The measurement of three cytokine transcripts in naïve and sensitized ovine peripheral blood mononuclear cells following in vitro stimulation with bluetongue virus serotype-23

Bluetongue (BT), a serious disease of sheep and some wild ruminants, is caused by bluetongue virus (BTV), a member of the family, Reoviridae. The current research thrust for controlling BT is on development of efficient vaccines, necessitating clear understanding of ovine immunology. At present, comparative studies on cytokine gene expression profiles of na?ve and BTV-sensitized peripheral blood mononuclear cells (PBMC) in sheep have not been clearly understood. In the present study, PBMC from na?ve and BEI-inactivated-saponin-adjuvanted BTV-1 vaccinated sheep were stimulated in vitro with heterologous BTV-23. At various intervals, RT-qPCR was carried out to estimate cytokine (interferon-gamma, interleukin-12 and interleukin-2) mRNA expressions that are linked to cell-mediated immunity. The results showed that PBMC cytokine profiles were relatively increased both temporally and quantitatively in immunized sheep PBMC compared to na?ve ones, suggesting that BTV-1 vaccination may prime immune system that can cross-react with BTV-23 antigens.     

Expression of matrix metalloproteinases in bovine luteal cells induced by prostaglandin F2α, interferon γ and tumor necrosis factor α

We recently demonstrated that luteal cells flow out from the ovary via lymphatic vessels during luteolysis. However, the regulatory mechanisms of the outflow of luteal cells are not known. Matrix metalloproteinases (MMPs) can degrade the extracellular matrix and basal membrane, and tissue inhibitors of matrix metalloproteinases (TIMPs) inhibit the activity of MMPs. To test the hypothesis that MMP expression in luteal cells is regulated by luteolytic factors, we investigated the effects of prostaglandin F2α (PGF), interferon γ (IFNG) and tumor necrosis factor α (TNF) on the mRNA expression of MMPs and TIMPs in cultured luteal cells. Luteal cells obtained from the CL at the mid-luteal stage (days 8–12 after ovulation) were cultured with PGF (0.01, 0.1, 1 μM), IFNG (0.05, 0.5, 5 nM) and TNF (0.05, 0.5, 0.5 nM) alone or in combination for 24 h. PGF and IFNG significantly increased the expression of MMP-1 mRNA. In addition, 1 μM PGF in combination with 5 nM IFNG stimulated MMP-1 and MMP-9 mRNA expression significantly more than either treatment alone. In contrast, IFNG significantly decreased the level of MMP-14 mRNA. The mRNA expression of TIMP-1, which preferentially inhibits MMP-1, was suppressed by 5 nM INFG. One μM PGF and 5 nM IFNG suppressed TIMP-2 mRNA expression. These results suggest a new role of MMPs: luteal MMPs stimulated by PGF and IFNG break down the extracellular matrix surrounding luteal cells, which accelerates detachment from the CL during luteolysis, providing an essential prerequisite for outflow of luteal cells from the CL to lymphatic vessels.     

Propofol attenuates LPS-induced tumor necrosis factor-α, interleukin-6 and nitric oxide expression in canine peripheral blood mononuclear cells possibly through down-regulation of nuclear factor (NF)-κB activation

Sepsis is a major cause of mortality in intensive care medicine. Propofol, an intravenous general anesthetic, has been suggested to have anti-inflammatory properties and able to prevent sepsis induced by Gram-positive and Gram-negative bacteria by down-regulating the gene expression of pro-inflammatory cytokines. However, propofol’s anti-inflammatory effects upon canine peripheral blood mononuclear cells (PBMCs) have not yet been clarified. Here, we isolate canine PBMCs and investigate the effects of propofol on the gene expressions of both lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) and tumor necrosis factor (TNF)-α and upon the production of nitric oxide (NO). Through real-time quantitative PCR and the Griess reagent system, we found that non-cytotoxic levels of propofol significantly inhibited the release of NO and IL-6 and TNF-α gene expression in LPS-induced canine PBMCs. Western blotting revealed that LPS does significantly increase the expression of inducible NO synthase (iNOS) protein in canine PBMCs, while pretreatment with propofol significantly decreases the LPS-induced iNOS protein expression. Propofol, at concentration of 25 µM and 50 µM, also significantly inhibited the LPS-induced nuclear translocation of nuclear factor (NF)-κB p65 protein in canine PBMCs. This diminished TNF-α, IL-6 and iNOS expression, and NO production was in parallel to the respective decreased NF-κB p65 protein nuclear translocation in the LPS-activated canine PBMCs pretreated with 25 µM and 50 µM propofol. This suggests that non-cytotoxic levels of propofol pretreatment can down-regulate LPS-induced inflammatory responses in canine PBMCs, possibly by inhibiting the nuclear translocation of the NF-κB p65 protein.     

Intratesticular expression of mRNAs of both interferon γ and tumor necrosis factor α is significantly increased in experimental autoimmune orchitis in mice

Experimental autoimmune orchitis (EAO) is one of the models of immunological male infertility. Murine EAO is CD4+T cell-dependent and classically induced by immunization with a testicular homogenate and adjuvants. We previously established that immunization with viable syngeneic testicular germ cells (TGC) can also induce murine EAO with no use of any adjuvant. Analyses of this EAO model have already revealed that cultured spleen cells of immunized mice secreted interferon (IFN)-γ and that treatment of the immunized mice with anti-IFN-γ monoclonal antibodies significantly suppressed the EAO. It is known that both IFN-γ and tumor necrosis factor (TNF)-α are representative cytokines of Th1 cells and exhibit local toxicity toward the seminiferous epithelium in vivo. However, changes in these two cytokines in EAO-affected testes have not yet been investigated. Therefore, in the present study, we investigated the expression of intratesticular IFN-γ and TNF- α mRNAs in TGC-induced EAO using real-time RT-PCR. The results demonstrated that the intratesticular mRNAs for both IFN-γ and TNF-α significantly increased, while other cytokines such as IL-1α, IL-1β, IL-6 and TGF-β did not show dramatic changes in the immunized mice. These results suggest that secretion of significant amounts of IFN-γ and TNF-α in situ contributes to the spermatogenic disturbance in EAO.     

Inhibin-α immunohistochemical expression in mature and immature canine Sertoli and Leydig cells

Formalin-fixed, paraffin-embedded sections from 32 canine pairs of testes were immunohistochemically examined for Inhibin-α (INHα). Samples were subdivided into two groups (group 1, neonates; group 2, puppies and adults) and results statistically compared. Inhibin-α was significantly expressed only in Sertoli cells of neonatal testes, while in Leydig cells it was expressed without significant difference between groups. These results suggest that, in dogs, INHα expression switches from Sertoli to Leydig cells during testicular maturation and that, in adult, Leydig cells represent the main source of INHα.     

Differential gene expression of antimicrobial peptides β defensins in the gastrointestinal tract of Salmonella serovar Pullorum infected broiler chickens

Salmonella enterica serovar Pullorum causes substantial mortality in chicks as well as results in persistent infection and vertical transmission in layer birds. An effective innate immune response in the early stages of infection could reduce bacterial colonization and mortality in chicks and persistency of infection in later stages. β Defensins (AvBDs) are now considered as one of the key components of innate immunity in avian species. In the present study, we quantified the mRNA expression levels of AvBDs (1–14) by real-time PCR in the gastrointestinal (GI) tissues (duodenum, jejunum, ileum and caecum) of 3-day-old broiler chicks after 24 h of oral infection with Salmonella Pullorum. Quantitative real-time PCR analysis revealed significant (P < 0.05) upregulation of AvBD3, 4, 5, 6 and 12 and a significant (P < 0.05) down regulation in the expressions of AvBD10, 11, 13 and 14 in one or few GI tissues, while no significant changes were observed for AvBD1, 2, 7, 8 and 9 gene expressions in any of the GI tissues investigated upon infection with S. Pullorum. Most substantial change in gene expression was found for AvBD5, being significantly (P < 0.01) upregulated in most of the GI tissues investigated. The differential expression levels of β defensins shed light on tailored innate immune response induced by S. Pullorum during the early stages of infection in chicks.     

11-Hydroxy-β-steroid dehydrogenase gene expression in canine adipose tissue and adipocytes: stimulation by lipopolysaccharide and tumor necrosis factor α

The enzyme 11β-hydroxysteroid dehydrogenase 1 (11β-HSD-1) is expressed in a number of tissues in rodents and humans and is responsible for the reactivation of inert cortisone into cortisol. Its gene expression and activity are increased in white adipose tissue (WAT) from obese humans and may contribute to the adverse metabolic consequences of obesity and the metabolic syndrome. The extent to which 11β-HSD-1 contributes to adipose tissue function in dogs is unknown; the aim of the present study was to examine 11β-HSD-1 gene expression and its regulation by proinflammatory and anti-inflammatory agents in canine adipocytes. Real-time PCR was used to examine the expression of 11β-HSD-1 in canine adipose tissue and canine adipocytes differentiated in culture. The mRNA encoding 11β-HSD-1 was identified in all the major WAT depots in dogs and also in liver, kidney, and spleen. Quantification by real-time PCR showed that 11β-HSD-1 mRNA was least in perirenal and falciform depots and greatest in subcutaneous, omental, and gonadal depots. Greater expression was seen in the omental depot in female than in male dogs (P = 0.05). Gene expression for 11β-HSD-1 was also seen in adipocytes, from both subcutaneous and visceral depots, differentiated in culture; expression was evident throughout differentiation but was generally greatest in preadipocytes and during early differentiation, declining as cells progressed to maturity. The inflammatory mediators lipopolysaccharide and tumor necrosis factor α had a main stimulatory effect on 11β-HSD-1 gene expression in canine subcutaneous adipocytes, but IL-6 had no significant effect. Treatment with dexamethasone resulted in a significant time- and dose-dependent increase in 11β-HSD-1 gene expression, with greatest effects seen at 24 h (2nM: approximately 4-fold; 20nM: approximately 14-fold; P = 0.010 for both). When subcutaneous adipocytes were treated with the peroxisome proliferator activated receptor γ agonist rosiglitazone, similar dose- and time-dependent effects were noted. However, no effects were seen when adipocytes from the gonadal WAT depot were treated with rosiglitazone. The induction of 11β-HSD-1 expression, by the pro-inflammatory cytokine tumor necrosis factor α and by lipopolysaccharide may have implications for the pathogenesis of obesity and its associated diseases in the dog.     

IFN-γ expression and infectivity of Toxoplasma infected tissues are associated with an antibody response against GRA7 in experimentally infected pigs

Toxoplasma gondii, an obligate intracellular parasite, can be transmitted to humans via the consumption of infected meat. However, there are currently no veterinary diagnostic tests available for the screening of animals at slaughter. In the current work, we investigated whether cytokine responses in the blood, and antibody responses against recombinant T. gondii GRA1, GRA7, MIC3 proteins and a chimeric antigen EC2 encoding MIC2-MIC3-SAG1, are associated with the infectivity of porcine tissues after experimental infection with T. gondii. Two weeks after experimental infection of conventional 5-week-old seronegative pigs, an IFN-γ response was detected in the blood, with a kinetic profile that followed the magnitude of the GRA7 antibody response. Antibody responses to GRA1, MIC3 and EC2 were very weak or absent up to 6 weeks post infection. Antibodies against GRA7 occurred in all infected animals and were associated with the presence of the parasite in tissues at euthanasia a few months later, as demonstrated by quantitative real-time PCR and isolation by bio-assay. Remarkably, although brain and heart tissue remained infectious, musculus gastrocnemius and musculus longissimus dorsi were found clear of infectious parasites 6 months after experimental infection. Seropositive response in a GRA7 ELISA indicates a Toxoplasma infection in pigs and is predictive of the presence of infectious cysts in pig heart and brain. This new ELISA is a promising tool to study the prevalence of Toxoplasma infection in pigs. Clearance of the infection in certain pig tissues suggests that the risk assessment of pig meat for human health needs further evaluation.     

Clinical and hematological signs associated with dogs naturally infected by Hepatozoon sp. and with other hematozoa: a retrospective study in Uberlândia, Minas Gerais, Brazil

An evaluation was made of the clinical and hematological aspects of 115 dogs infected naturally by Hepatozoon sp. and treated at the Hospital School of the Faculty of Veterinary Medicine in Uberlandia, MG, Brazil. Of the 115 dogs for whom peripheral blood films were evaluated, 89 (77.39%) presented parasitemia by Hepatozoon sp. solely, while 26 (22.61%) had combination of Hepatozoon sp., Babesia sp. and Ehrlichia sp. Young male dogs less than a year old, of undefined breed (UB), were the most commonly affected. Thirty-nine (33.92%) of the dogs were asymptomatic while 76 (66.08%) presented varied clinical symptoms, the most frequent being anorexia, pulmonary alterations, hyperthermia, pale mucosae, apathy and/or prostration, and diarrhea. The majority of hematological alterations were normochromic-normocytic anemia, leukocytosis, neutrophilia, and nuclear deviation of neutrophils to the left (NDNL). The findings of this study confirm that Hepatozoon sp. causes clinical and hematological alterations of varied intensity, which, albeit not specific to canine hepatozoonosis, reinforce the notion that the discovery of the agent in dogs, even with low parasitemia, should be taken into consideration.     

Effects of gibberellin and its inhibitors on growth and gene expression in Poa pratensis北大核心CSCD

研究了不同浓度的赤霉素及其抑制剂烯效唑和多效唑对草地早熟禾生长及赤霉素相关基因表达的影响,为外源生长调节剂在草坪养护管理中的高效利用提供理论依据。以1/2 Hogland营养液培养的草地早熟禾为材料,随机分成7组,分别施用不同浓度的赤霉素、烯效唑、多效唑,测定根及叶片的生长量、叶绿体分布、叶绿素含量以及赤霉素相关基因的表达量。结果显示：与对照比较,外源烯效唑和多效唑均显著抑制草地早熟禾的伸长生长以及干/鲜重,但提高了根冠比、叶绿素a、叶绿素b及类胡萝卜素含量（P<0.05）;增加了叶肉细胞层数,使叶绿体充满在叶片上下表皮之间的叶肉细胞中;赤霉素合成代谢途径关键酶基因PpKAO、PpKS1、PpGA₃ox、PpGA₂ox表达量显著下调,赤霉素信号转导受体蛋白基因PpGID1显著上调（P<0.05）。外源赤霉素对草地早熟禾的伸长生长具有促进作用,但降低了根、叶的干/鲜重,提高根冠比,显著提高了赤霉素分解基因PpGA₂ox表达量（P<0.05）。总之,烯效唑、多效唑对草地早熟禾生长发育及基因表达的调控作用与赤霉素相反,但无明显浓度效应;在草坪修剪后立即喷施1000倍的烯效唑或多效唑,对减少用工成本,增加草坪观赏性、提高园林绿化中的草坪草管护水平具有重要意义。     

Dietary betaine activates hepatic VTGⅡ expression in laying hens associated with hypomethylation of GR gene promoter and enhanced GR expression

Background: Vitellogenin(VTG) is a precursor of egg yolk proteins synthesized within the liver of oviparous vertebrates. Betaine is an important methyl donor that is reported to improve egg production of laying hens with an unclear mechanism. In the present study, we fed betaine-supplemented diet(0.5%) to laying hens for 4 wk and investigated its effect on VTGII expression in the liver.Results: Betaine did not affect chicken weight, but significantly(P 0.05) increased egg laying rate accompanied with a significant(P 0.05) increase in hepatic concentration and plasma level of VTGI. Plasma estrogen level did not change,but the hepatic expression of estrogen receptor α(ERα) mR NA was significantly(P 0.05) up-regulated. Betaine did not affect the protein content of ERα, but significantly(P 0.05) increased hepatic expression of glucocorticoid receptor(GR)at both mR NA and protein levels. Also, ERα/GR interaction tended to be enhanced in the liver nuclear lysates of betainesupplemented hens as determined by co-immunoprecipitation. Furthermore, dietary betaine supplementation significantly increased(P 0.05) the hepatic expression of methyl-transfer enzymes, such as BHMT, GNMT, and DNMT1,which was associated with higher SAM/SAH ratio and hypomethylation of GR promoter regions.Conclusions: Betaine activates hepatic VTGII expression in association with modified DNA methylation of GR gene promoter, GR expression and ERα/GR interaction. Activation of hepatic VTGII expression may contribute, at least partly, to improved egg production in betaine-supplemented hens.     

A stochastic model accommodating the FAMACHA© system for estimating worm burdens and associated risk factors in sheep naturally infected with Haemonchus contortus

A previously developed multiple regression algorithm was used as the basis of a stochastic model to simulate worm burdens in sheep naturally infected with Haemonchus contortus over five consecutive Haemonchus seasons (November to January/February) on a farm in the summer rainfall region in South Africa, although only one season is discussed. The algorithm associates haemoglobin levels with worm counts in individual animals. Variables were represented by distributions based on FAMACHA(?) scores and body weights of sheep, and Monte Carlo sampling was used to simulate worm burdens. Under conditions of high disease risk, defined as the sampling event during the worm season with the lowest relative mean haemoglobin level for a class of sheep, the model provided a distribution function for mean class H. contortus burdens and the probability of these occurring. A mean H. contortus burden for ewes (n=130 per sample) of approximately 1000 (range 51-28,768) and 2933 (range 78-44,175) for rams (n=120 per sample) was predicted under these conditions. At the beginning of the worm season when the risk of disease was lowest (i.e. when both classes had their highest estimated mean haemoglobin levels), a mean worm burden of 525 (range 39-4910) for ewes and 651 (range 37-17,260) for rams was predicted. Model indications were that despite being selectively drenched according to FAMACHA(?) evaluation, 72% of the ewes would maintain their mean worm burden below an arbitrarily selected threshold of 1000 even when risk of disease was at its highest. In contrast, far fewer rams (27%) remained below this threshold, especially towards the end of the worm season. The model was most sensitive to changes in haemoglobin value, and thus by extrapolation, the haematocrit, which is used as the gold standard for validating the FAMACHA(?) system. The mean class haemoglobin level at which there was a 50% probability of worm burdens being ≤ 1000 worms was 7.05 g/dl in ewes and 7.92 g/dl in rams.