Expression of toll‐like receptor 2 mRNA in bronchial epithelial cells is not induced in RAO‐affected horses

Reasons for performing study: Airway inflammation in recurrent airway obstruction (RAO) is triggered by housing affected horses in stables. It has been suggested that RAO is an allergic condition, but innate immune mechanisms are also involved. Fungal products activate innate immune mechanisms through toll‐like receptor 2 (TLR2). In human airway epithelium, TLR2 activation leads to interleukin (IL)‐8 production. This pathway is negatively regulated by the zinc finger protein A20. This study was performed to enhance understanding of innate immune mechanisms in RAO. Hypothesis: TLR2 and IL‐8 mRNA are elevated in RAO during stabling compared with controls. A20 mRNA is negatively associated with the numbers of airway inflammatory cells. Objectives: To determine TLR 2 , IL‐8 and A20 mRNA expression in lungs of stabled and pastured RAO‐affected and control horses. Methods: Airway obstruction and inflammatory cell counts in bronchoalveolar lavage were measured, and TLR 2 , IL‐8 and A20 mRNA expression quantified by qRT‐PCR in 6 RAO‐affected and 6 control horses, during and after exposure to hay and straw. Results: Airway obstruction and neutrophils were increased in RAO‐affected horses during stabling. While stabling increased IL‐ 8 , TLR2 and A20 mRNA were unaffected. TLR2 and A20 were significantly correlated (r = 0.83) and A20 mRNA was negatively associated with inflammatory cells. Potential relevance: Stabling does not lead to an increase in TLR2 expression. Other molecules or processes in the TLR2 cascade might be important in fungal‐induced airway inflammation. Equine epithelial‐derived A20 may be involved in modulation of airway inflammation.     

Leptospiral lipopolysaccharide stimulates the expression of toll‐like receptor 2 and cytokines in pig fibroblasts

Pigs throughout the world are afflicted with leptospirosis, causing serious economic losses and potential hazards to human health. Although it has been known that leptospiral lipopolysaccharide (L‐LPS) is involved in an immunological reaction between an antigen and a host cell, little is known about how the immune system of pigs can respond to L‐LPS. Here, we stimulated pig fibroblasts by L‐LPS and then quantitatively measured gene and protein expression levels of two toll‐like receptors (TLRs), TLR2 and TLR4, by real‐time PCR and Western blotting. As a result, expression of TLR2 was found to be significantly up‐regulated within 24 h after L‐LPS stimulation whereas induction of TLR4 expression was relatively weak. We also revealed that of myeloid differentiation primary response gene 88 (MyD88), interleukin 6 (IL‐6) and IL‐8 gene expressions were markedly up‐regulated by L‐LPS stimulation. These results may suggest that the pig cell can activate TLR2 rather than TLR4 by L‐LPS stimulation, thereby inducing expression of cytokines.     

固有免疫相关模式识别受体研究进展

固有免疫是机体免疫系统防御微生物感染的第1道防线。模式识别受体(PRRs)中的Toll样受体(TLRs)、核苷酸寡聚结合域样受体(NLRs)和视黄酸诱导基因1样受体(RLRs)等识别不同或重叠的微生物组成成分,形成相应的信号转导产生免疫应答。近些年对于固有免疫相关PRRs的研究也取得了较大的进展,为免疫相关疾病的治疗提供了思路。本文对固有免疫相关PRRs的特征及不同PRRs的信号传导途径之间相互作用进行综述。     

Differential expression of genes encoding neurotrophic factors and their receptors along the septal‐temporal axis of the rat hippocampus

The hippocampus plays a key role in learning and emotional regulation. The hippocampus’ function varies along its septotemporal axis, with the septal pole being more frequently involved in spatial learning and memory, and the temporal pole playing a greater role in emotional behaviors. In this study, we present findings aimed at checking the expression level of the genes encoding neurotrophins and their receptors, including nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), and their receptors (TrkA, TrkB and TrkC) in the hippocampus along the septotemporal axis. Using real‐time PCR, several different expression patterns were observed. Remarkably, the expression of both NT‐3 and TrkA genes in the septal hippocampus was higher than in the middle and temporal hippocampus. Higher expression of NT‐3 and TrkA may implicate active neurogenesis in the dentate gyrus (DG) of the septal hippocampus because more neurogenesis occurs in the septal than the temporal DG of rats. Finally, the results obtained in this study emphasize the importance of choosing the hippocampal portion along its septotemporal axis for any hippocampal molecular and biochemical experimental studies.     

Oxyntomodulin reduces expression of glucagon‐like peptide 1 receptor in the brainstem of chickens

Oxyntomodulin (OXM) is a peptide released from the gut and attenuates food intake by acting on hypothalamus. However, its role at the molecular level is not well studied. In the first section of this study, we analysed the effect of OXM on food intake behaviour after injecting into the lateral ventricle of chickens. The outcome showed that food intake decreased significantly after administering 4 nmol of OXM. In the second part, the expression of glucagon‐like peptide 1 receptor (GLP‐1R) in the brainstem was analysed by real‐time RT‐PCR. The results showed that expression of GLP‐1R was reduced to 27% and 16% at 30 and 90 mins after injection of OXM respectively. In saline‐injected chickens, no reduction in GLP‐1R was seen. It can be concluded that OXM has a down regulatory effect on the responding receptor, GLP‐1R and OXM in chicks has the same reductive effect on food intake as in the mammals.     

Functional insulin‐like factor 3 (INSL3) hormone‐receptor system in the testes and spermatozoa of domestic ruminants and its potential as a predictor of sire fertility

Insulin‐like factor 3 (INSL3) is essential for fetal testis descent, and has been implicated in the testicular and sperm functions in adult males; however, similar functions in domestic ruminants remain largely unknown. This study investigated the functional INSL3 hormone‐receptor system in adult ruminant testes and spermatozoa, and explored its potential to diagnose the fertility of sires. Testes and spermatozoa were obtained from fertile bulls, rams and he‐goats, whereas subfertile testes and spermatozoa were obtained only from bulls. As expected, INSL3 was visualized in Leydig cells, while we clearly demonstrated that the functional receptor, relaxin family peptide receptor 2 (RXFP2), enabling INSL3 to bind was identified in testicular germ cells and in the sperm equatorial segment of bulls, rams and he‐goats. In comparison to fertile bulls, the percentage of INSL3‐ and RXFP2‐expressing cells and their expression levels per cell were significantly reduced in the testes of subfertile bulls. In addition, the population of INSL3‐binding spermatozoa was also significantly reduced in the semen of subfertile bulls. These results provide evidence for a functional INSL3 hormone‐receptor system operating in ruminant testes and spermatozoa, and its potential to predict subfertility in sires.     

传染性法氏囊病病毒对鸡法氏囊组织中干扰素和p53转录的影响

为了分析传染性法氏囊病病毒(IBDV)感染对鸡体抗病毒天然免疫能力的影响,本研究应用IBDV弱毒(B87)和强毒(QL/12)分别感染4周龄SPF鸡,每隔12h(12~84h)取3只鸡的法氏囊组织作为1个pool,用荧光定量PCR(qRT-PCR)检测其感染前后干扰素(IFN)和p53的mRNA水平,同时分析法氏囊组织的病理变化和病毒载量。结果显示:在QL/12组,IFN-α和IFN-βmRNA水平随着时间的增加逐渐升高,48h达到峰值,随后降低;IFN-γmRNA水平随着时间的增加逐渐升高,60h达到峰值,随后逐渐降低;p53mRNA含量迅速升高,60h达到峰值,随后降低。在B87组,IFN-αmRNA水平先降低后升高(48h达到最低值),而IFN-βmRNA水平变化不规则,72h含量最高;IFN-γmRNA水平先逐渐升高后降低(72h达到峰值);p53mRNA含量变化较小,36h含量最高。QL/12组诱导法氏囊组织产生的IFN mRNA(IFN-α、IFN-β和IFN-γmRNA)以及p53mRNA水平均显著高于B87组。病理组织学检查,QL/12组法氏囊淋巴滤泡呈不同程度的损伤,而B87组法氏囊组织未见明显的组织病变。qRT-PCR检测,QL/12组法氏囊组织中病毒含量48h达到峰值(8.4×106拷贝/mg),而B87组法氏囊组织中病毒含量72h达到峰值(6.6×104拷贝/mg)。本研究结果表明IBDV感染严重干扰了鸡体IFN和p53介导的抗病毒天然免疫反应。     

Protein and mRNA expression of follicle‐stimulating hormone receptor and luteinizing hormone receptor during the oestrus in the yak (Bos grunniens)

Follicle‐stimulating hormone (FSH) and luteinizing hormone (LH) have a central role in follicle growth, maturation and oestrus, but no clear pathway in the seasonal oestrus of yak (Bos grunniens) has been found. To better understand the role of FSH and LH in seasonal oestrus in the yak, six yaks were slaughtered while in oestrus, and the pineal gland, hypothalamus, pituitary gland, and gonads were collected. Using real‐time PCR and immunohistochemical assays, we determined the mRNA and protein expression of the FSH and LH receptors (FSHR and LHR) in these organs. The analysis showed that the FSHR mRNA expression level was higher in the pituitary gland tissue compared with LHR (p < .01) during oestrus. By contrast, there was low expression of FSHR and LHR mRNA in the pineal gland and hypothalamus. FSHR mRNA expression was higher than that of LHR (p < .05) in the ovary, whereas LHR mRNA expression was higher than that of FSHR (p < .01) in the uterus. FSHR and LHR proteins were located in the pinealocyte, synaptic ribbon and synaptic spherules of the pineal gland and that FSH and LH interact via nerve fibres. In the hypothalamus, FSHR and LHR proteins were located in the magnocellular neurons and parvocellular neurons. FSHR and LHR proteins were localized in acidophilic cells and basophilic cells in the pituitary gland, and in surface epithelium, stromal cell and gland epithelium in the uterus. In the ovary, FSHR and LHR protein were present in the ovarian follicle. Thus, we concluded that FSHR and LHR are located in the pineal gland, hypothalamus, pituitary and gonad during oestrus in the yak. However, FSHR was mainly expressed in the pituitary gland and ovaries, whereas LHR was mainly expressed in the pituitary gland and uterus.     

Association of toll‐like receptor 2 polymorphisms with somatic cell score in Xinjiang Brown cattle

This study aims to identify single nucleotide polymorphisms (SNPs) and haplotypes in the TLR2 gene, and analyze the association of SNPs or haplotypes and somatic cell scores in 151 Xinjiang Brown cattle and 138 Holsteins to evaluate the role of TLR2 during intramammary infections. TLR2 coding region was amplified by PCR and screened for SNP sequencing. Genotypes and frequencies of SNPs were identified. Finally, the associations of genotypes or haplotypes and somatic cell scores (SCS) were analyzed. The results showed that: (i) 15 SNPs (E+653, E+945, E+978, E+1010, E+1250, E+1688, E+1707, E+1779, E+1782, E+1891, E+1995, E+2025, E+2055, E+2214 and E+2295) were observed and detected from 289 cows; (ii) distribution of the 14 SNPs were significantly different from Xinjiang Brown cattle and Holstein (P < 0.001) except for the E+945 (P > 0.05); (iii) in 11 SNPs (E+945, E+978, E+1010, E+1688, E+1707, E+1779, E+1782, E+1995, E+2025, E+2055 and E+2214), the SCS of AB genotype was lower than AA (P < 0.05) in Xinjiang Brown cattle; and (iv) haplotypes composed of the above‐mentioned 11 SNPs were constructed. The SCS of cattle with Hap5 was lower than that of Hap3 (P < 0.05). This suggests that Hap5 might play an important role in sub‐mastitis resistance in Xinjiang Brown cattle.     

Antimicrobial activity of a bovine myeloid antimicrobial peptide (BMAP‐28) against methicillin‐susceptible and methicillin‐resistant Staphylococcus aureus

A bovine myeloid antimicrobial peptide (BMAP‐28) is a member of the cathelicidin family which is included in the innate immune system of mammals. Recently, there have been many studies about antimicrobial peptides. This study aims to clarify whether BMAP‐28 has bactericidal activity against methicillin‐resistant Staphylococcus aureus (MRSA) and compares its activity against methicillin‐susceptible S. aureus (MSSA) and MRSA. We found that the peptide was effective in killing MRSA (minimal inhibitory concentration (MIC) range; 5–20 µg/mL). It was also revealed that MSSA (MIC range; 1.25–20 µg/mL) had two levels of susceptibility to BMAP‐28. We also examined the effect of BMAP‐28 on bacterial shape to visually show its activity. After exposure to the peptide, both MSSA and MRSA cells showed the morphological changes on their surfaces. Our results indicate that BMAP‐28 is a promising candidate for medicine against drug‐resistant bacteria.     

An overview of recent developments in corneal immunobiology: potential relevance in the etiogenesis of corneal disease in the horse

This paper overviews some recent developments in mammalian corneal immunobiology, and discusses how these may act as pointers towards understanding the immunology underlying some common corneal diseases in the horse, including infectious ulceration and presumptively immune-mediated non-ulcerative disease. Specifically, three aspects of corneal immunobiology are examined: the role of Toll-like receptors in surface immunity and in the etiogenesis of microbial ulceration, the relationship between conjunctiva associated lymphoid tissue (CALT) and immunoprotection of the corneal surface, and the mechanisms determining corneal immune privilege (IP) and how down regulation of IP may be an important factor in the genesis of corneal immunoinflammatory disease.     

猪输卵管和子宫中EGF表达的研究

研究EGF mRNA在猪输卵管、子宫中的表达。采用RT—PCR技术检测猪卵泡期、排卵期和孕期的输卵管伞部、壶腹部、峡部和子宫中EGF的mRNA的表达。结果表明：各期卵管伞部、壶腹部、峡部和子宫中EGF的mRNA都强烈表达，但看不出明显的强弱变化，进步证实EGF在猪雌性生殖程中起着重要的作用。     

Fungal phytases: characteristics and amelioration of nutritional quality and growth of non‐ruminants

Fungal phytases are histidine acid phosphatases, a subclass of acid phosphatases, which catalyse the hydrolysis of phytic acid resulting in the release of phosphate moieties and thus mitigate its antinutritional properties. The supplementation of feed with phytases increases the bioavailability of phosphorus and minerals in non‐ruminant animals and reduces the phosphorus pollution due to phosphorus excretion in the areas of intensive livestock production. Although phytases are reported in plants, animals and micro‐organisms, fungal sources are used extensively for the production of phytases on a commercial scale. Phytases have been produced by fungi in both solid‐state fermentation (SSF) and submerged fermentation (SmF). The fungal phytases are high molecular weight proteins ranging from 35 to 500 kDa. They are optimally active within pH and temperature ranges between 4.5 and 6.0, and 45 and 70 °C respectively. Phytate degradation leads to amelioration in the nutritional status of foods and feeds by improving the availability of minerals, phosphorus and proteins in non‐ruminant animals and human beings and thus mitigates the environmental phosphorus pollution. Our article focuses on the role of fungal phytases in improving nutritional value of foods and feeds with concomitant increase in growth of non‐ruminant animals and mitigating environmental phosphorus pollution.     

Effect of dietary methionine on growth performance and insulin‐like growth factor‐I mRNA expression of growing meat rabbits

An experiment was conducted to determine the effects of different amounts of dietary methionine on growth performance, serum protein, growth hormone (GH), insulin‐like growth factor‐I (IGF‐I) concentrations and IGF‐I mRNA expression of growing meat rabbits. One hundred weaned growing meat rabbits were allocated to individual cages and randomly divided into five groups. The methionine addition concentrations of the five groups were 0, 2, 4, 6 and 8 g/kg diet (as‐fed basis) and sulphur amino acids (SAA) concentrations ranging from 3.8 to 11.6 g/kg diet, respectively. The results obtained were as follows: the average daily gain of 2, 4 and 6 g/kg diet groups was higher than that of 0 g/kg diet group (p < 0.01). The feed gain ratio of the 4 g/kg diet group was lower than those of 0 and 8 g/kg diet group (p < 0.01). Methionine concentrations did not affect serum urea nitrogen, total protein, insulin and IGF‐I concentration (p > 0.05). The quadratic effects of methionine on the serum concentration of albumin (Alb) and GH were obtained (p = 0.013, p = 0.018). The quadratic effect of methionine amount on IGF‐I mRNA expression was obtained (p = 0.045). The serum concentration of Alb of the 4 g/kg diet group was higher than those of 0 and 8 g/kg diet group (p < 0.01). The serum concentration of GH of 8 g/kg diet group was higher than that of the 0 g/kg diet group (p < 0.05). The liver IGF‐I mRNA expression of 4 g/kg diet group was higher than those of the 0 and 8 g/kg diet group (p < 0.05). Providing a diet mainly consisted of corn, wheat bran and peanut vine, the optimum dietary methionine addition concentration and SAA concentration for a weaner to 2‐month‐old growing meat rabbits were shown to be 2 and 5.7 g/kg diet respectively.     

机体识别病毒核酸的几种分子模式及途径

近些年机体病原相关模式受体及其识别分子机制的研究,极大地促进了先天性分子免疫学的发展,并成为现代免疫学研究的重点和热点领域。作者通过机体识别病毒核酸的Toll样受体(Toll-like receptors,TLRs)、RIG-I样受体(RIG-I like receptors,RLRs)、NOD样受体(NOD-like receptors,NLRs)和DAI(DNA-dependent activatorof interferon-regulatory factors,DAI)等模式识别分子的细胞定位、分子结构及其识别病毒核酸途径的介绍,系统、全面地探讨了宿主机体如何全方位识别和消除入侵病毒的分子免疫防御途径,为抗病毒药物和疫苗的设计以及抗病育种提供了理论依据。     

Real‐time RT‐PCR for the detection and quantitative analysis of equine rhinitis viruses

Reasons for performing study: Equine rhinitis viruses (ERV) cause respiratory disease and loss of performance in horses. It has been suggested that the economic significance of these viruses may have been underestimated due to insensitive methods of detection. Objectives: To develop a sensitive, rapid, real‐time RT‐PCR (rRT‐PCR) assay suitable for the routine diagnosis and epidemiological surveillance of the A and B variants of ERV. Methods: TaqMan primer probe sets for ERAV and ERBV were designed from conserved regions of the 5′ UTR of the ERV genome. Over 400 samples from both clinically affected and asymptomatic horses were employed for validation of the assays. ERAV samples positive by rRT‐PCR were verified by virus isolation and ERBV positive samples were verified by rRT‐PCR using a different set of primers. Results: The detection limit of the rRT‐PCR for both viruses was 10–100 genome copies. Of 250 archival nasal swabs submitted for diagnostic testing over a 7 year period, 29 were ERAV positive and 3 were ERBV positive with an average incidence rate per year of 10 and 1.5%, respectively. There was evidence of co‐circulation of ERAV and ERBV with equine influenza virus (EIV). Of 100 post race urine samples tested, 29 were ERAV positive by rRT‐PCR. Partial sequencing of 2 ERBV positive samples demonstrated that one was 100% identical to ERBV1 from a 270 bp sequence and the other was more closely related to ERBV2 than ERBV1 (95% compared to 90% nucleotide identity in 178 bp). Conclusions: The rRT‐PCR assays described here are specific and more sensitive than virus isolation. They have good reproducibility and are suitable for the routine diagnosis of ERAV and ERBV. Potential relevance: These assays should be useful for investigating the temporal association between clinical signs and rhinitis virus shedding.