Molecular characterization of cytokine TWEAK and its receptor Fn14 in pig (Sus scrofa)

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor superfamily (TNFSF). The interaction of TWEAK with its receptor fibroblast growth factor-inducible 14 (Fn14) regulates multiple cellular responses, including stimulation of proliferation, migration, apoptosis, angiogenesis, and induction of proinflammatory cytokines. This paper reports for the first time the molecular cloning of porcine TWEAK and Fn14 by EST and RACE strategies. The full-length cDNA of porcine TWEAK is 1327bp, including an open reading frame (ORF) of 747bp. Its genomic DNA consists of seven exons and six introns and is approximately 10kb in size by computer-assisted analysis. Sequence similarity at the amino acid level between porcine TWEAK and human or mouse was 95 and 92%, respectively. The full-length cDNA of porcine Fn14 contains 691bp, of which 390bp are the ORF. Sequence similarity at the amino acid level between porcine Fn14 and human, or mouse, or frog was 95, 93 and 64%, respectively. Real-time quantitative PCR (Q-PCR) analysis revealed that both TWEAK and Fn14 are constitutively expressed in various tissues in pig. Our results suggest that the TWEAK-Fn14 pathway is evolutionarily highly conserved. It will be helpful for investigation on the biological role of the TWEAK/Fn14 system in this important animal model. Furthermore, it provides insight into the molecular evolution of the emerging TWEAK and Fn14 families.     

猪Cathepsin B 基因cDNA分子克隆、序列分析及遗传多态性分析

采用 RT-PCR 结合克隆测序的方法,从猪肌肉组织总 RNA 中克隆出了猪组织蛋白酶 B(Cathepsin B,CTSB)基因的 cDNA 序列,并推导出其编码的氨基酸序列.猪 CTSB 基因开放阅读框(ORF)全长1 008 bp,编码335 个氨基酸.同源性分析结果表明,猪与人、鼠、牛的 CTSB 基因 cDNA 编码区(CDS)同源性分别为 85%、81%、90%,推测的氨基酸序列同源性分别为 81%、79%、91%.利用同源性结合序列特征预测表明该蛋白具有信号肽和前肽序列.蛋白质结构同源建模分析表明,该蛋白具有木瓜蛋白酶家族的典型空间结构,包括1个底物结合凹槽和3个相互靠近的活性位点.另外采用 PCR-SSCP 方法,在147头个体中分析了CTSB基因第6内含子内的SS-CP位点多态性,检测到3个等位基因6种基因型.FF基因型个体的各项嫩度指标最高,其最大剪切力与硬度值分别为6.56 kg和23.55 kg·s,极显著地高于 EE 和 EF 基因型个体(P<0.01),平均剪切力为4.85 kg,显著地高于EF基因型个体(P<0.05).     

Histomorphologic and Histochemical Characteristics of Carpal Glands (Glandulae Carpeae) in Domestic Swine (Sus scrofa domesticus) and Wild Swine (Sus scrofa ferus)

Investigations were carried out on samples from carpal glands of domestic and wild swine of different ages. The anatomic position of carpal glands is identical in domestic and wild swine. Glandular acini in domestic swine are bigger and more scarce than in wild swine. LDH activity is more intense in wild swine, while the activity of the investigated diaphorases is almost even. The activity alkaline and acid phosphatase is stronger in domestic swine, while the activity of non-specific esterases is present only in wild animals. The more or less manifest presence of all investigated enzymes indicates that carpal glands in domestic and wild porcine specimens are very active metabolically. Regarding the intensity of enzymatic reactions, some differences are probably due to different sexual maturity, as well as to the method of feeding and housing in these groups of animals.     

Molecular cloning and functional characterization of porcine IFN-beta promoter stimulator 1 (IPS-1)

The IFN-beta promoter stimulator 1 (IPS-1), also known as MAVS/VISA/Cardif, is an adaptor molecule for the retinoic-acid-inducible protein I (RIG-I) or melanoma-differentiation-associated gene 5 (MDA5) that recognizes intracellular double-stranded RNA (dsRNA) and triggers a signal for producing type I IFN. In the present study, porcine IPS-1 cDNA was cloned, using RT-PCR coupled with rapid amplification of cDNA ends (RACE)-PCR, from porcine peripheral blood mononuclear cells. The open reading frame of porcine IPS-1 consists of 1575bp encoding 524 amino acids. The putative porcine IPS-1 protein contains a N-terminal CARD-like domain, a central proline-rich domain, a C-terminal transmembrane domain, and exhibits similarity to mouse, rat, monkey, human and cattle counterparts, ranging from 59% to 79%. Semi-quantitative RT-PCR showed that porcine IPS-1 mRNA was widely expressed in different tissues. Porcine kidney (PK-15) cells transfected with a DNA construct encoding porcine IPS-1 produced type I IFN, and activated IRF3 and NF-kappaB. Deletion mutant analyses further revealed that both the CARD-like domain and transmembrane domain are essential for these functions. In addition, poly(I:C)-induced porcine IFN-beta promoter activation in PK-15 cells was significantly reduced by siRNA targeting IPS-1, indicating that IPS-1 is an important immunoregulator in the porcine innate immune system. The availability of porcine IPS-1 and establishment of its function in the type I IFN signaling pathway provides a useful molecule for defining its role during the course of pig infectious diseases.     

Homology of DNA sequences encompassing malignant hyperthermia mutation site in the ovine (Ovis aries) and porcine (Sus scrofa) ryr1 gene

The PCR products of ryr1 gene amplified from genomic DNA of Sus scrofa and synthetic prolific lines of Ovis aries were analysed. The polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), and sequencing analysis for research on ovine, ryanodine receptor ( ryr1 ) gene. The ovine fragment of ryr1 gene did not differ from exon 17 of the porcine ryr1 gene.     

The Electrocardiogram of Conventional and Miniature Swine (Sus scrofa)

Standard, augmented limb leads and lead V₁₀ (representing the Z axis) taken in sequence and three semi-orthogonal leads (I, aVF, and V₁₀) taken simultaneously were recorded from 43 healthy pigs. Records were analyzed for rate, rhythm, interval duration and component amplitudes. The wave form of QRS complexes were analyzed in all leads studied. P, QRS, and T vectors were calculated for the mean dorsal (frontal), sagittal, and transverse planes. study of orthogonal leads in right sternal recumbency indicated that ventricular activation is spatially oriented dorsad, sinistrad and slightly caudad. No essential difference was noticed in the conventional and miniature swine. Ventricular fibrillation induced electrically in four older pigs was a progressive, terminal event; in two newborn piglets, ventricular fibrillation induced several times was terminated in each case by spontaneous recovery to sinus rhythm.     

Morphological Investigations of the Glandulae Profundae Plicae Semilunares Conjuctivae in the Domestic Swine (Sus scrofa domesticus) and the Wild Hog (Sus scrofa ferus)

Samples of glandulae profundae plicae semilunares (Harderian glands) from five domestic swine and five wild hogs were used for this research. The gland samples were fixed in Bouin solution and mounted in paraffin. The paraffin slices were stained with haematoxylin and eosin, according to the periodic-acid-Schiff (PAS) method, with alcian blue (pH 2.5), with toluidine blue (pH 4.0), and applying a combination of staining with alcian blue (pH 2.5) and the PAS method. In domestic swine and wild hogs, these glands are tubular-alveolar with wide glandular lumina. A great deal of acid mucopolysaccharides and PAS-positive substances were noted within swines’ glandular cells, while, in wild hogs, PAS-positive substances were not frequent, acid mucopolysaccharides being noted in only a few glandular-acini cells. The appearance of the metachromatic phenomenon was not noted either in domestic swine or in wild hogs. In domestic swine, the level of acid mucopolysaccharides is probably due either to the housing method or to the influence of the alkaline substances that may appear on an eye's mucous conjunctive membrane during intensive breeding, such that the Harderian glands protect the mucous membrane by extracting acid mucopolysaccharides.     

Molecular detection of Leptospira spp. in wild boar (Sus scrofa) hunted in Liguria region (Italy)

Leptospirosis is a re-emerging and widespread zoonosis, worldwide distributed, due to a wide variety of wild and domestic animal species able to act as natural or accidental hosts. During last years, in Europe, as in Italy, wild boar (Sus scrofa) population is increased. This animal represents a reservoir for different etiological agents, such as Leptospira. The aim of this investigation was to evaluate the prevalence of Leptospira spp. in wild boar hunted in Liguria region (Italy) during two-year hunting seasons. From 611 hunted wild boar, kidneys were collected. DNA was extracted from each organ and different targets were used to detect pathogenic (lipL32 gene), intermediate (16S rRNA gene) and saprophytic (23S rRNA gene) Leptospira by Taqman-based RealTime-PCR assays. Overall, kidneys were sampled from 282 adults, 155 sub-adults and 174 young wild boar (in total 314 males and 298 females). By RealTime PCR 77 kidneys were positive and, among these, 74 resulted positive for pathogenic (96.10%) and 3 (3.90%) for intermediate Leptospira. No significant differences in pathogenic Leptospira infection ratio were detected between male (11.50%) and female (12.75%). Only 13 sub-adult animals (8.39%) resulted infected by pathogenic Leptospira; 23 young animals (13.22%) and 38 adult animals (13.47%) were positive. The results of this study confirmed the importance of wild boar in the epidemiology of leptospirosis, which is able to infect other animal species (domestic and wild) including humans. Rarely, intermediate Leptospira could be able to infect wild boar with a renal localization that can contribute to their shedding and circulation.     

野猪消化能力及其消化道结构的研究

本研究以八眉猪为对照,分别对体质量在53 kg±2.3 kg的野猪消化能力和体质量在33kg~43 kg的野猪消化道结构进行了研究。结果表明,野猪日采食量(2.44 kg±0.15 kg)显著低于八眉猪(3.43 kg±0.28 kg)(P<0.05);八眉猪对粗蛋白质的日消化量(0.309 kg±0.084 kg)显著高于野猪(0.231 kg±0.126 kg)(P<0.05),但野猪对粗蛋白质的消化率(75.89±1.26)却显著高于八眉猪(72.3±0.45);野猪对粗纤维的日消化量(0.059 kg±0.028 kg)和消化率(45.72±1.82)显著低于八眉猪,而对磷的利用率(27.67±1.91)显著高于八眉猪(19.99±1.79)。野猪的大肠长度(3.36±0.38 m)、大肠容积(5.91±1.04 L)、胃容积(1.82±1.36 L)、小肠长度(14.15±0.31L)、均极显著小于八眉猪(P<0.01)。     

Molecular detection of Mycoplasma suis in captive white-lipped peccaries (Tayassu pecari) and wild boars (Sus scrofa) in Brazil

Mycoplasma suis, the etiological agent of swine hemoplasmosis, is an epicellular bacterium that adheres to the surface of pig erythrocytes leading to deformations of the target cells. Little is known about the occurrence of M. suis in wild swine populations around the world, its economic impact on swine herds, and the risk of human infection. The aim of this study was to investigate, by quantitative real-time PCR (qPCR) based on the 16S rRNA gene, the occurrence of M. suis in a captive population of white-lipped peccaries (100 Tayassu pecari) and in free-living wild boars (14 Sus scrofa) in Brazil. None of the white-lipped peccaries were positive for M. suis, whereas seven (50%) wild boars were positive in qPCR assays. The quantification of M. suis-16S rRNA copies/μL ranged from 1.42 × 10° to 3.906 × 10¹ in positive animals, indicating a low bacteremia and a chronic carrier status in free-living wild boars. In conclusion, M. suis might be a non-frequent pathogen in wild suids maintained in captivity. Despite the low bacteremia, the prevalence of M. suis in wild boar population in Brazil seems to be high.     

Dietary Vitamin D Toxicity in a Household of Pot-Beliied Pigs (Sus scrofa)

Vitamin D intoxication developed in Vietnamese Pot-Bellied Pigs ( Sus scrofa ) fed 2 commercially available swine rations. Pronounced hypercalcemia and a history incompatible with other causes of hypercalcemia led to confirmation of this diagnosis by plasma vitamin D metabolite analysis in 2 affected animals as compared to a control animal. Feed sample analysis suggested the diet as the likely source of toxicity.     

Molecular cloning and characterization of chicken tumor necrosis factor (TNF)-superfamily ligands, CD30L and TNF-related apoptosis inducing ligand (TRAIL)

CD30 ligand (CD30L) and tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) are members of the TNF-superfamily that have many important biological activities in cell proliferation and apoptotic death. In this study, both genes in the chicken were cloned and their expression was analyzed. Complementary DNA fragments were obtained from a suppressive subtractive hybridization library with or without lipopolysaccharide (LPS)-stimulation. Chicken CD30L consists of 1,152 base pairs (bp) with an open reading frame (ORF) of 720 bp having 36.4% identity with human CD30L, whereas chicken TRAIL is 1,134 bp long with an ORF of 912 bp having 54.4% identity with human TRAIL. Chicken CD30L was expressed at high levels in the spleen, bursa of Fabricius and in the chicken monocytic leukemia cell line, IN24. Stimulation with LPS in the spleen, bursa of Fabricius and the IN24 cell line did not affect CD30L expression. The gene expression of chicken TRAIL was essentially to the same level in all tissues examined. The time course of expression was not significantly altered by LPS-stimulation in the spleen, thymus and bursa of Fabricius, but reached a maximal level 8 hr after stimulation in the IN24 cell line. The high level expression of both genes in lymphoid organs and IN24 cell line indicates that chicken CD30L and TRAIL may also play an important role in apoptotic signal transduction and the regulation of cell proliferation in the immune system.     

Indirect estimation of porcine parvovirus maternal immunity decay in free-living wild boar (Sus scrofa) piglets by capture-recapture data

Linear mixed regression and logspline density estimation were performed to estimate the survival curve and half life of passively acquired antibodies against porcine parvovirus (PPV) in 44 wild boar piglets captured in the Northern Apennines, Italy. One piglet had no detectable maternal antibodies at 2.5 months post partum and no antibodies were detected in any of the remaining piglets by 4 months of age. Fitted survival curves indicated that maternal antibodies were undetectable from 2.5 to 6 months of age, with a median value at 3 months and a low probability of persistent maternal antibodies after 4 months of age. The estimated half life was 23 days (95% confidence interval 22-26 days). The results agree with previous data for decay of maternally acquired antibodies against PPV in the domestic pig and indicate the value of capture-recapture analysis for the estimation of infection parameters in free-living animals.     

猪akirin2基因的克隆与真核表达

Trizol法从猪脾脏中提取总RNA,RT-PCR方法得到猪Akirin2的完整编码区,软件分析猪Akirin2的核酸序列与蛋白序列,进行染色体定位。将Akirin2构建到带有荧光标记的真核表达载体pEGFP-C1中,通过脂质体转染法将pEGFP-Akirin2转入猪脐静脉血管内皮细胞系(SUVEC)中进行表达。结果表明,该序列编码203个氨基酸,猪akirin2基因定位于猪1号染色体,含有4个外显子,猪与牛的Akirin2蛋白高度同源。重组表达质粒pEGFP-Akirin2经双酶切鉴定和测序分析,表明构建成功,pEGFP-Akirin2转染SUVEC细胞后,经荧光检测证实转入的akirin2基因得到表达。研究结果为探讨猪Aki-rin2蛋白在免疫调控中的功能提供了基础数据。     

Notomelia in a piglet (Sus scrofa): Case report and review of the literature

This report describes a case of notomelia in a female piglet. The extra limb was attached at the interscapular region and was significantly undergrown. Its appendage was bifurcated with a unilateral keratinized tip. This is probably the first report of notomelia in a pig. The observations were compared with the bovine and ovine cases of notomelia reported so far.     

Surgical correction of periocular fat pads and entropion in a potbellied pig (Sus scrofa)

A 16-year-old Vietnamese potbellied pig was examined because of recurrent ocular discharge and reduced visual ability. Bilateral upper eyelid entropion and impaired vision secondary to periocular fat deposition were diagnosed. Surgical correction with excision of subdermal fat and redundant skin was performed to address both issues. Surgery restored vision and resolved ocular irritation. More than 1 year following surgical therapy the pig is visual and comfortable with no evidence of recurrent fat deposition or entropion.     

First isolation of Trichinella britovi from a wild boar (Sus scrofa) in Belgium

Since 1992, when the European Union Council Directive requires that wild boars (Sus scrofa) hunted in EU for commercial purpose should be examined for Trichinella, the infection has not been detected in wild boars from Belgium, despite serological evidence of the presence of anti-Trichinella antibodies in wildlife and previous reports of Trichinella larvae in this host species. In November 2004, Trichinella larvae were detected in a wild boar hunted near Mettet, Namur province (Southern Belgium). Larvae were identified as Trichinella britovi by polymerase chain reaction methods. This is the first report of the identification of Trichinella larvae from Belgium at the species level. The detection of T. britovi in wildlife in Belgium is consistent with findings of this parasite in other European countries and confirms the need to test game meat for Trichinella to prevent its transmission to humans.