Ovine CD14- an Immune Response Gene Has a Role Against Gastrointestinal Nematode Haemonchus contortus-A Novel Report.

CD14 (also known as the monocyte differentiation antigen) is an important immune response gene known to be primarily responsible for innate immunity against bacterial pathogens, and as a pattern recognition receptor (PRR), binds with LPS (endotoxin), lipoproteins, and lipotechoic acid of bacteria. So far very limited work has been conducted in parasitic immunology. In the current study, we reported the role of CD14 in parasitic immunology in livestock species (sheep) for the first time. Ovine CD14 is characterized as a horse-shoe shaped bent solenoid with a hydrophobic amino-terminal pocket for CD14 along with domains. High mutation frequency was observed, out of total 41 mutations identified, 23 mutations were observed to be thermodynamically unstable and 11 mutations were deleterious in nature, causing major functional alteration of important domains of CD14, an indication of variations in individual susceptibility for sheep against Haemonchus contortus infestations. In silico studies with molecular docking reveal a role of immune response against Haemonchus contortus in sheep, which is later confirmed with experimental evidence through differential mRNA expression analysis for sheep, which revealed better expression of CD14 in Haemonchus contortus infected sheep compared to that of non-infected sheep. We confirmed the above findings with supportive evidence through haematological and biochemical analyses. Phylogenetic analysis was conducted to assess the evolutionary relationship with respect to humans and it was observed that sheep may well be used as model organisms due to better genetic closeness compared to that of mice.

Developing ovine mammary terminal duct lobular units have a dynamic mucosal and stromal immune microenvironment.

The human breast and ovine mammary gland undergo striking levels of postnatal development, leading to formation of terminal duct lobular units (TDLUs). Here we interrogate aspects of sheep TDLU growth as a model of breast development and to increase understanding of ovine mammogenesis. The distributions of epithelial nuclear Ki67 positivity differ significantly between younger and older lambs. Ki67 expression is polarised to the leading edge of the developing TDLUs. Intraepithelial ductal macrophages exhibit periodicity and considerably increased density in lambs approaching puberty. Stromal macrophages are more abundant centrally than peripherally. Intraepithelial T lymphocytes are more numerous in older lambs. Stromal hotspots of Ki67 expression colocalize with immune cell aggregates that exhibit distinct organisation consistent with tertiary lymphoid structures. The lamb mammary gland thus exhibits a dynamic mucosal and stromal immune microenvironment and constitutes a valuable model system that provides new insights into postnatal breast development.

Genes involved in immune, gene translation and chromatin organization pathways associated with Mycoplasma ovipneumoniae presence in nasal secretions of domestic sheep.

Mycoplasma ovipneumoniae contributes to polymicrobial pneumonia in domestic sheep. Elucidation of host genetic influences of M. ovipneumoniae nasal detection has the potential to reduce the incidence of polymicrobial pneumonia in sheep through implementation of selective breeding strategies. Nasal mucosal secretions were collected from 647 sheep from a large US sheep flock. Ewes of three breeds (Polypay n = 222, Rambouillet n = 321, and Suffolk n = 104) ranging in age from one to seven years, were sampled at three different times in the production cycle (February, April, and September/October) over four years (2015 to 2018). The presence and DNA copy number of M. ovipneumoniae was determined using a newly developed species-specific qPCR. Breed (P<0.001), age (P<0.024), sampling time (P<0.001), and year (P<0.001) of collection affected log10 transformed M. ovipneumoniae DNA copy number, where Rambouillet had the lowest (P<0.0001) compared with both Polypay and Suffolk demonstrating a possible genetic component to detection. Samples from yearlings, April, and 2018 had the highest (P<0.046) detected DNA copy number mean. Sheep genomic DNA was genotyped with the Illumina OvineHD BeadChip. Principal component analysis identified most of the variation in the dataset was associated with breed. Therefore, genome wide association analysis was conducted with a mixed model (EMMAX), with principal components 1 to 6 as fixed and a kinship matrix as random effects. Genome-wide significant (P<9x10-8) SNPs were identified on chromosomes 6 and 7 in the all-breed analysis. Individual breed analysis had genome-wide significant (P<9x10-8) SNPs on chromosomes 3, 4, 7, 9, 10, 15, 17, and 22. Annotated genes near these SNPs are part of immune (ANAPC7, CUL5, TMEM229B, PTPN13), gene translation (PIWIL4), and chromatin organization (KDM2B) pathways. Immune genes are expected to have increased expression when leukocytes encounter M. ovipneumoniae which would lead to chromatin reorganization. Work is underway to narrow the range of these associated regions to identify the underlying causal mutations.

IMGT® Biocuration and Comparative Analysis of Bos taurus and Ovis aries TRA/TRD Loci.

The adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens to generate immunity, and mount stronger attacks each time the pathogen is encountered. T cell receptors are the antigen receptors of the adaptive immune response expressed by T cells, which specifically recognize processed antigens, presented as peptides by the highly polymorphic major histocompatibility (MH) proteins. T cell receptors (TR) are divided into two groups, αß and γδ, which express distinct TR containing either α and ß, or γ and δ chains, respectively. The TRα locus (TRA) and TRδ locus (TRD) of bovine (Bos taurus) and the sheep (Ovis aries) have recently been described and annotated by IMGT® biocurators. The aim of the present study is to present the results of the biocuration and to compare the genes of the TRA/TRD loci among these ruminant species based on the Homo sapiens repertoire. The comparative analysis shows similarities but also differences, including the fact that these two species have a TRA/TRD locus about three times larger than that of humans and therefore have many more genes which may demonstrate duplications and/or deletions during evolution.

Whole transcriptome approach to evaluate the effect of aluminium hydroxide in ovine encephalon.

Aluminium hydroxide adjuvants are crucial for livestock and human vaccines. Few studies have analysed their effect on the central nervous system in vivo. In this work, lambs received three different treatments of parallel subcutaneous inoculations during 16 months with aluminium-containing commercial vaccines, an equivalent dose of aluminium hydroxide or mock injections. Brain samples were sequenced by RNA-seq and miRNA-seq for the expression analysis of mRNAs, long non-coding RNAs and microRNAs and three expression comparisons were made. Although few differentially expressed genes were identified, some dysregulated genes by aluminium hydroxide alone were linked to neurological functions, the lncRNA TUNA among them, or were enriched in mitochondrial energy metabolism related functions. In the same way, the miRNA expression was mainly disrupted by the adjuvant alone treatment. Some differentially expressed miRNAs had been previously linked to neurological diseases, oxidative stress and apoptosis. In brief, in this study aluminium hydroxide alone altered the transcriptome of the encephalon to a higher degree than commercial vaccines that present a milder effect. The expression changes in the animals inoculated with aluminium hydroxide suggest mitochondrial disfunction. Further research is needed to elucidate to which extent these changes could have pathological consequences.

Ovine viperin inhibits bluetongue virus replication.

Viral infections can lead to interferon production, which achieves its antiviral function primarily by activating the JAK/STAT pathway and inducing multiple interferon-stimulated genes (ISGs). Although considerable ISGs have been identified in antiviral researches, little is known about ISGs in bluetongue virus (BTV) infection. Viperin is the most highly induced ISG following BTV infection, which suggests that it may play a critical role in the anti-BTV immune response. The aim of this study was to characterize ovine Viperin (oViperin) and explore whether it can inhibit BTV replication. We cloned the coding sequences (CDS) of sheep Viperin, and the sequence analysis showed that oViperin displayed a high similarity with other species. oViperin has a leucine zipper in the N-terminal, a CxxxCxxC motif in the SAM domain, and a conservative C-terminus. We found that oViperin mRNA expression was significantly up-regulated in a time- and multiplicity of infection (MOI)-dependent manner following BTV infection. oViperin overexpression resulted in a significant inhibition in BTV replication, whereas an oViperin knockdown in MDOK cells increased BTV replication. This study shows for the first time, that oViperin has antiviral activity towards BTV infection and provides important information to research the interaction between BTV and oViperin.

The impact of dietary supplementation with guava (Psidium guajava L.) agroindustrial waste on growth performance and meat quality of lambs.

The aim of this study was to evaluate the growth performance and meat quality of lambs (40 animals) fed with increasing levels (0; 7.5; 15.0; 22.5 and 30.0%) of guava agroindustrial waste (GAW). The pH, cooking loss, colour and chemical composition were measured in the Longissimus lumborum (LL) muscle. A sensory analysis was performed with a panel of eight trained testers using a quantitative descriptive method in order to define sensory descriptors and their intensities. The lambs obtained a mean weight gain of 316 g/day consuming 1.32 kg/day of dry matter. The inclusion of GAW in the diet of lambs did not affect pH, L*, a* and b*. However, significant difference (P < .05) among the treatments were obtained for the chemical composition of LL: moisture decreased, while ash increased and intramuscular fat increased until 15% and then decreased at 22.5% with the inclusion of GAW. However, the sensorial characteristics of lamb meat (lamb odour and flavour, tenderness and juiciness) were not affect by the inclusion of GAW. Therefore, GAW can be included up to 30% in lamb feed without compromising the performance, physic-chemical and sensory characteristics of meat.

Effects of spirulina supplementation on lipid metabolism disorder, oxidative stress caused by high-energy dietary in Hu sheep.

The aim of this study was to investigate the effect of spirulina supplementation in a high-energy (HE) diet on lipid metabolism, oxidative status and immunity in Hu lambs. The lambs were assigned to two groups receiving either a standard diet (ST) or a HE diet. Each group was divided into three subgroups: no spirulina supplementation (control), 1% spirulina supplementation, or 3% spirulina supplementation. The body fat, serum cholesterol, triacylglycerol and oxidative stress increased in lambs fed the HE diet. However, 3% spirulina supplementation in the HE diet reduced above parameters and enhanced antioxidant capacity, including increased SOD activity and T-AOC content in serum and Longissimus thoracis et lumborum (LTL). Additionally, lambs receiving 3% spirulina supplementation showed an improvement in immunity-related parameters, including increased IgG concentration in serum and red and white blood cell counts. In conclusion, 3% spirulina supplementation in HE diet ameliorated lipid metabolic disorder and oxidative stress caused by a HE diet.

Molecular and functional characterization of ovis aries IFN-epsilon.

Interferon-epsilon (IFN-ε) is a type I IFN playing an essential role in innate and adaptive immunity against viral infection. Ovis aries IFN-ε (OvIFN-ε), consisting of 582 bp and which encodes a protein of 193 amino acids containing a signal peptide of 21 amino acids, was cloned and characterized. OvIFN-ε shares 51.6∼ 86.5% similarity to other species of IFN-ε, and evolves from the IFN-ε branch but not the other types of IFN. Additionally, OvIFN-ε gene is well conserved during evolution, and is highly transcribed in the liver, lung, brain, skin, ovary and uterus. Recombinant protein of OvIFN-ε was expressed in Escherichia coli and purified with nickel chelated column, which exhibited broad antiviral activity in vitro, sensitivity of trypsin, and stability of pH and temperature to some extent. Furthermore, OvIFN-ε could induce the transcription of ISG15, Mx1 and OAS in a time-dependent manner, as well as inhibit the VSV and BVDV replication in Ovis aries peripheral blood lymphocyte cells and MDBK cells. This study revealed OvIFN-ε has the typical characterization of type I IFNs and exerts antiviral activity against VSV and BVDV, and induces the expression of ISGs, which not only enriches the understanding of IFN-ε, but also facilitates further research on the antiviral defense responses of Ovis aries.

RIG-I Has a Role in Immunity Against Haemonchus contortus, a Gastrointestinal Parasite in Ovis aries: A Novel Report.

Retinoic acid inducible gene I (RIG-I) is associated to the DExD/H box RNA helicases. It is a pattern recognition receptor (PRR), playing a crucial role in the system and is a germ line encoded host sensor to perceive pathogen-associated molecular patterns (PAMPs). So far, reports are available for the role of RIG-I in antiviral immunity. This is the first report in which we have documented the role of RIG-I in parasitic immunity. Haemonchus contortus is a deadly parasite affecting the sheep industry, which has a tremendous economic importance, and the parasite is reported to be prevalent in the hot and humid agroclimatic region. We characterize the RIG-I gene in sheep (Ovis aries) and identify the important domains or binding sites with Haemonchus contortus through in silico studies. Differential mRNA expression analysis reveals upregulation of the RIG-I gene in the abomasum of infected sheep compared with that of healthy sheep, further confirming the findings. Thus, it is evident that, in infected sheep, expression of RIG-I is triggered for binding to more pathogens (Haemonchus contortus). Genetically similar studies with humans and other livestock species were conducted to reveal that sheep may be efficiently using a model organism for studying the role of RIG-I in antiparasitic immunity in humans.

Comparative metabolome analysis of serum changes in sheep under overgrazing or light grazing conditions.

BACKGROUND: Overgrazing is a primary contributor to severe reduction in forage quality and production in Inner Mongolia, leading to extensive ecosystem degradation, sheep health impairment and growth performance reduction. Further studies to identify serum biomarkers that reflect changes in sheep health and nutritional status following overgrazing would be beneficial. We hereby hypothesize that reduced sheep growth performance under overgrazing conditions would be associated with metabolic and immune response alterations. This study used an untargeted metabolomics analysis by conducting ultra-high-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) of sheep serum under overgrazing and light grazing conditions to identify metabolic disruptions in response to overgrazing. RESULTS: The sheep body weight gains as well as serum biochemical variables associated with immune responses and nutritional metabolism (immunoglobulin G, albumin, glucose, and nonesterified fatty acids) were significantly decreased with overgrazing compared with light grazing condition. In contrast, other serum parameters such as alanine and aspartate aminotransferase, alkaline phosphatase, total bilirubin, blood urea nitrogen, and interleukin-8 were markedly higher in the overgrazing group. Principal component analysis discriminated the metabolomes of the light grazing from the overgrazing group. Multivariate and univariate analyses revealed changes in the serum concentrations of 15 metabolites (9 metabolites exhibited a marked increase, whereas 6 metabolites showed a significant decrease) in the overgrazing group. Major changes of fatty acid oxidation, bile acid biosynthesis, and purine and protein metabolism were observed. CONCLUSIONS: These findings offer metabolic evidence for putative biomarkers for overgrazing-induced changes in serum metabolism. Target-identification of these particular metabolites may potentially increase our knowledge of the molecular mechanisms of altered immune responses, nutritional metabolism, and reduced sheep growth performance under overgrazing conditions.

Effect of maternal cottonseed feed on the immune and antioxidant status of Santa Ines lambs.

Cottonseed has been used as a nutritional alternative in animal production. However, consequences of this nutrient in the progeny is not well characterized. Thus, this work evaluated the effect on the immune and antioxidant status of the progeny of feeding Santa Ines ewe with or without cottonseed. Twenty-four Santa Ines ewes were distributed in two feeding regimes: cottonseed (CS) concentrate (n = 12) and soybean (SB) concentrate (n = 12). After birth, lambs remained with their mothers and blood samples were collected at 1st, 3rd, 7th, 15th, 30th and 60th day of life of 24 lambs born from mothers fed with (CS, n = 12) or without (SB, n = 12) cottonseed. Serum total protein, albumin, alpha beta globulin, gamma globulin, immunoglobulin G and M, activity of glutathione peroxidase (GPx), catalase (CAT), oxygen radical absorbance capacity (ORAC) and variables related to iron metabolism were affected only by sampling times (P < 0.05). The concentration of serum total protein, alpha beta globulin, gamma globulin and immunoglobulin G and M, GPx activity and ORAC values decreased as lamb age increased. Serum albumin concentration and CAT activity, in turn, increased as lamb age increased. In this work, maternal feeding with cottonseed did not affect the serum protein profile and antioxidant status of progeny during the lactation period, indicating no transfer of gossypol effects by milk secretions. Thus, the alternative in ruminants feeding with cottonseed can be used without maternal-descendant effects to immunity and oxidative stress in lambs.

Kisspeptin recombinant oral vaccine: A master gene vaccine inhibiting the reproductive physiology and behavior of ram lambs.

The KISS1 gene product, kisspeptin, stimulates gonadotrophic steroid hormone (GNRH) neuronal signaling through the G-protein coupled receptor, kiss1r. Disturbance of this signaling pathway causes hypogonadotropic hypogonadism in mammals. As part of this cutting-edge research project, we analyzed the efficacy of an oral kisspeptin recombinant vaccine on the reproductive physiology and behavior of ram lambs. Ten 56-day old ram lambs were randomly divided into treatment and control groups to receive the experimental recombinant vaccines, C500/pKS-asd or C500/pVAX-asd (aspartate-ß semialdehyde dehydrogenase), respectively. The vaccines were orally administered at day 0, 28 and 56 and blood samples were taken and scrotal circumference data recorded at 14-day intervals (days 0, 14, 28, 42, 56, 70, and 84). At the end of the experimental period, day 98, sexual behaviors were assessed, scrotal circumferences were measured, and blood samples were collected. Testicular samples were also collected after the animals were sacrificed. Anti-kisspeptin antibody and testosterone serum levels were measured by indirect ELISA. Results demonstrated that the levels of anti-kisspeptin antibodies were significantly higher in the treatment group compared to controls (P<0.05, P<0.01 and P<0.001). However, serum testosterone levels were lower in the treatment group (P<0.01). Interestingly, vaccine administration contributed to a significant reduction (P<0.01) in sexual behavior propensity. These results suggest that the kisspeptin recombinant oral vaccine regulates and inhibits the reproductive physiology and behavior of ram lambs.

Molecular Signature of Aluminum Hydroxide Adjuvant in Ovine PBMCs by Integrated mRNA and microRNA Transcriptome Sequencing.

There have been few in vivo studies on the effect of aluminum hydroxide adjuvant and its influence on the immune response to vaccination. In this study, lambs received a parallel subcutaneous treatment with either commercial vaccines containing aluminum hydroxide or an equivalent dose of this compound only with the aim of identifying the activated molecular signature. Blood samples were taken from each animal at the beginning and at the end of the experiment and PBMCs isolated. Total RNA and miRNA libraries were prepared and sequenced. After alignment to the Oar3.1 reference genome and differential expression with 3 programs, gene enrichment modeling was performed. For miRNAs, miRBase and RNAcentral databases were used for detection and characterization. Three expression comparisons were made: vaccinated animals at the beginning and at the end of the treatment, adjuvanted animals at the same times, and animals of both treatments at the end of the experiment. After exposure to both treatments, a total of 2,473; 2,980 and 429 differentially expressed genes were identified in vaccinated animals, adjuvanted animals and animals at the end of both treatments, respectively. In both adjuvant and vaccine treated animals the NF-κB signaling pathway was enriched. On the other hand, it can be observed a downregulation of cytokines and cytokine receptors in the adjuvanted group compared to the vaccinated group at the final time, suggesting a milder induction of the immune response when the adjuvant is alone. As for the miRNA analysis, 95 miRNAs were detected: 64 previously annotated in Ovis aries, 11 annotated in Bos taurus and 20 newly described. Interestingly, 6 miRNAs were differentially expressed in adjuvant treated animals, and 3 and 1 in the other two comparisons. Lastly, an integrated miRNA-mRNA expression profile was developed, in which a miRNA-mediated regulation of genes related to DNA damage stimulus was observed. In brief, it seems that aluminum-containing adjuvants are not simple delivery vehicles for antigens, but also induce endogenous danger signals that can stimulate the immune system. Whether this contributes to long-lasting immune activation or to the overstimulation of the immune system remains to be elucidated.

Influence of Nigella sativa seeds, Rosmarinus officinalis leaves and their combination on growth performance, immune response and rumen metabolism in Dorper lambs.

The objective of this study was to determine the effects of dietary supplementation of Nigella sativa L. seeds, Rosmarinus officinalis L. leaves and their combination on rumen metabolism, nutrient intake and digestibility, growth performance, immune response and blood metabolites in Dorper lambs. Twenty-four entire male Dorper lambs (18.68 ± 0.6 kg, 4-5 months old) were randomly assigned to a concentrate mixture containing on a dry matter basis either, no supplement (control, T1), 1% R. officinalis leaves (T2), 1% N. sativa seeds (T3) or 1% R. officinalis leaves +1% N. sativa seeds (T4). The lambs had ad libitum access to urea-treated rice straw (UTRS) and were raised for 90 days. Supplemented lambs had greater (P < 0.05) intake of DM and UTRS than the control lambs. The T4 lambs had lower (P < 0.05) nutrient digestibility than those fed other treatments. Total and daily weight gain was greater (P < 0.05) in T2 lambs than those fed other diets. The T3 and T4 lambs had greater (P < 0.05) ruminal pH than the T1 and T2 lambs. Supplemented lambs had lower (P < 0.05) ruminal total volatile fatty acids, acetate, propionate, NH3-N and C18:0 than the control lambs. The T4 lambs had lower (P < 0.05) population of Fibrobacter succinogenes, Ruminococcus albus, methanogens and total protozoa compared with those fed other diets. Supplemented lambs had lower (P < 0.05) neutrophils, basophils and serum urea and greater (P < 0.05) serum IgA and IgG compared with the control lambs. The current results emphasised the variation in the efficacy of medicinal plants in ruminant nutrition.

Four-segmented Rift Valley fever virus-based vaccines can be applied safely in ewes during pregnancy.

Rift Valley fever virus (RVFV) causes severe and recurrent outbreaks on the African continent and the Arabian Peninsula and continues to expand its habitat. This mosquito-borne virus, belonging to the genus Phlebovirus of the family Bunyaviridae contains a tri-segmented negative-strand RNA genome. Previously, we developed four-segmented RVFV (RVFV-4s) variants by splitting the M-genome segment into two M-type segments each encoding one of the structural glycoproteins; Gn or Gc. Vaccination/challenge experiments with mice and lambs subsequently showed that RVFV-4s induces protective immunity against wild-type virus infection after a single administration. To demonstrate the unprecedented safety of RVFV-4s, we here report that the virus does not cause encephalitis after intranasal inoculation of mice. A study with pregnant ewes subsequently revealed that RVFV-4s does not cause viremia and does not cross the ovine placental barrier, as evidenced by the absence of teratogenic effects and virus in the blood and organs of the fetuses. Altogether, these results show that the RVFV-4s vaccine virus can be applied safely in pregnant ewes.

Modelling the Dynamics of Post-Vaccination Immunity Rate in a Population of Sahelian Sheep after a Vaccination Campaign against Peste des Petits Ruminants Virus.

BACKGROUND: Peste des petits ruminants (PPR) is an acute infectious viral disease affecting domestic small ruminants (sheep and goats) and some wild ruminant species in Africa, the Middle East and Asia. A global PPR control strategy based on mass vaccination-in regions where PPR is endemic-was recently designed and launched by international organizations. Sahelian Africa is one of the most challenging endemic regions for PPR control. Indeed, strong seasonal and annual variations in mating, mortality and offtake rates result in a complex population dynamics which might in turn alter the population post-vaccination immunity rate (PIR), and thus be important to consider for the implementation of vaccination campaigns. METHODS: In a context of preventive vaccination in epidemiological units without PPR virus transmission, we developed a predictive, dynamic model based on a seasonal matrix population model to simulate PIR dynamics. This model was mostly calibrated with demographic and epidemiological parameters estimated from a long-term follow-up survey of small ruminant herds. We used it to simulate the PIR dynamics following a single PPR vaccination campaign in a Sahelian sheep population, and to assess the effects of (i) changes in offtake rate related to the Tabaski (a Muslim feast following the lunar calendar), and (ii) the date of implementation of the vaccination campaigns. RESULTS: The persistence of PIR was not influenced by the Tabaski date. Decreasing the vaccination coverage from 100 to 80% had limited effects on PIR. However, lower vaccination coverage did not provide sufficient immunity rates (PIR < 70%). As a trade-off between model predictions and other considerations like animal physiological status, and suitability for livestock farmers, we would suggest to implement vaccination campaigns in September-October. This model is a first step towards better decision support for animal health authorities. It might be adapted to other species, livestock farming systems or diseases.

Vaccination of Sheep with a Methanogen Protein Provides Insight into Levels of Antibody in Saliva Needed to Target Ruminal Methanogens.

Methane is produced in the rumen of ruminant livestock by methanogens and is a major contributor to agricultural greenhouse gases. Vaccination against ruminal methanogens could reduce methane emissions by inducing antibodies in saliva which enter the rumen and impair ability of methanogens to produce methane. Presently, it is not known if vaccination can induce sufficient amounts of antibody in the saliva to target methanogen populations in the rumen and little is known about how long antibody in the rumen remains active. In the current study, sheep were vaccinated twice at a 3-week interval with a model methanogen antigen, recombinant glycosyl transferase protein (rGT2) formulated with one of four adjuvants: saponin, Montanide ISA61, a chitosan thermogel, or a lipid nanoparticle/cationic liposome adjuvant (n = 6/formulation). A control group of sheep (n = 6) was not vaccinated. The highest antigen-specific IgA and IgG responses in both saliva and serum were observed with Montanide ISA61, which promoted levels of salivary antibodies that were five-fold higher than the second most potent adjuvant, saponin. A rGT2-specific IgG standard was used to determine the level of rGT2-specific IgG in serum and saliva. Vaccination with GT2/Montanide ISA61 produced a peak antibody concentration of 7 × 1016 molecules of antigen-specific IgG per litre of saliva, and it was estimated that in the rumen there would be more than 104 molecules of antigen-specific IgG for each methanogen cell. Both IgG and IgA in saliva were shown to be relatively stable in the rumen. Salivary antibody exposed for 1-2 hours to an in vitro simulated rumen environment retained approximately 50% of antigen-binding activity. Collectively, the results from measuring antibody levels and stablility suggest a vaccination-based mitigation strategy for livestock generated methane is in theory feasible.

Differences in leukocyte differentiation molecule abundances on domestic sheep (Ovis aries) and bighorn sheep (Ovis canadensis) neutrophils identified by flow cytometry.

Although both domestic sheep (DS) and bighorn sheep (BHS) are affected by similar respiratory bacterial pathogens, experimental and field data indicate BHS are more susceptible to pneumonia. Cross-reactive monoclonal antibodies (mAbs) for use in flow cytometry (FC) are valuable reagents for interspecies comparative immune system analyses. This study describes cross-reactive mAbs that recognize leukocyte differentiation molecules (LDMs) and major histocompatibility complex antigens on DS and BHS leukocytes. Characterization of multichannel eosinophil autofluorescence in this study permitted cell-type specific gating of granulocytes for evaluating LDMs, specifically on neutrophils, by single-label FC. Evaluation of relative abundances of LDMs by flow cytometry revealed greater CD11a, CD11b, CD18 (ß2 integrins) and CD 172a (SIRPα) on DS neutrophils and greater CD14 (lipopolysaccharide receptor) on BHS neutrophils. Greater CD25 (IL-2) was identified on BHS lymphocytes following Concavalin A stimulation. While DS and BHS have similar total peripheral blood leukocyte counts, BHS have proportionately more neutrophils.

Evidence for the existence of regulatory and effector B cell populations in Peyer's patches of sheep.

IL-10 secreting CD21(+) B cells exist in sheep Peyer's patches (PP). It's not known however, whether all PP B cells are regulatory or whether an effector population also exists in this tissue. To further characterize the subpopulations of B cells in PP's, highly purified B cells were negatively sorted from jejunal PP and fractionated according to co-expression of CD72(+)CD21(+)or CD72(+)CD21(-) molecules and then stimulated with the TLR9-agonist, CpG ODN. IL-10, IL-12, IFN-γ, and IgM production were then assayed. We observed that only highly purified CD72(+)CD21(+) B cells spontaneously secreted high levels of IL-10, but they did not produce any IL-12, IFN-γ or IgM suggesting that this cell population contains regulatory B cells. In contrast, CD72(+)CD21(-) B cells did not secrete IL-10, but secreted IL-12, IFN-γ, and IgM, suggesting they include effector cells. In addition, B cells expressing surface IgA, IgM and IgG1 all secreted similar levels of IL-10. We further confirmed that only B cells produce IL-10, while other cells in the PP including DCs and T cells do not. Our investigations may provide evidence for the existence of two sub-populations in sheep PP; IL-10 secreting regulatory (CD72(+)CD21(+)) cells, and IL-12/IFN-γ/IgM-secreting effector (CD72(+)CD21(-)) cells.