Plasminogen activator urokinase interacts with the fusion protein and antagonizes the growth of Peste des petits ruminants virus.

Peste des petits ruminants is an acute and highly contagious disease caused by the Peste des petits ruminants virus (PPRV). Host proteins play a crucial role in viral replication. However, the effect of fusion (F) protein-interacting partners on PPRV infection is poorly understood. In this study, we found that the expression of goat plasminogen activator urokinase (PLAU) gradually decreased in a time- and dose-dependent manner in PPRV-infected goat alveolar macrophages (GAMs). Goat PLAU was subsequently identified using co-immunoprecipitation and confocal microscopy as an F protein binding partner. The overexpression of goat PLAU inhibited PPRV growth and replication, whereas silencing goat PLAU promoted viral growth and replication. Additionally, we confirmed that goat PLAU interacted with a virus-induced signaling adapter (VISA) to antagonize F-mediated VISA degradation, increasing the production of type I interferon. We also found that goat PLAU reduced the inhibition of PPRV replication in VISA-knockdown GAMs. Our results show that the host protein PLAU inhibits the growth and replication of PPRV by VISA-triggering RIG-I-like receptors and provides insight into the host protein that antagonizes PPRV immunosuppression.IMPORTANCEThe role of host proteins that interact with Peste des petits ruminants virus (PPRV) fusion (F) protein in PPRV replication is poorly understood. This study confirmed that goat plasminogen activator urokinase (PLAU) interacts with the PPRV F protein. We further discovered that goat PLAU inhibited PPRV replication by enhancing virus-induced signaling adapter (VISA) expression and reducing the ability of the F protein to degrade VISA. These findings offer insights into host resistance to viral invasion and suggest new strategies and directions for developing PPR vaccines.

Effects of topical application of resveratrol on tight junction barrier and antimicrobial compound production in lactating goat mammary glands.

In mammary glands, the formation of less-permeable tight junctions (TJs) and the production of antimicrobial compounds like lactoferrin and defensins are important for preventing mastitis. Resveratrol, a polyphenol contained in red grapes, is known to protect mammary epithelial cells (MECs) from oxidative stress; however, oral administration of resveratrol causes a decrease in certain biological processes through conjugation and metabolic conversion. In this study, we determined the beneficial effects of resveratrol on TJs and antimicrobial compounds in cultured goat MECs by adding it to the medium, and in lactating goat mammary glands by topical application for percutaneous absorption. TJ barrier function was evaluated by transepithelial resistance and expression or localization pattern of claudins for culture model in vitro and by somatic cell count, Na+, albumin, and IgG in milk for topical application in vivo. Concentrations of antimicrobial compounds and cytokines were measured using ELISA. Activation of STAT3 was evaluated by Western blotting. Resveratrol strengthened TJ barrier function by upregulating claudin-3 in cultured MECs and topical application to udders reduced somatic cell count, Na+, albumin, and IgG in milk. Resveratrol increased ß-defensin and S100A7 levels in cultured MECs and milk. In addition, resveratrol down-regulated cytokine production and STAT3 pathway. These findings suggest that the topical application of resveratrol to udders may be effective in preventing mastitis.

In vitro evaluation of immunomodulatory activities of goat milk Extracellular Vesicles (mEVs) in a model of gut inflammation.

Gut represents a major immunological defense barrier with mucosal immune system and intestinal epithelial cells (IECs). In all intestinal diseases, in particular inflammatory bowel disease (IBD), both the absorption and the local immune system are compromised and alternative effective therapies are sought after. Extracellular Vesicles (EVs) have the capability to regulate immune cells within the inflammatory microenvironment, by dampening inflammation and restoring intestinal barrier integrity. Recently, the immune-modulatory role of EVs has also been confirmed for milk EVs (mEVs), notable for their easy production, high sample volumes, cost-effective scalable production and non-toxic and non-immunogenic behavior. In this context, the aim of this study was to evaluate goat mEV anti-inflammatory and immuno-modulating effects on an in vitro model (IPEC-J2) of intestinal inflammation through gene expression evaluation with RT-qPCR and cytokine release dosage with ELISA test. After the establishment of a pro-inflammatory environment due to LPS stimuli, IL6, CXCL8, IL12p35, IL12p40, IFNB, IL18, TLR7 and NOS2 resulted significantly up-regulated in stimulated IPEC-J2 cells compared to those of the basal culture. After 48 h of mEV treatment in inflamed IPEC-J2 a partial restoration of initial conditions was detected, with the IL18 and IL12p40 significant down-regulation, and IL12p35, EBI3, TLR7, BD1 and BD3 up-regulation. IL-18 reduced protein production was also detected in supernatants. Moreover, a decrease of MMP9 and NOS2 together with a strong up-regulation of MUC2 indicated a recovery of cellular homeostasis and, therefore, potential beneficial effects on the intestinal mucosa. Nevertheless, 48 h post-treatment, an increased gene expression and protein release of IL-8 was observed. This paper is one of the firsts to assess the effect of goat mEVs and the first one, in particular, of doing this on an in vitro model of gut inflammation. The obtained results show a potential capability of goat mEVs to modulate inflammation and to play beneficial effects on the intestinal mucosa.

Evaluation of antioxidant and oxidant status of goats (Capra aegagrus hircus) naturally infected with Haemonchus contortus.

The present study aimed to assess the antioxidant and oxidant status of goats naturally infected with Haemonchus contortus. Based upon the parasite burden, infection in goats was categorized as heavy (> 500 worms), mild (100-500 worms) or low (< 100 worms). Abomasal tissues from non-infected and infected goats were used for the determination of catalase (CAT), glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), aspartate (AST) and alanine (ALT) aminotransferases, acid (ACP) and alkaline (ALP) phosphatases, superoxide content (O2-), protein carbonyl (PC), malondialdehyde (MDA) and reduced glutathione (GSH). A significantly higher level of CAT, GST and GR activity and a lower level of GPx activity were recorded in infected compared to non-infected tissue. A significant increase in the level of AST, ALT, ALP and ACP was found in the abomasal tissue of the infected animals, which was related to the worm burden. The oxidative stress markers were also altered, with a significant decline in GSH levels, whereas MDA, PC and O2- concentrations showed a marked increase. In conclusion, it has been demonstrated that haemonchosis in goats resulted in considerable oxidative stress, which was directly related to the worm burden.

Characterization of inflammatory responses by cervical cytology, cytokine expression and ultrastructure changes in a goat subclinical endometritis model.

Endometritis is a common puerperal disease in livestock. Thus, a practical clinical test for the establishment of diagnosis is needed. We developed a goat model for subclinical endometritis diagnosis by examining cytological, ultrastructural features and molecular expression in cervical discharge collected by cervical cytobrush method. A suspension of E. coli was infused into the uterine horn of goats, and cervical discharge, peripheral blood and endomertrial biopsy samples were collected before inoculation and at 3, 6, 12, 24, 72, 120 and 168 hr post-inoculation (pi). In experimental goats, total leukocytes increased at 6-12 hr pi in hematological examinations, abundant neutrophils were observed in cervical discharge smears, and some micro-villi shortened or were lost from the epithelium at 3 hr pi, with some inflammatory cells infiltrated into the uterine glands and lamina propria. The mRNA expression levels of the Toll-like receptor 4 (TLR4), cytokines and ß-defensin-2 increased significantly within 12 hr pi and returned to pre-inoculation levels at 7 day pi. The results showed that subclinical endometritis was simulated in this bacterial infusion goat model, and the evaluation of cervical cytology was in good agreement with molecular changes of cytokines in cervical discharge, hematology and histology. We thus conclude that the simple cervical cytobrush technique can effectively obtain cervical discharge samples for cytology examination in the early diagnosis of subclinical endometritis in goats.

Evaluation of serum concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-10, and nitric oxide (NO) during the estrous cycle, early pregnancy and abortion in goats.

The aim of this study was to establish the serum concentrations, ranges, and trends of Th1 type cytokine (tumor necrosis factor (TNF)-α and interleukin (IL)-2), Th2 type cytokine (IL-10), and nitric oxide (NO) during the estrous cycle, early pregnancy and abortion in goats. Boer goats (n=25) having symptoms of normal estrous cycles were selected, 20 were mated and 15 conceived a pregnancy, and the remaining five were not mated and served as estrous controls. On the Day 60 of pregnancy, all 15 pregnant goats were induced to abort the pregnancy by intramuscular injection of prostaglandin (PG). Serum samples were collected on Days 1, 7, 14, and 19 of the estrous cycle, at Days 0, 10, 20, 30, 40, 50, and 60 of pregnancy, and at Days 1, 3, 8, 10 over the period when abortion were occurring. Results of the present study indicated that during the estrous cycle the balance between Th1 and Th2 cytokines slightly shifted toward Th1 cytokine production (TNF-α and IL-2). The NO may have a direct positive role in inducing a Th1 response. During early pregnancy, TNF-α and IL-2 serum concentrations markedly increased from Days 0 to 10, and gradually decreased from Days 10 to 60, while IL-10 and NO serum concentrations remained elevated from Days 0 to 60. The increased concentrations of IL-10 and decreased concentrations of TNF-α and IL-2 are characteristic of a Th2-enhanced response, which may be related to increased concentrations of NO. These changes may be essential to maintain a normal pregnancy. In addition, the serum concentrations of TNF-α, IL-2 and NO at Days 1, 3, 8 and 10 of the period of induced abortion were markedly greater than that on Day 60 of pregnancy. Conversely, IL-10 concentrations at these four time points of abortion were markedly less than that on Day 60 of pregnancy. After abortion, the Th2 response shifted to a Th1-enhanced response. Thus, NO concentrations increase and the Th1-enhanced response may function synergistically to be involved in physiologic responses that lead to abortion of the pregnancy. It is concluded that the serum concentrations of the Th1/Th2 cytokine and NO changed temporally as the estrous cycle, pregnancy and abortion progressed advanced. A Th2-enhanced state promoted normal pregnancy, while increased concentrations of Th1 were observed during the period of fetal abortion. The concentrations of NO varied in regulation of the Th1/Th2 cytokine concentration balance during the three phases (estrous cycle, pregnancy, and fetal abortion) of goats.

Acute phase proteins increase with sarcoptic mange status and severity in Iberian ibex (Capra pyrenaica, Schinz 1838).

Sarcoptic mange is a contagious skin disease caused by Sarcoptes scabiei, affecting both domestic and wild mammals, including the Iberian ibex (Capra pyrenaica), a medium-sized mountain ungulate almost endemic to the Iberian Peninsula. Acute phase proteins (APPs) could be an indicator of sarcoptic mange disease and severity in Iberian ibex. Serum samples from 131 healthy and sarcoptic mange-affected Iberian ibexes were collected from 2005 to 2012 in Sierra Nevada Natural Space in southern Spain. Serum alpha-1-acid glycoprotein (AGP), serum amyloid A (SAA) and haptoglobin (Hp) concentrations were quantified, and statistically significant differences according to sarcoptic mange disease and severity were assessed. Both AGP and SAA were significantly higher in the sarcoptic mange-affected ibexes than in the healthy ones as well as in the severely affected ibexes as compared to those with less than 50 % of the body surface affected. For the first time, changes in APP are reported in relation to sarcoptic mange in Iberian ibex. It is also reported for the first time that the intensity of APP increase depends on the severity of sarcoptic mange, which could be related with the pathological secondary amyloidosis, leading to organ dysfunction in severely mange-affected animals. Species and population differences in the increase of APP in response to sarcoptic mange could indicate individual and population differences in the immune capability of each population to deal with mange, population prevalence and mortality being the last indicators of such sensitivity.

Toxicity of white snakeroot (Ageratina altissima) and chemical extracts of white snakeroot in goats.

White snakeroot (Ageratina altissima) is a sporadically toxic plant that causes trembles in livestock and milk sickness in humans that drink tainted milk. The putative toxin in white snakeroot is tremetone and possibly other benzofuran ketones, even though it has not been demonstrated in vivo. Toxic white snakeroot was dosed to goats, and they developed clinical signs of poisoning, exercise intolerance, significant increases in serum enzyme activities, and histological changes. Tremetone and the other benzofuran ketones were extracted with hexane; the extracts and residues were analyzed for tremetone and dosed to goats at tremetone and benzofuran ketone concentrations similar to the original plant material. However, none of the dosed goats developed the disease. The results demonstrate for the first time that white snakeroot is a potent myotoxin in goats and that other compound(s), which may be lost or modified during the extraction process, could be involved in causing trembles and milk sickness.

Differential cytokine expression in natural and experimental mastitis induced by Mycoplasma agalactiae in dairy goats.

Cytokines, primarily produced by macrophages and lymphocytes, mobilize the immune system in response to infection, particularly at mucosal surfaces. Knowledge of the pathogenesis and persistence of Mycoplasma agalactiae (Ma) in the mammary gland is still insufficient. The aim of this study was to elucidate the role of cytokines in the pathogenesis of caprine mastitis caused by Ma. Cytokine expression was evaluated by immunohistochemical methods in the inflammatory lesions of 10 (5 naturally and 5 experimentally infected) goats with Ma-induced mastitis. Immunolabelling for IL-10, IFN-γ, IL-4 and TNF-α was observed in inflammatory cells within the lumen of acini and ducts and in the interstitial spaces and was usually associated with the presence of Ma antigen. The results suggest that cytokines play a role in the pathophysiological processes during Ma infection as differential expression of these cytokines was detected in relation to the course of the infection.

Intestinal strictures, fibrous adhesions and high local interleukin-10 levels in goats infected naturally with Mycobacterium avium subsp. paratuberculosis.

This study describes pathological findings and their association with the production of interferon (IFN)-γ and interleukin (IL)-10 in goats infected naturally with Mycobacterium avium subsp. paratuberculosis (MAP). Twenty-seven goats were subjected to pathological examination. More than half of the animals had severe, diffuse, transmural granulomatous enteritis, often with abundant acid-fast bacilli (AFB), which was most evident in the proximal jejunum. Jejunal strictures and fibrous, peritoneal adhesions were findings that are not often reported in animals with paratuberculosis. Immunohistochemical labelling of IL-10 was seen within diffuse, granulomatous lesions and this may have prevented optimal local IFN-γ production and exacerbated the disease. However, since IFN-γ production was detected in cells from blood, jejunum and jejunal lymph nodes of goats with severe lesions by enzyme-linked immunosorbent assay, intracellular labelling and in-situ hybridization, the up-regulation of IL-10 might have been a consequence rather than a cause of the severe disease. The IL-10 labelling was co-localized with major histocompatibility complex (MHC) class II(+) cells, but rarely with CD4(+) cells. Comparable numbers of CD4(+) and CD8(+) T cells were recruited to both severe, diffuse lesions and small to moderate granulomatous lesions, while few T cells expressing the γδ form of the T-cell receptor were associated with both types of lesions.

Evaluation of drying methods and toxicity of rayless goldenrod ( Isocoma pluriflora ) and white snakeroot ( Ageratina altissima ) in goats.

White snakeroot and rayless goldenrod cause "trembles" and "milk sickness" in livestock and humans, respectively. The toxin in white snakeroot and rayless goldenrod was identified in 1927 and 1930, respectively, as tremetol. It was reported that the toxin in white snakeroot disappears as it is dried and that completely dried plants were incapable of producing trembles or milk sickness. Conversely, it has been reported that the rayless goldenrod toxin was not destroyed by drying and that the plant is toxic either fresh or dry. In this study the concentrations of tremetone, dehydrotremetone, and structurally similar compounds were determined in white snakeroot and rayless goldenrod before and after various drying conditions. Tremetone, dehydrotremetone, and structurally similar compounds in rayless goldenrod and white snakeroot are most stable upon freeze-drying, followed by air-drying, and least stable upon oven-drying (60 °C). Also demonstrated is that tremetone is stable and that dried white snakeroot and rayless goldenrod are capable of inducing toxicosis in livestock.

Peripartal hormonal changes in Alpine goats: a comparison between physiological and pathological parturition.

In this study, 31 pregnant Alpine does were used to investigate the peripartal plasma profiles of progesterone, estradiol-17ß, 15-ketodihydro-PGF(2α) and cortisol, assessing differences between goats with physiological and pathological parturition. The goats were observed around the time of parturition; all peripartum abnormalities were recorded, and veterinary assistance was provided if necessary. Blood samples were collected every 12 h from 7 days before to 7 days after delivery, and plasma used for hormonal analysis by radioimmunoassay. Two animals died during the study, and their data were excluded from the study. Of the remaining 29 animals, 23 goats had a spontaneous and physiological delivery, while six goats showed pathological parturition, including dystocia and retained placenta. The 65 alive kids were viable at birth and at 7 days of age. The results concerning the hormonal concentrations in the normal parturition confirm and define more precisely the patterns already described in the goat, while the comparison between physiological and pathological parturition has never been previously reported in this species. Highest peripartum levels of cortisol were found in the pathological group at delivery (30.6 vs 15.9 ng/ml) (p<0.01) and 12 h later (26.2 vs 11.1 ng/ml) (p<0.05); the greater cortisol concentrations found in goats with dystocia and retained placenta could suggest a higher level of stress. No significant differences between the two groups were found with respect to the circulating values of the other hormones, but the individual variability and the small number of goats enrolled in the pathological delivery group could have masked possible differences.

Sparse PrP(Sc) accumulation in the placentas of goats with naturally acquired scrapie.

BACKGROUND: Domestic goats (Capra hircus) are a natural and experimental host of scrapie and bovine spongiform encephalopathy, the transmissible spongiform encephalopathies (TSE) of sheep and cattle. Goats are also susceptible to experimental infection with the agents of TSEs of deer and elk (chronic wasting disease) and humans (Creutzfeldt Jakob disease). Distribution of PrPSc, the abnormal prion protein, is similar in the tissues of scrapie-infected sheep and goats but no data are available on the potential shedding of the agent through the placenta, the presumed route of transmission of ovine scrapie. We describe the sparse accumulation of PrPSc in the placentas of goats with naturally acquired classical scrapie in comparison to field cases of classical ovine scrapie. RESULTS: PrPSc was detected in the shed placentas from a sample of U.S. goats with naturally occurring scrapie, diagnosed by antemortem lymphoid tissue biopsy or identified as high risk progeny of infected dams. PrPSc accumulation patterns in the intact placentome and western blot banding was similar in the caprine and ovine samples. However, levels of PrPSc estimated from ELISA and immunohistochemistry assays were generally lower in goats than in sheep, although wide variation was noted in both species. CONCLUSIONS: PrPSc accumulates in the shed placentas of goats with naturally acquired scrapie. Although these levels were low in most caprine samples, the caprine placenta may contribute to prion contamination of kidding facilities and transmission to co-housed sheep or goats.

Detection of typical and atypical bovine spongiform encephalopathy and scrapie prion strains by prion protein motif-grafted antibodies.

To evaluate further the reactivity of prion-specific monoclonal antibodies containing the 89-112 or 136-158 prion protein (PrP) polypeptides, immunoprecipitations were performed on brain extracts from Italian bovines, sheep and goats with transmissible spongiform encephalopathies. No binding of IgG 89-112 or IgG 136-158 to PrP in normal brain extracts was detected. Conversely, both reagents immunoprecipitated PrP from bovine and bovine amyloidotic spongiform encephalopathies, and from typical and atypical scrapie brain extracts. The immunoprecipitated PrP bands mirrored the Western blot (WB) profile of the different prion strains, indicating universal affinity of two independent PrP regions for disease-associated PrP conformers regardless of species source and strain properties. Immunoprecipitation with motif-grafted antibodies increased the sensitivity of conventional detection methods based on centrifugation followed by WB, which was confirmed by assay of diluted samples using both methods. These reagents or derivative molecules may thus find broad applications in prion detection and research.

KIT-positive gastrointestinal stromal tumours in two Spanish ibex (Capra pyrenaica hispanica).

Gastrointestinal mesenchymal tumours from two Spanish ibex (Capra pyrenaica hispanica) were examined grossly, histologically and immunohistochemically. One neoplasm was a 1.5 kg tan multinodular cavitated mass in the forestomach. The other tumour was a firm mural mass 1.2 cm in diameter in the colon. Microscopically, both tumours were formed mainly by spindle shaped cells arranged in closely packed interlacing fascicles. Neoplastic cells in both tumours labelled positively for KIT (CD117), vimentin and alpha-smooth muscle actin. These findings suggest that both neoplasms were gastrointestinal stromal tumours and most likely to be derived from the interstitial cells of Cajal or their progenitor cells.

Malignant peripheral nerve sheath tumour (malignant schwannoma) in the diaphragm of a goat.

This report describes a malignant schwannoma in the diaphragm of a 2-year-old goat. The immunohistochemical and histological features indicated a diagnosis of malignant schwannoma rather than neurofibrosarcoma. The diaphragm represents an unusual location for neoplasia in both domestic animals and human beings. A possible anatomical origin from the phrenic nerve is discussed.

Diversity in neuroanatomical distribution of abnormal prion protein in atypical scrapie.

Scrapie is a transmissible spongiform encephalopathy (TSE) in sheep and goats. In recent years, atypical scrapie cases were identified that differed from classical scrapie in the molecular characteristics of the disease-associated pathological prion protein (PrP(sc)). In this study, we analyze the molecular and neuropathological phenotype of nine Swiss TSE cases in sheep and goats. One sheep was identified as classical scrapie, whereas six sheep, as well as two goats, were classified as atypical scrapie. The latter revealed a uniform electrophoretic mobility pattern of the proteinase K-resistant core fragment of PrP(sc) distinct from classical scrapie regardless of the genotype, the species, and the neuroanatomical structure. Remarkably different types of neuroanatomical PrP(sc) distribution were observed in atypical scrapie cases by both western immunoblotting and immunohistochemistry. Our findings indicate that the biodiversity in atypical scrapie is larger than expected and thus impacts on current sampling and testing strategies in small ruminant TSE surveillance.

Intestinal glandular inclusions (glandular choristoma) in the mesenteric lymph node of a goat.

A glandular choristoma found in the mesenteric lymph node of a goat would appear to represent the first reported case of non-neoplastic glandular inclusions in domestic animals. The origin of this type of lesion may be difficult to determine, but in the present case cytokeratin expression patterns suggested that the inclusions had an intestinal origin.

A biochemical study of cystic stage fluids of Echinococcus granulosus of animal origin in Libya.

The biochemical analyses of hydatid cyst fluids of sheep, goats and camels slaughtered in abattoir of Sirt, Libya showed the same constituents of elements and organic compounds. But, sheep hydatid cyst fluids contained significantly more triglycerides (P<0.05), phosphorus (P<0.05) and protein (P<0.001) than those in camels and goats. Urea was significantly more (P<0.05) in sheep and goats compared to camels.

Stress associated with road transportation in desert sheep and goats, and the effect of pretreatment with xylazine or sodium betaine.

The present work investigates some clinical, endocrinological, biochemical and haematological variables in desert sheep and goats stressed in the course of individual road transportation, and the influence thereon of pretreatment with an established anti-stressor drug, xylazine HCl, and a test compound, sodium betaine (trimethylglycine). Road transportation for 2h resulted in variable and statistically insignificant increases in heart, pulse and respiratory rates in both control and experimental animals. Transportation stress significantly increased the concentrations of plasma cortisol, and glucose, and decreased that of magnesium. The endogenous thiocyanate concentration was unaffected. The stress also insignificantly decreased the haematocrit (PCV), and the number of lymphocytes, and increased the concentration of haemoglobin. Pretreatment of sheep and goats with xylazine at a single dose of 0.01 mg/kg by the intravenous route significantly ameliorated the effects induced by the stressful stimulus. The effects of pretreatment of the two species with sodium betaine (10 mg/kg) produced variable and insignificant effects.