Characterization of the Hepatic Transcriptome for Divergent Immune-Responding Sheep Following Natural Exposure to Gastrointestinal Nematodes.

Infections with gastrointestinal nematodes (GINs) reduce the economic efficiency of sheep operations and compromise animal welfare. Understanding the host's response to GIN infection can help producers identify animals that are naturally resistant to infection. The objective of this study was to characterize the hepatic transcriptome of sheep that had been naturally exposed to GIN parasites. The hepatic transcriptome was studied using RNA-Sequencing technology in animals characterized as high (n = 5) or medium (n = 6) based on their innate immune acute-phase (AP) response phenotype compared with uninfected controls (n = 4), and with biased antibody-mediated (AbMR, n = 5) or cell-mediated (CMR, n = 5) adaptive immune responsiveness compared to uninfected controls (n = 3). Following the assessment of sheep selected for innate responses, 0, 136, and 167 genes were differentially expressed (DE) between high- and medium-responding animals, high-responding and uninfected control animals, and medium-responding and uninfected control animals, respectively (false discovery rate (FDR) < 0.05, and fold change |FC| > 2). When adaptive immune responses were assessed, 0, 53, and 57 genes were DE between antibody- and cell-biased animals, antibody-biased and uninfected control animals, and cell-biased and uninfected control animals, respectively (FDR < 0.05, |FC| > 2). Functional analyses identified enriched gene ontology (GO) terms and metabolic pathways related to the innate immune response and energy metabolism. Six functional candidate genes were identified for further functional and validation studies to better understand the underlying biological mechanisms of host responses to GINs. These, in turn, can potentially help improve decision making and management practices to increase the overall host immune response to GIN infection.

Variants of NLRP3 Protein in Haemonchus contortus Infected Sheep: Impact on Immune Cell Responsiveness to LPS In Vitro.

Pathogen recognition is an essential component to achieve the desired outcome of host protection. Nod-like receptor pyrin containing domain 3 (NLRP3) is a cytoplasmic pattern recognition receptor (PRR) with a wide array of agonists, such as PAMPs, DAMPs, ATP, bacterial product and viral products. Stimulation of the NLRP3 inflammasome results in proteolytic activation of IL-1ß and IL-18, cell pyroptosis and classically, the induction of proinflammatory responses. St. Croix (STC) sheep have resistance traits exhibiting the appropriate T-helper type 2 immune response ensuing protection during helminth parasitic infection whereas parasite-susceptible Suffolk (SUF) sheep have an impaired response resulting in parasite establishment and adverse symptoms. The objective of these experiments was to determine if NLRP3 protein in H. contortus-infected SUF sheep was defective using the classical activation pathway of NLRP3 inflammasome. Peripheral blood mononuclear cells (PBMCs) derived from H. contortus-infected STC and SUF sheep were isolated from whole blood and treated (MCC950 treatment for 2 h followed by LPS treatment for 3 h, 1400 W treatment for 2 h followed by LPS treatment for 3 h, LPS treatment for 3 h or culture media for 3 h). qPCR analysis of LPS-stimulated PBMC revealed an upregulation in inflammatory associated genes IL-1ß, TLR4, TNFα and NFκB (p < 0.0001) in STC PBMC and downregulation in IFNγ, IL-6 and iNOS for SUF PBMC. Pharmacological inhibition of iNOS in SUF PBMC resulted in an upregulation in the expression of IFNγ. These preliminary data begin to discover a relationship between NLRP3 activation and TLR4 signalling in PBMC of STC and SUF sheep.

Evaluation of the immunogenicity and protective efficacy of an inactivated vaccine candidate for sheep infected with ovine parainfluenza virus type 3.

Respiratory diseases constitute a major health problem for ruminants, resulting in considerable economic losses throughout the world. Parainfluenza type 3 virus (PIV3) is one of the most important respiratory pathogens of ruminants. The pathogenicity and phylogenetic analyses of PIV3 virus have been reported in sheep and goats. However, there are no recent studies of the vaccination of sheep or goats against PIV3. Here, we developed a purified inactivated ovine parainfluenza virus type 3 (OPIV3) vaccine candidate. In addition, we immunized sheep with the inactivated OPIV3 vaccine and evaluated the immune response and pathological outcomes associated with OPIV3 TX01 infection. The vaccinated sheep demonstrated no obvious symptoms of respiratory tract infection, and there were no gross lesions or pathological changes in the lungs. The average body weight gain significantly differed between the vaccinated group and the control group (P < 0.01). The serum neutralization antibody levels rapidly increased in sheep post-vaccination and post-challenge with OPIV3. Furthermore, viral shedding in nasal swabs and viral loads in the lungs were reduced. The results of this study suggest that vaccination with this candidate vaccine induces the production of neutralizing antibodies and provides significant protection against OPIV3 infection. These results may be helpful for further studies on prevention and control strategies for OPIV3 infections.

Characterization of intestinal mononuclear phagocyte subsets in young ruminants at homeostasis and during Cryptosporidium parvum infection.

Introduction: Cryptosporidiosis is a poorly controlled zoonosis caused by an intestinal parasite, Cryptosporidium parvum, with a high prevalence in livestock (cattle, sheep, and goats). Young animals are particularly susceptible to this infection due to the immaturity of their intestinal immune system. In a neonatal mouse model, we previously demonstrated the importance of the innate immunity and particularly of type 1 conventional dendritic cells (cDC1) among mononuclear phagocytes (MPs) in controlling the acute phase of C. parvum infection. These immune populations are well described in mice and humans, but their fine characterization in the intestine of young ruminants remained to be further explored. Methods: Immune cells of the small intestinal Peyer's patches and of the distal jejunum were isolated from naive lambs and calves at different ages. This was followed by their fine characterization by flow cytometry and transcriptomic analyses (q-RT-PCR and single cell RNAseq (lamb cells)). Newborn animals were infected with C. parvum, clinical signs and parasite burden were quantified, and isolated MP cells were characterized by flow cytometry in comparison with age matched control animals. Results: Here, we identified one population of macrophages and three subsets of cDC (cDC1, cDC2, and a minor cDC subset with migratory properties) in the intestine of lamb and calf by phenotypic and targeted gene expression analyses. Unsupervised single-cell transcriptomic analysis confirmed the identification of these four intestinal MP subpopulations in lamb, while highlighting a deeper diversity of cell subsets among monocytic and dendritic cells. We demonstrated a weak proportion of cDC1 in the intestine of highly susceptible newborn lambs together with an increase of these cells within the first days of life and in response to the infection. Discussion: Considering cDC1 importance for efficient parasite control in the mouse model, one may speculate that the cDC1/cDC2 ratio plays also a key role for the efficient control of C. parvum in young ruminants. In this study, we established the first fine characterization of intestinal MP subsets in young lambs and calves providing new insights for comparative immunology of the intestinal MP system across species and for future investigations on host-Cryptosporidium interactions in target species.

Brucella melitensis Rev1Δwzm: Placental pathogenesis studies and safety in pregnant ewes.

One of the main causes of human brucellosis is Brucella melitensis infecting small ruminants. To date, Rev1 is the only vaccine successfully used to control ovine and caprine brucellosis. However, it is pathogenic for pregnant animals, resulting in abortions and vaginal and milk shedding, as well as being infectious for humans. Therefore, there is an urgent need to develop an effective vaccine that is safer than Rev1. In efforts to further attenuate Rev1, we recently used wzm inactivation to generate a rough mutant (Rev1Δwzm) that retains a complete antigenic O-polysaccharide in the bacterial cytoplasm. The aim of the present study was to evaluate the placental pathogenicity of Rev1Δwzm in trophoblastic cells, throughout pregnancy in mice, and in ewes inoculated in different trimesters of pregnancy. This mutant was evaluated in comparison with the homologous 16MΔwzm derived from a virulent strain of B. melitensis and the naturally rough sheep pathogen B. ovis. Our results show that both wzm mutants triggered reduced cytotoxic, pro-apoptotic, and pro-inflammatory signaling in Bewo trophoblasts, as well as reduced relative expression of apoptosis genes. In mice, both wzm mutants produced infection but were rapidly cleared from the placenta, in which only Rev1Δwzm induced a low relative expression of pro-apoptotic and pro-inflammatory genes. In the 66 inoculated ewes, Rev1Δwzm was safe and immunogenic, displaying a transient serological interference in standard RBT but not CFT S-LPS tests; this serological response was minimized by conjunctival administration. In conclusion, these results support that B. melitensis Rev1Δwzm is a promising vaccine candidate for use in pregnant ewes and its efficacy against B. melitensis and B. ovis infections in sheep warrants further study.

Comparative analysis of host immune responses to Hydatid cyst in human and ovine hepatic cystic Echinococcosis.

BACKGROUND: Hydatid disease is caused by the larval stages of the canine tapeworm Echinococcus granulosus. It is one of the most critical helminthic diseases, representing worldwide public health and socio-economic concern. AIM: This study aimed to investigate the expression of apoptosis and immune response within hepatic tissues of humans and sheep infected with the Hydatid cyst. METHODS: Paraffin-embedded tissue was prepared from each tissue sample and used for histopathological examination by Haematoxylin- Eosin. Also, toluidine blue staining was used for mast cell detection, while an immunohistochemical study was performed to assess CD3 T lymphocytes, CD4 helper T lymphocytes, CD8 cytotoxic T lymphocytes, CD20 memory B lymphocytes, CD68 macrophage, and caspase-3 antibodies. RESULTS: The histological examination revealed significant changes, including the infiltration of inflammatory cells, predominantly lymphocytes with scattered giant cells, necrotic hepatic tissue, and fibrosis. Toluidine blue stain revealed a higher number of mast cells (5 cells/field) in humans compared to sheep (3.6 cells/field). The immunohistochemical analysis confirmed that the CD3 were the most predominant inflammatory cell in the hepatic tissue of humans (intensive 70%), and sheep (moderate 38.47%). Caspase-3 was observed in all samples in different grades and mostly in human liver tissue. CONCLUSION: This data could aid in recognizing immunological markers for differentiating disease progression, as well as enhance the understanding of local immune responses to cystic Echinococcosis (CE). The findings could provide preliminary data for future studies on immune responses associated with Hydatid cysts.

The Development of Toxoplasma gondii Recombinant Trivalent Chimeric Proteins as an Alternative to Toxoplasma Lysate Antigen (TLA) in Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Immunoglobulin G (IgG) in Small Ruminants.

This study presents an evaluation of seventeen newly produced recombinant trivalent chimeric proteins (containing the same immunodominant fragment of SAG1 and SAG2 of Toxoplasma gondii antigens, and an additional immunodominant fragment of one of the parasite antigens, such as AMA1, GRA1, GRA2, GRA5, GRA6, GRA7, GRA9, LDH2, MAG1, MIC1, MIC3, P35, and ROP1) as a potential alternative to the whole-cell tachyzoite lysate (TLA) used in the detection of infection in small ruminants. These recombinant proteins, obtained by genetic engineering and molecular biology methods, were tested for their reactivity with specific anti-Toxoplasma IgG antibodies contained in serum samples of small ruminants (192 samples of sheep serum and 95 samples of goat serum) using an enzyme-linked immunosorbent assay (ELISA). The reactivity of six recombinant trivalent chimeric proteins (SAG1-SAG2-GRA5, SAG1-SAG2-GRA9, SAG1-SAG2-MIC1, SAG1-SAG2-MIC3, SAG1-SAG2-P35, and SAG1-SAG2-ROP1) with IgG antibodies generated during T. gondii invasion was comparable to the sensitivity of TLA-based IgG ELISA (100%). The obtained results show a strong correlation with the results obtained for TLA. This suggests that these protein preparations may be a potential alternative to TLA used in commercial tests and could be used to develop a cheaper test for the detection of parasite infection in small ruminants.

Evaluation of Th1/Th2, regulatory cytokines and transcriptional factor FoxP3 in sheep immunized with a partially protective and non-protective vaccine and challenged with Fasciola hepatica.

Gene expression for Th1/Th2 cytokines (IL-4 and IFN-É£), regulatory cytokines (TGF-ß and IL-10) and the transcriptional factor FoxP3 was analyzed in the liver and hepatic lymph nodes (HLN) from sheep immunized with partially protective and non-protective vaccine candidates and challenged with Fasciola hepatica. FoxP3 T cells were also evaluated by immunohistochemistry (IHQ). The most remarkable difference between the partially protected vaccinated (V1) group and the non-protected vaccinated (V2) group was a more severe expansion of FoxP3 T cells recorded by IHQ in both the liver and HLN of the V2 group as compared to the V1 group, whereas no differences were found between the V2 group and the infected control (IC) group. Similar results were recorded for FoxP3 gene expression although significant differences among V1 and V2 groups were only significant in the HLN, while FoxP3 gene expression was very similar in the V2 and IC groups both in the liver and HLN. No significant differences for the remaining cytokines were recorded between the V1 and V2 groups, but in the liver the V2 group shows significant increases of IFN-É£ and IL-10 as compared to the uninfected control (UC) group whereas the V1 group did not. The lower expansion of FoxP3 T cells and lower increase of IFN-É£ and IL-10 in the partially protected vaccinated group may be related with lower hepatic lesions and fluke burdens recorded in this group as compared to the other two infected groups. The most relevant change in regulatory cytokine gene expression was the significant increase of TGF-ß in the liver of IC, V1 and V2 groups as compared to the UC group, which could be related to hepatic lesions.

Reducing fecal egg count through selective breeding alters dorper lamb response to Haemonchus contortus in an artificial challenge trial.

Infection by gastrointestinal nematodes (GIN), particularly Haemonchus contortus, can be detrimental to sheep health and performance. Genetic susceptibility to GIN varies between breeds, with those lacking high levels of natural resistance often requiring frequent anthelmintic treatment when facing parasitic challenge. Genetic technology can serve as a tool to decrease GIN susceptibility via selection for sheep with reduced fecal egg count (FEC) estimated breeding values (EBVs). However, the physiological changes that result from implementation of this strategy are not well described. Additionally, there is a need for comparison of animals from recent selective breeding against breeds with inherent GIN resistance. In this study we administered a challenge of H. contortus to Dorper x White Dorper (DWD; n = 92) lambs that have been genetically selected for either low (DWD-) or high (DWD+) FEC EBVs and Barbados Blackbelly x Mouflon (BBM; n = 19) lambs from a genetically resistant breed backgrounds. Lamb FEC, packed-cell volume (PCV) and serum IgG were measured at intermittent levels over 5 weeks. At day 21 and day 35, the selectively bred DWD- had a lower mean FEC compared to DWD+, but were higher than BBM. Reductions in both PCV and serum IgG from initial day 0 levels were observed in DWD lambs, but not in BBM. Furthermore, from a subset of lambs (n = 24) harvested at day 21, DWD- only tended (p = 0.056) to have lower mean worm counts than DWD+, with BBM having the lowest mean worm count. Differentially expressed genes (DEGs) identified via RNA-sequencing of abomasal tissue at day 21 indicate a more pronounced Th2 immune response and more rapid worm expulsion occurred in iBBM than iDWD- and iDWD+ lambs. However, gene expression in DWD- suggests an association between reduced FEC EBV and gastric acid secretion and the ability to limit worm fecundity. Ultimately, selection of Dorper sheep for low FEC EBV can reduce susceptibility to GIN, but it will likely require multiple generations with this trait as a breeding priority before presenting a similar resistance level to Caribbean breeds.

Moniezia benedeni infection increases IgE+ cells in sheep (Ovis aries) small intestine.

The concentration of immunoglobulin (Ig) E is the lowest among serum Igs, but it can induces type I hypersensitivity and plays an important role in anti-parasitic infection. The present study aimed to explore the residence characteristics of IgE+ cells in the sheep small intestine and the impact of Moniezia benedeni infection on them. The recombinant plasmids pET-28a-IgE were constructed and induced and expressed in Escherichia coli. BL21 (DE3). The rabbit anti-sheep IgE polyclonal antibody was prepared using the obtained recombinant protein as antigen. Finally, the levels of IgE+ cells in the small intestine of healthy (Control group) and naturally M. benedeni-infected (Infected group) sheep were detected analyzed. The results showed that the rabbit anti-sheep IgE polyclonal antibody with good immunogenicity (titer = 1: 128000) could specifically bind to the heavy chain of natural sheep IgE. In the Control group, the IgE+ cells were mainly distributed in lamina propria of the small intestine, and the densities were significantly decreased from duodenum to ileum (P<0.05), with respective values of (4.28 cells / 104 µm2, 1.80 cells / 104 µm2, and 1.44 cells / 104 µm2 in duodenum, jejunum, and ileum. In the Infected group, IgE+ cells density were 6.26 cells / 104 µm2, 3.01 cells / 104 µm2, and 2.09 cells / 104 µm2 in duodenum, jejunum and ileum respectively, which were significantly higher in all segments compared to the Control group (P<0.05), increasing by 46.26%, 67.22% and 45.14%, respectively. In addition, compared with the Control group, the IgE protein levels were significantly increased in all intestinal segments of the Infected group (P<0.01), however, there was no significant differences among the different intestinal segments within the same group (P>0.05). The results demonstrated that M. benedeni infection could significantly increase the content of IgE and the distribution density of its secreting cells in sheep small intestine. The intestinal mucosal immune system of sheep presented obvious specificity against M. benedeni infection. This lays a good foundation for further exploring molecular mechanisms of the intestinal mucosal immune system monitoring and responding to M. benedeni infection.

Evaluation of the Role of Galectins in Parasite Immunity.

Galectin-11 (LGALS-11) and galectin-14 (LGALS-14) are ruminant specific galectins, first reported in sheep. Although their roles in parasite immunity are still being elucidated, it appears that they influence protection against parasites. In gastrointestinal infections with the nematode Haemonchus contortus, both galectin-11 and galectin-14 appear to be protective. However, in a chronic infection of liver fluke, Fasciola hepatica, these galectins may aid parasite survival. To unravel the structural, functional, and ligand profile of galectin-11 and galectin-14, recombinant production of these proteins is vital. Here we present the recombinant production of soluble galectin-11 and galectin-14 from domestic sheep for in vitro and structural biology studies. These methods include parasite cultivation and infection, galectin staining of host and parasite tissue, surface staining of parasites with recombinant galectins, pull-down assays to identify endogenous galectin binding proteins, and in vitro assays to monitor the effect of galectins on parasite development.

Determination of macrophage types by immunohistochemical methods in the local immune response to liver hydatid cysts in sheep.

Cystic echinococcosis is a zoonotic parasitic disease caused by Echinococcus granulosus. The main hosts in the life cycle of this parasite are dogs and other carnivores; The intermediate hosts are human, sheep, goat, cattle, pig, buffalo, horse and camel. The parasite damages the tissue by forming lesions in the form of fluid-filled cysts in the liver. These lesions are bounded by a layer of local inflammatory cells formed by the host. In the layer formed by this inflammatory response, there are lymphocytes, neutrophils and eosinophil leukocytes, including macrophages. Samples taken from sheep with hydatid cysts in their livers were followed for pathological analysis, and then histopathological and immunohistochemical examinations were performed. After histopathological examinations, the types of macrophages involved in the local immune response against cysts in the liver were determined by immunohistochemical methods using anti-INOS and anti-IL-10 antibodies. INOS and IL-10 immunopositivity were detected in all samples. Statistically, no significant difference was observed between these immunopositivity. This showed that both macrophage types are involved in the local immune response to hydatid cyst, and that Th1 and Th2 immune response stimulation continues together. It was concluded that in future studies that will be planned and experimentally, it will be possible to reveal more clearly how these macrophage types take part in the local immune response.

Tuft Cells Increase Following Ovine Intestinal Parasite Infections and Define Evolutionarily Conserved and Divergent Responses.

Helminth parasite infections of humans and livestock are a global health and economic problem. Resistance of helminths to current drug treatment is an increasing problem and alternative control approaches, including vaccines, are needed. Effective vaccine design requires knowledge of host immune mechanisms and how these are stimulated. Mouse models of helminth infection indicate that tuft cells, an unusual type of epithelial cell, may 'sense' infection in the small intestine and trigger a type 2 immune response. Currently nothing is known of tuft cells in immunity in other host species and in other compartments of the gastrointestinal (GI) tract. Here we address this gap and use immunohistochemistry and single cell RNA-sequencing to detail the presence and gene expression profile of tuft cells in sheep following nematode infections. We identify and characterize tuft cells in the ovine abomasum (true stomach of ruminants) and show that they increase significantly in number following infection with the globally important nematodes Teladorsagia circumcincta and Haemonchus contortus. Ovine abomasal tuft cells show enriched expression of tuft cell markers POU2F3, GFI1B, TRPM5 and genes involved in signaling and inflammatory pathways. However succinate receptor SUCNR1 and free fatty acid receptor FFAR3, proposed as 'sensing' receptors in murine tuft cells, are not expressed, and instead ovine tuft cells are enriched for taste receptor TAS2R16 and mechanosensory receptor ADGRG6. We also identify tuft cell sub-clusters at potentially different stages of maturation, suggesting a dynamic process not apparent from mouse models of infection. Our findings reveal a tuft cell response to economically important parasite infections and show that while tuft cell effector functions have been retained during mammalian evolution, receptor specificity has diverged. Our data advance knowledge of host-parasite interactions in the GI mucosa and identify receptors that may potentiate type 2 immunity for optimized control of parasitic nematodes.

First study of Brucella ovis antibodies in purebred sheep flocks in the State of Parana, Brazil / [Primeiro estudo de anticorpos para Brucella ovis em rebanhos de ovinos puros de Origem, no estado do Paraná, Brasil]

Brucella ovis, a non-zoonotic species, is the etiological agent of ovine brucellosis, an infectious disease of clinical or subclinical occurrence in sheep flocks. Until then, there is no serological study of anti-Brucella ovis antibodies in purebred sheep herds. This study aimed to determine the presence of anti-Brucella ovis antibodies in purebred sheep flocks with breeding purposes from Parana State. Blood samples from 728 animals, of which 563 were females and 165 males, between 8 and 56 months of age from the six major sheep producing mesoregions of Parana, were submitted to detection of anti-Brucella ovis antibodies by the Agar Gel Immunodiffusion technique using an antigen from the bacteria Brucella ovis (Reo 198). The results indicate the presence of this disease in purebred sheep from Parana State in a low occurrence of 0.27% (2/728). The only two positive animals were rams, Santa Inês breed, from the same flock in the East Center region of Parana, without clinical disease. In conclusion, Brucella ovis is present in purebred sheep in Parana State, Brazil, and this low occurrence may have occurred due to rigorous breeding systems that may contribute to reduce the transmission of this disease.(AU)

Brucella ovis, espécie não zoonótica, é o agente etiológico da brucelose ovina, doença infecciosa de ocorrência clínica ou subclínica. Atualmente, não existe estudo sorológico de anticorpos anti-Brucella ovis em rebanhos de ovinos puros de origem. Este estudo teve como objetivo determinar a presença de anticorpos anti-Brucella ovis em rebanhos ovinos de raça pura de origem, com fins reprodutivos do estado do Paraná. Amostras de sangue de 728 animais, sendo 563 fêmeas e 165 machos, entre oito e 56 meses de idade, pertencentes a seis principais mesorregiões produtoras de ovinos no Paraná, foram submetidas à detecção de anticorpos anti-Brucella ovis pela técnica de imunodifusão em ágar gel usando-se um antígeno da bactéria Brucella ovis (Reo 198). Os resultados indicam a presença da doença em ovinos puros de origem do estado do Paraná em baixa ocorrência de 0,27% (2/728). Os dois únicos animais positivos foram reprodutores da raça Santa Inês, do mesmo rebanho da região Centro Leste do Paraná, sem manifestação clínica. Em conclusão, Brucella ovis está presente em ovinos puros de origem no estado do Paraná, e essa baixa ocorrência pode ter ocorrido devido a sistemas rigorosos de criação, que podem contribuir para a redução da transmissão dessa doença.(AU)

Immunization With Bovine Herpesvirus-4-Based Vector Delivering PPRV-H Protein Protects Sheep From PPRV Challenge.

The Morbillivirus peste des petits ruminants virus (PPRV) is the causal agent of a highly contagious disease that mostly affects sheep and goats and produces considerable losses in developing countries. Current PPRV control strategies rely on live-attenuated vaccines, which are not ideal, as they cannot differentiate infected from vaccinated animals (DIVA). Recombinant vector-based vaccines expressing viral subunits can provide an alternative to conventional vaccines, as they can be easily paired with DIVA diagnostic tools. In the present work, we used the bovine herpesvirus-4-based vector (BoHV-4-A) to deliver PPRV hemagglutinin H antigen (BoHV-4-A-PPRV-H-ΔTK). Vaccination with BoHV-4-A-PPRV-H-ΔTK protected sheep from virulent PPRV challenge and prevented virus shedding. Protection correlated with anti-PPRV IgGs, neutralizing antibodies and IFN-γ-producing cells induced by the vaccine. Detection of antibodies exclusively against H-PPRV in animal sera and not against other PPRV viral proteins such as F or N could serve as a DIVA diagnostic test when using BoHV-4-A-PPRV-H-ΔTK as vaccine. Our data indicate that BoHV-4-A-PPRV-H-ΔTK could be a promising new approach for PPRV eradication programs.

Timing of Transcriptomic Peripheral Blood Mononuclear Cell Responses of Sheep to Fasciola hepatica Infection Differs From Those of Cattle, Reflecting Different Disease Phenotypes.

Infection with the zoonotic trematode Fasciola hepatica, common in many regions with a temperate climate, leads to delayed growth and loss of productivity in cattle, while infection in sheep can have more severe effects, potentially leading to death. Previous transcriptomic analyses revealed upregulation of TGFB1, cell death and Toll-like receptor signalling, T-cell activation, and inhibition of nitric oxide production in macrophages in response to infection. However, the differences between ovine and bovine responses have not yet been explored. The objective of this study was to further investigate the transcriptomic response of ovine peripheral blood mononuclear cells (PBMC) to F. hepatica infection, and to elucidate the differences between ovine and bovine PBMC responses. Sixteen male Merino sheep were randomly assigned to infected or control groups (n = 8 per group) and orally infected with 120 F. hepatica metacercariae. Transcriptomic data was generated from PBMC at 0, 2 and 16 weeks post-infection (wpi), and analysed for differentially expressed (DE) genes between infected and control animals at each time point (analysis 1), and for each group relative to time 0 (analysis 2). Analysis 2 was then compared to a similar study performed previously on bovine PBMC. A total of 453 DE genes were found at 2 wpi, and 2 DE genes at 16 wpi (FDR < 0.1, analysis 1). Significantly overrepresented biological pathways at 2 wpi included role of PKR in interferon induction and anti-viral response, death receptor signalling and RIG-I-like receptor signalling, which suggested that an activation of innate response to intracellular nucleic acids and inhibition of cellular apoptosis were taking place. Comparison of analysis 2 with the previous bovine transcriptomic study revealed that anti-inflammatory response pathways which were significantly overrepresented in the acute phase in cattle, including IL-10 signalling, Th2 pathway, and Th1 and Th2 activation were upregulated only in the chronic phase in sheep. We propose that the earlier activation of anti-inflammatory responses in cattle, as compared with sheep, may be related to the general absence of acute clinical signs in cattle. These findings offer scope for "smart vaccination" strategies for this important livestock parasite.

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.

First Survey of SNPs in TMEM154, TLR9, MYD88 and CCR5 Genes in Sheep Reared in Italy and Their Association with Resistance to SRLVs Infection.

Maedi-visna virus (MVV) and caprine arthritis encephalitis virus (CAEV), referred to as small ruminant lentiviruses (SRLVs), belong to the genus Lentivirus of the Retroviridae family. SRLVs infect both sheep and goats, causing significant economic losses and animal welfare damage. Recent findings suggest an association between serological status and allelic variants of different genes such as TMEM154, TLR9, MYD88 and CCR5. The aim of this work was to investigate the role of specific polymorphisms of these genes in SRLVs infection in some sheep flocks in Italy. In addition to those already known, novel variants in the TMEM154 (P7H, I74V, I105V) gene were detected in this study. The risk of infection was determined finding an association between the serological status and polymorphisms P7H, E35K, N70I, I74V, I105V of TMEM154, R447Q, A462S and G520R in TLR9 gene, H176H* and K190K* in MYD88 genes, while no statistical association was observed for the 4-bp deletion of the CCR5 gene. Since no vaccines or treatments have been developed, a genetically based approach could be an innovative strategy to prevent and to control SRLVs infection. Our findings are an important starting point in order to define the genetic resistance profile towards SRLVs infection.

Diagnosis and Pathogenesis of Nairobi Sheep Disease Orthonairovirus Infections in Sheep and Cattle.

Nairobi sheep disease orthonairovirus (NSDV) is a zoonotic tick-borne arbovirus, which causes severe gastroenteritis in small ruminants. To date, the virus is prevalent in East Africa and Asia. However, due to climate change, including the spread of transmitting tick vectors and increased animal movements, it is likely that the distribution range of NSDV is enlarging. In this project, sheep and cattle (hitherto classified as resistant to NSDV) were experimentally infected with NSDV for a comparative study of the species-specific pathogenesis. For this purpose, several new diagnostic assays (RT-qPCR, ELISA, iIFA, mVNT, PRNT) were developed, which will also be useful for future epidemiological investigations. All challenged sheep (three different doses groups) developed characteristic clinical signs, transient viremia and virus shedding-almost independent on the applied virus dose. Half of the sheep had to be euthanized due to severe clinical signs, including hemorrhagic diarrhea. In contrast, the course of infection in cattle was only subclinical. However, all ruminants showed seroconversion-implying that, indeed, both species are susceptible for NSDV. Hence, not only sheep but also cattle sera can be included in serological monitoring programs for the surveillance of NSDV occurrence and spread in the future.

A recent update about seroprevalence of ovine neosporosis in Northern Egypt and its associated risk factors.

Neospora caninum (Family: Sarcocystidae) is an obligate intracellular protozoan. It is one of the most critical abortifacients in ruminants. The seroprevalence of antibodies against N. caninum and its risk factors was investigated among 430 sheep from four North Egyptian governorates, Alexandria, Gharbia, Menofia, and Qalyubia, during the period from 2017 to 2018. Generally, the overall prevalence rate of N. caninum among sheep was 8.6%. The logistic regression analysis for the obtained data revealed that N. caninum increased significantly with age (OR = 2.4, 95% CI: 8.4-18.7) of the ewe (OR = 3.3, 95% CI: 7.6-14.9), particularly among sheep in contact with dogs (OR = 4.9, 95% CI: 7.5-14.3). Besides, locality, season, and pregnancy status of examined sheep had no significant effect on the appearance of N. caninum infection. the present findings confirm the presence of N. caninum among sheep in Egypt which probably play a role in reproductive failure in sheep. Therefore, sanitary measures and monitoring of the infection should be implemented to reduce the spreading of the infection.