks. Two control groups were fed either standard diet (AIN-93G food) or AIN-93G amino acids (AA, containing amino acids equivalent to casein but with no intact protein) and with water provided ad libitum. Three treatment groups were fed AIN-93G AA and supplemented with either bovine ultra-heat treatment (UHT) milk or soy or almond UHT beverages as their sole liquid source. At trial end, DNA was extracted from caecum contents, and microbial abundance and diversity assessed using high throughput sequencing of the V3 to V4 variable regions of the 16S ribosomal RNA gene. Results: Almost all phyla (91%) differed significantly (FDR < 0.05) in relative abundance according to treatment and there were distinct differences seen in community structure between treatment groups at this level. At family level, forty taxa showed significantly different relative abundance (FDR < 0.05). Bacteroidetes (Bacteroidaceae) and Firmicutes populations (Lactobacillaceae, Clostridiaceae and Peptostreptococcaceae) increased in relative abundance in the AA almond supplemented group. Supplementation with milk resulted in increased abundance of Actinobacteria (Coriobacteriaceae and Bifidobacteriaceae) compared with other groups. Soy supplementation increased abundance of some Firmicutes (Lactobacilliaceae) but not Actinobacteria, as previously reported by others. Conclusion: Supplementation with milk or plant-based drinks has broad impacts on the intestinal microbiome of young rats. Changes induced by cow milk were generally in line with previous reports showing increased relative abundance of Bifidobacteriacea, whilst soy and almond beverage did not. Changes induced by soy and almond drink supplementation were in taxa commonly associated with carbohydrate utilisation. This research provides new insight into effects on the microbiome of three commercially available products marketed for similar uses.

Surface-reacted calcium carbonate microparticles as templates for lactoferrin encapsulation.

Microencapsulation helps to improve bioavailability of a functional whey protein, lactoferrin (Lf), in adults. Herein, we report the Lf loading capacity (LC) and retention efficiency (RE) in the microparticles of surface-reacted calcium carbonate (SRCC) of different types and compare them to those of widely used vaterite microparticles. The LCs and REs are analyzed in connection to the total surface area and the volume of intraparticle pores. The best performing SRCC3 demonstrates Lf LC of 11.00 wt% achieved in a single absorption step and 74% RE after two cycles of washing with deionized water. A much larger surface area of SRCC templates and a lower pH required to release Lf do not affect its antitumor activity in MCF-7 assay. Layer-by-Layer assembly of pepsin-tannic acid multilayer shell around Lf-loaded microparticles followed by acidic decomposition of the inorganic core produces microencapsulated Lf with a yield ~36 times higher than from vaterite templates reported earlier, while the scale of encapsulated Lf production is ~12,000 times larger. In vitro digestion tests demonstrate the protection of ~65% of encapsulated Lf from gastric digestion. The developed capsules are prospective candidates for functional foods fortified with Lf.

Supplementation with Bovine Milk or Soy Beverages Recovers Bone Mineralization in Young Growing Rats Fed an Insufficient Diet, in Contrast to an Almond Beverage.

BACKGROUND: Nondairy beverages, produced from soy, rice, oat, almond, or coconut, are increasingly being used as alternatives to dairy milk, with the perception that they are healthier and/or more sustainable products than dairy products. OBJECTIVE: The aim of this study was to compare the effects of supplementing either bovine milk, soy, or almond-based beverages to young, growing rats fed an intact-protein diet or a diet that had protein substituted with amino acids (AA-diet). METHODS: Three-week-old male Sprague-Dawley rats were randomly assigned to 5 groups (n = 10/group) and fed ad libitum for 4 wk. Two control groups were fed either standard AIN-93G food [20% casein (CN) protein] or AIN-93G with amino acids (AAs) equivalent to CN protein, and water to drink. Three treatment groups were fed AIN-93G AA and supplemented with either bovine ultra-heat treatment (UHT) milk or soy or almond UHT beverages. Rat weight gain and food intakes were recorded. During week 4, body composition was assessed using DEXA to determine lean soft tissue, fat, and bone mass. At trial end, bone biomechanical properties and blood plasma mineral concentrations were measured. RESULTS: At the end of the trial, animals supplemented with almond beverage were lightest (P > 0.05), with higher plasma calcium concentrations (P > 0.05) and lower bone mineral content (BMC) and bone density (P > 0.05) than animals supplemented with milk or soy beverage. Soy-supplemented animals had similar BMC and bone density compared with milk-supplemented animals, although the soy group gained most weight (P > 0.05) and had the highest fat:lean ratio (P > 0.05) compared with other groups. CONCLUSIONS: In the model tested, supplementing rats with bovine UHT milk and soy UHT beverage provided favorable bone health outcomes. Conversely, almond UHT beverage was not an effective supplement and could be detrimental to bone mineralization and strength outcomes.

Gastric digestion of cow and goat milk: Peptides derived from simulated conditions of infant digestion.

Infant formula products are predominantly manufactured using cow milk protein; goat milk also provides a suitable protein source. In this study, we directly compared cow and goat milk protein digestion using pH and enzyme conditions to simulate infant gastric conditions. Generated peptides, identified using liquid chromatography coupled to a mass spectrometer, show both similarities and differences in cow and goat milk post-digestion profiles. The majority of peptides were from casein proteins, 50% representing ß-casein, with many peptides unique to each species. Low or no peptides for ß-Lactoglobulin and α-Lactalbumin, respectively, suggest these proteins were highly resistant to infant gastric digestion, as reported by others. Minor milk proteins, comprising 5% of peptides, were represented by different proteins from cow and goat. Peptides with known bioactivities were also observed, both in common and unique to each species. Together these data may explain reported differences in digestion characteristics of cow and goat milk.

Bovine milk derived skimmed milk powder and whey protein concentrate modulates Citrobacter rodentium shedding in the mouse intestinal tract.

Skimmed milk powder (SMP) and whey protein concentrate (WPC) were manufactured from fresh milk collected from cows producing high or low Immunoglobulin (Ig) A levels in their milk. In addition commercial products were purchased for use as diluent or control treatments. A murine enteric disease model (Citrobacter rodentium) was used to assess whether delivery of selected bioactive molecules (IgA, IgG, Lactoferrin (Lf)) or formulation delivery matrix (SMP, WPC) affected faecal shedding of bacteria in C. rodentium infected mice. In trial one, faecal pellets collected from mice fed SMP containing IgA (0.007-0.35 mg/mL), IgG (0.28-0.58 mg/mL) and Lf (0.03-0.1 mg/mL) contained fewer C. rodentium (cfu) compared to control mice fed water (day 8, p < 0.04, analysis of variance (ANOVA) followed by Fisher's unprotected least significant difference (ULSD)). In trial two, WPC containing IgA (0.35-1.66 mg/mL), IgG (0.58-2.36 mg/mL) and Lf (0.02-0.45 mg/mL) did not affect C. rodentium shedding, but SMP again reduced faecal C. rodentium levels (day 12, p < 0.04, ANOVA followed by Fisher's ULSD). No C. rodentium was detected in sham phosphate-buffered saline inoculated mice. Mice fed a commercial WPC shed significantly greater numbers of C. rodentium over 4 consecutive days (Fishers ULSD test), compared to control mice fed water. These data indicate that SMP, but not WPC, modulates faecal shedding in C. rodentium-infected mice and may impact progression of C. rodentium infection independently of selected bioactive concentration. This suggests that food matrix can impact biological effects of foods.

Effect of the dietary delivery matrix on vitamin D3 bioavailability and bone mineralisation in vitamin-D3-deficient growing male rats.

This study assessed bioavailability and utilisation of vitamin D3 in two feeding trials using young, growing Sprague-Dawley male rats. Trial one fed animals standard AIN-93G diet (casein protein) containing no vitamin D3 and goat or cow skimmed milk supplemented with vitamin D3. Trial two fed animals modified dairy-free AIN-93G diet (egg albumin) containing no vitamin D3 and goat or cow skimmed or full-fat milk supplemented with vitamin D3. Control groups received AIN-93G diets with or without vitamin D, and water. At 8 weeks of age, blood samples were collected for vitamin and mineral analysis, and femurs and spines were collected for assessment of bone mineralisation and strength. In both trials, analyses showed differences in bioavailability of vitamin D3, with ratios of serum 25-hydroxyvitamin D3 to vitamin D3 intake more than 2-fold higher in groups drinking supplemented milk compared with groups fed supplemented solid food. Bone mineralisation was higher in groups drinking supplemented milk compared with groups fed supplemented solid food, for both trials (P<0·05). There was no difference in the parameters tested between skimmed milk and full-fat milk or between cow milk and goat milk. Comparison of the two trials suggested that dietary protein source promoted bone mineralisation in a growing rat model: modified AIN-93G with egg albumin produced lower bone mineralisation compared with standard AIN-93G with casein. Overall, this study showed that effects of vitamin D3 deficiency in solid diets were reversed by offering milk supplemented with vitamin D3, and suggests that using milk as a vehicle to deliver vitamin D is advantageous.

Gastric digestion of cow and goat milk: Impact of infant and young child in vitro digestion conditions.

Many infants and young children are fed nutritional milk formulas. Although products are commonly based on cow milk, goat milk provides an alternative. We directly compared digestion of cow and goat milk proteins, varying pH, enzyme concentrations and incubation times to simulate infant and young child gastric conditions. Protein digestion and peptide formation were evaluated using electrophoresis and chromatography. Digestion of higher molecular weight whey proteins increased with decreased pH and higher enzyme concentrations of young child gastric digestion conditions compared to infant conditions. ß-lactoglobulin was poorly digested under all gastric digestion conditions. Caseins reacted to pH changes differently compared to whey proteins, with less digestion of casein at pH 3.0 than at pH 5.0. Caseins from goat milk tended to be more efficiently digested compared to caseins from cow milk and peptide profiles from goat milk were distinct from cow milk.

Canine distemper virus neutralization activity is low in human serum and it is sensitive to an amino acid substitution in the hemagglutinin protein.

Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions, which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies.

Transfer of intestinal bacterial components to mammary secretions in the cow.

Results from large multicentre epidemiological studies suggest an association between the consumption of raw milk and a reduced incidence of allergy and asthma in children. Although the underlying mechanisms for this association are yet to be confirmed, researchers have investigated whether bacteria or bacterial components that naturally occur in cow's milk are responsible for modulating the immune system to reduce the risk of allergic diseases. Previous research in human and mice suggests that bacterial components derived from the maternal intestine are transported to breast milk through the bloodstream. The aim of our study was to assess whether a similar mechanism of bacterial trafficking could occur in the cow. Through the application of culture-independent methodology, we investigated the microbial composition and diversity of milk, blood and feces of healthy lactating cows. We found that a small number of bacterial OTUs belonging to the genera Ruminococcus and Bifidobacterium, and the Peptostreptococcaceae family were present in all three samples from the same individual animals. Although these results do not confirm the hypothesis that trafficking of intestinal bacteria into mammary secretions does occur in the cow, they support the existence of an endogenous entero-mammary pathway for some bacterial components during lactation in the cow. Further research is required to define the specific mechanisms by which gut bacteria are transported into the mammary gland of the cow, and the health implications of such bacteria being present in milk.

Development of a duplex real-time RT-qPCR assay to monitor genome replication, gene expression and gene insert stability during in vivo replication of a prototype live attenuated canine distemper virus vector encoding SIV gag.

Advancement of new vaccines based on live viral vectors requires sensitive assays to analyze in vivo replication, gene expression and genetic stability. In this study, attenuated canine distemper virus (CDV) was used as a vaccine delivery vector and duplex 2-step quantitative real-time RT-PCR (RT-qPCR) assays specific for genomic RNA (gRNA) or mRNA have been developed that concurrently quantify coding sequences for the CDV nucleocapsid protein (N) and a foreign vaccine antigen (SIV Gag). These amplicons, which had detection limits of about 10 copies per PCR reaction, were used to show that abdominal cavity lymphoid tissues were a primary site of CDV vector replication in infected ferrets, and importantly, CDV gRNA or mRNA was undetectable in brain tissue. In addition, the gRNA duplex assay was adapted for monitoring foreign gene insert genetic stability during in vivo replication by analyzing the ratio of CDV N and SIV gag genomic RNA copies over the course of vector infection. This measurement was found to be a sensitive probe for assessing the in vivo genetic stability of the foreign gene insert.

Membrane-bound SIV envelope trimers are immunogenic in ferrets after intranasal vaccination with a replication-competent canine distemper virus vector.

We are investigating canine distemper virus (CDV) as a vaccine vector for the delivery of HIV envelope (Env) that closely resembles the native trimeric spike. We selected CDV because it will promote vaccine delivery to lymphoid tissues, and because human exposure is infrequent, reducing potential effects of pre-existing immunity. Using SIV Env as a model, we tested a number of vector and gene insert designs. Vectors containing a gene inserted between the CDV H and L genes, which encoded Env lacking most of its cytoplasmic tail, propagated efficiently in Vero cells, expressed the immunogen on the cell surface, and incorporated the SIV glycoprotein into progeny virus particles. When ferrets were vaccinated intranasally, there were no signs of distress, vector replication was observed in the gut-associated lymphoid tissues, and the animals produced anti-SIV Env antibodies. These data show that live CDV-SIV Env vectors can safely induce anti-Env immune responses following intranasal vaccination.

The Germinative Epithelium of Sheep Vibrissae and Wool Follicles has Extensive Proliferative Potential but is Dependent on the Dermal Papilla.

AIM: To investigate the growth potential of keratinocytes derived from the germinative epithelium (GE) of ovine hair follicles. Stem cells from the outer root sheath (ORS) of hair follicles migrate to the GE in the lower follicle where they proliferate and differentiate to form the hair fiber. It has been suggested that the GE comprises transit-amplifying cells and that the duration of anagen is determined by their limited proliferative potential. However, we show here that keratinocytes derived from the GE of ovine follicles grow extensively in vitro, arguing against this hypothesis. MATERIALS AND METHODS: Primary cultures of keratinocytes were initiated from microdissected GE tissue from ovine vibrissae and wool follicles. Clonal lines of keratinocytes were derived by limiting dilution. Their growth potential was determined by exhaustive serial passaging. Expression of differentiation markers was evaluated by real-time polymerase chain reaction. RESULTS: Initiation of these cultures required that interaction between the GE and dermal papilla was maintained. However, the keratinocytes could subsequently be cloned and were grown as pure cell populations for 26-52 cell doublings. This proliferative potential is several orders of magnitude greater than required to maintain a single anagen phase. The keratinocytes were indistinguishable from ORS keratinocytes from the same follicles, expressing K14 while undifferentiated, and upregulating the epidermal and inner root sheath markers, loricrin and KRT27 on differentiation. Thus, these cells initially depend on papilla-derived signals to grow, but can revert to an ORS-like phenotype in vitro. Their extensive proliferative capacity shows that the GE is not an exclusively transit-amplifying cell population.

Annotation of sheep keratin intermediate filament genes and their patterns of expression.

Keratin IF (KRT) and keratin-associated protein genes encode the majority of wool and hair proteins. We have identified cDNA sequences representing nine novel sheep KRT genes, increasing the known active genes from eight to 17, a number comparable to that in the human. However, the absence of KRT37 in the type I family and the discovery of type II KRT87 in sheep exemplify species-specific compositional differences in hair KRT genes. Phylogenetic analysis of hair KRT genes within type I and type II families in the sheep, cattle and human genomes revealed a high degree of consistency in their sequence conservation and grouping. However, there were differences in the fibre compartmentalisation and keratinisation zones for the expression of six ovine KRT genes compared with their human orthologs. Transcripts of three genes (KRT40, KRT82 and KRT84) were only present in the fibre cuticle. KRT32, KRT35 and KRT85 were expressed in both the cuticle and the fibre cortex. The remaining 11 genes (KRT31, KRT33A, KRT33B, KRT34, KRT36, KRT38-39, KRT81, KRT83 and KRT86-87) were expressed only in the cortex. Species-specific differences in the expressed keratin gene sets, their relative expression levels and compartmentalisation are discussed in the context of their underlying roles in wool and hair developmental programmes and the distinctive characteristics of the fibres produced.