丹系长白猪恶性高温综合症（pMHS）基因的检测分析

采集91头丹系长白种猪耳组织样品，用高盐法提取DNA，特异性扩增包含C1843在内的CRC／RYR1660bp片段，用HhaⅠ进行直接酶切，根据电泳图谱确定MHS基因座位上的基因型。结果表明91头丹系长白猪中，杂合猪（MHSN／MHSn）为10头，占10.98％，纯合阴性猪81头占89.02％，没有发现纯合阳性猪。MHSN和MHSn的基因频率分别为0.9451和0.0549。结合国内已有的测定结果，认为目前我国丹系长白猪群中MHSN及MHSn的基因频率分别为0.9086和0.0914。     

Porcine malignant hyperthermia susceptibility: halothane-induced increase in cytoplasmic free calcium in lymphocytes

We tested the hypothesis that lymphocytes from swine with susceptibility to malignant hyperthermia (MH) had increased sensitivity to the membrane-perturbing effects of halothane that increase cytoplasmic calcium. Cytoplasmic concentration of ionized calcium in lymphocytes isolated from blood was determined in the presence and absence of halothane for 10 Pietrain x Poland China swine that were susceptible to MH and 20 Yorkshire swine that were resistant to MH. Calcium was determined by dual-emission spectrofluorometry and by measuring the ratio of free to calcium-bound form of the fluorescent calcium dye Indo-1. Mean values for calcium concentrations in lymphocytes from MH-susceptible (MHS) swine were 80% less than control values (40.5 +/- 38.8 and 185.3 +/- 91.6 nmol/L; P less than 0.01). Untreated lymphocytes from MHS swine accumulated calcium at half the rate observed for controls. Exposure to 1 mmol/L halothane resulted in a 3-fold increase of free calcium concentration to 127.9 +/- 81.3 nmol/L in the lymphocytes of MHS swine, but had no significant effect on lymphocytes from control swine (225.0 +/- 91.4; P less than 0.01). Exposure to 2 mmol/L halothane resulted in a 6-fold increase of free calcium concentration to 255.9 +/- 91.4 nmol/L in lymphocytes from MHS swine and a 63% increase in lymphocytes from controls (303.8 +/- 116). The rate of halothane-induced increase in cytosolic calcium was 13 times greater in lymphocytes from MHS swine, compared with controls. These data indicated that the molecular defect that results in halothane-hypersensitivity and is characteristic of muscle of MHS swine also occurs in lymphocytes from MHS swine.     

红三叶根际溶磷菌的筛选与培养基优化

为从岷山红三叶根际土壤中筛选出大量高效溶磷菌株,本研究采用Pikovaskaia’s、PKOC1和PKOC2三种溶磷培养基进行溶磷菌株的初步筛选和优良溶磷菌株16S rRNA基因序列鉴定,并对分离筛选效果较好的PKOC2培养基进行响应面优化。结果表明:采用Pikovaskaia’s、PKOC1和PKOC2三种溶磷培养基,从红三叶根际分离出26株溶磷菌株;经16S rRNA基因序列对7株优良溶磷菌株进行初步比对鉴定,初步鉴定结果为:菌株MHS4为蜡样芽胞杆菌(Bacillus cereus);菌株MHS7和MHS19为枯草芽胞杆菌(Bacillus subtilis);菌株MHS27为苏云金杆菌(Bacillus thuringiensis);菌株MHS30为荧光假单胞菌(Pseudomonas fluorescens);菌株MHS31为盖氏假单胞菌(Pseudomonas gessardii);菌株MHS49为产酸克雷伯菌(Klebsiella oxytoca)。PKOC2溶磷培养基优化配方为:葡萄糖18.19 g·L^-1,Ca₃(PO₄)₂ 5 g·L^-1,MgCl₂·6H₂O 3.21 g·L^-1,MgSO₄·7H₂O 0.25 g·L^-1,KCl 0.2 g·L^-1,(NH₄)₂SO₄ 0.08 g·L^-1。红三叶根际溶磷菌资源丰富,优化后的溶磷培养基为高效溶磷菌资源筛选提供资源支持。     

Phospholipid content of subcellular structures in skeletal muscles after halothane loading in Landrace swine in relation to mating variants and genotype

Membraneous phospholipids of subcellular structures were determined from the musculature of German Landrace pigs of the GDR, following exposure to halothane. Mating variants A (H+ male X H+ female), B (H+ male X H- female), and C (H- male X H+ female) were used for positive responders (MHS), while variants B, C, and D (H- male X H- female) were used for negative responders (MHN). Four phospholipid fractions were recorded from the muscle samples for mitochondria and microsomes (according to SR section). Differences between the MHS and MHN groups for the above fractions and without consideration of mating variants and genotype were not observed, although unambiguous responses were exhibited by all animals, either positive or negative to halothan. Significant differences with regard to the above phospholipid fractions were recordable only for variant A (MHS group) as compared to D (MHN), in other words, for the homozygous genotypes, once the above results had been rearranged within MHS and MHN along with different mating variants and genotypes. However, no unambiguous results were obtainable for the heterozygous genotypes of mating variants B and C. Possible underlying reasons are discussed in some detail. The results obtained from mating variants A and D are likely to confirm earlier findings and seem to suggest that the sarcoplasmic reticulum is the primary site of origin of susceptibility to halothane or malignant hyperthermia.     

红三叶根际溶磷菌株分泌有机酸与溶磷能力的相关性研究

为初步探究红三叶根际溶磷菌株分泌有机酸与溶磷能力的相关性。利用改良PKOC2液体培养基对筛选自红三叶根际的4株优良溶磷菌进行液体培养,采用钼蓝比色法和高效液相色谱法动态监测溶磷菌培养期间发酵液pH、溶磷量及分泌有机酸的种类和数量。结果表明:菌株MHS7、MHS27、MHS30和MHS49均能分泌乳酸、草酸、苹果酸、富马酸和丁二酸,其中菌株MHS30还能够分泌酒石酸;菌株MHS7溶磷量与pH呈负相关(P>0.05);菌株MHS27溶磷量与pH呈正相关(P>0.05),与分泌乳酸量呈显著正相关(P<0.05);菌株MHS30溶磷量与pH呈显著负相关(P<0.05),与分泌有机酸总量呈极显著正相关(P<0.01),与分泌酒石酸呈显著正相关(P<0.05);菌株MHS49溶磷量与分泌丁二酸呈显著正相关(P<0.05);各菌株分泌有机酸的种类和数量差异较大,溶磷菌的溶磷量与总有机酸量存在一定相关性,但有效磷增量并不完全由总有机酸量来决定,有机酸的种类和数量对溶磷量也有一定的影响。不同菌株溶磷能力与其分泌的有机酸种类和含量之间的关系存在多样性,不同溶磷菌株存在不同的溶磷途径。     

Intravenous Administration of Azumolene to Reverse Malignant Hyperthermia in Swine

Background: The efficacy of intravenous (IV) administration of azumolene (Az), an analogue 30‐fold more soluble than dantrolene, on pigs susceptible to malignant hyperthermia (MH) is incompletely understood. Objective: To evaluate efficacy of Az on MH crisis in pigs. Animals: Eight normal (MHN) and 7 susceptible to MH (MHS) pigs (Landrace × Large White × Pietran). Methods: Prospective, laboratory trial. Hypermetabolic crisis was observed in MHS pigs, but not in MHN pigs, after a combined administration of inhaled halothane (1.5%) and IV injection of succinylcholine (SCh; 2.5 mg/kg). Susceptibility was confirmed using a caffeine and halothane contracture test. Az was administered 15 minutes after administration of SCh. Results: Respiratory acidosis (pH 7.16 ± 0.02; Pco ₂, 46.2 ± 9.1 mmHg, HCO₃, 22.5 ± 2.3 mmol/L), fever (38.2 ± 1.1°C), cardiac arrhythmias, and muscle contracture were observed in MHS pigs. MHS pigs (n = 5) treated with Az (2 mg/kg IV) survived the crisis with attenuation of signs (pH 7.30 ± 0.10; Pco ₂, 36.3 ± 4.5 mmHg; HCO₃, 22.9 ± 2.3 mmol/L) and recovery of normal muscle tone and cardiac rhythm. Conclusions and Clinical Importance: Az represents a possible substitute for dantrolene to reverse MH crisis in susceptible pigs.     

Influence of MHS genotype and feeding regime on allometric and temporal growth of pigs assessed by magnetic resonance imaging

A total of 68 barrows, 4-way-crosses with a Piétrain × Hampshire sire and a Large White × German Landrace dam were used in this study. The pigs were divided into 4 groups regarding the MHS (malignant hyperthermia syndrome) genotype (NN and Nn) and feeding regime (intensive and restrictive). The piglets were genotyped by DNA test; data on muscle and fat growth were obtained by repeated measurements of body composition at 4 weeks intervals by magnetic resonance imaging (MRI). Differential growth analysis showed that muscle tissue grew proportionally with the increase of live weight (allometric coefficient, b ≈ 1); fat grew faster in relation to live weight (b > 1) in all investigated groups of pigs. Significant differences in growth coefficients for fat were found only between feeding groups. The analysis by asymmetric S-function showed different patterns of live weight growth of pigs from two feeding regimes. Within the feeding regimes, no significant differences in live weight growth patterns between the pigs of different MHS genotype (NN and Nn) were found. Muscle growth pattern significantly differed between the groups of investigated pigs: Nn pigs from the intensive feeding group were significantly superior to NN pigs in the same group and to Nn pigs fed restrictively (p < 0.05), but only in the phase of progressive growth (b-coefficient of the S-function). In this respect Nn pigs performed better under intensive feeding than under restrictive feeding regime, while no difference was found between NN pigs from two feeding regimes. By combining information on live weight and muscle growth, the optimal slaughter weight of pigs was calculated: 130 and 126 kg for intensively fed NN and Nn pigs, respectively, and about 114 kg for both genotypes from the restricted group of pigs. From fat growth analysis by asymmetric S-function, no influence of genotype on fat deposition in both feeding systems could be observed. Growth patterns of fat differed significantly only between the feeding groups. This study showed that the growth of body components fits a sigmoid curve and that the asymmetric S-function proved to be a more accurate and informative model than a simple allometric function, providing a base for important decisions in fattening of pigs. A practical consequence of this study is that the more cost-effective restrictive feeding regime can be recommended as more appropriate in fattening of pigs, since intensive feeding generally failed to improve their growth traits. Similarly, inclusion of MHS-gene did not enhance muscle growth characteristics of investigated pigs, so MHS-negative pigs (NN) can be considered as more desirable fatteners, especially when fed restrictively.     

Pathogenesis of BSE

Before the emergence of bovine spongiform encephalopathy (BSE) and recognition of its zoonotic potential, the major example of the transmissible spongiform encephalopathies (TSEs) of animals was scrapie of sheep. But there is no evidence that scrapie transmits naturally to any species other than sheep and goats. The pathogenesis of scrapie has been studied most in experimental laboratory rodent species. In most experimental models of scrapie, after peripheral non-neural routes of infection, replication of the agent can first be detected in lymphoreticular system (LRS) tissue. When the route of introduction of agent into the body is localized, initial involvement will be in LRS tissue draining the infection site. Thereafter, there is a striking amplification of the agent in the LRS and spread by lymphatic/haematogenous routes, giving widespread dissemination in the LRS. This precedes replication in the CNS, but is not the means by which infection reaches the CNS. There is now substantial evidence from experimental models of scrapie that involvement of the CNS is by peripheral nervous system (PNS) pathways. In some models employing oral exposure the earliest localized LRS replication is in the gut-associated lymphoid tissue (GALT) and autonomic PNS routing to the CNS has been implicated. However, the relative importance of different routes of spread of TSEs within the body is determined by a number of host- and agent-dependent factors and, therefore, generalizations from an experimental model to a natural disease across a species barrier may not be appropriate. With the occurrence of BSE and recognition of its food-borne route of transmission via meat and bone meal, has come greater awareness of the probable importance of the oral route of infection in ruminant species affected by TSEs. In consequence, studies have increasingly focused on the natural host species to examine pathogenetic events.     

Pathogenesis of BSE

Before the emergence of bovine spongiform encephalopathy (BSE) and recognition of its zoonotic potential, the major example of the transmissible spongiform encephalopathies (TSEs) of animals was scrapie of sheep. But there is no evidence that scrapie transmits naturally to any species other than sheep and goats. The pathogenesis of scrapie has been studied most in experimental laboratory rodent species. In most experimental models of scrapie, after peripheral non-neural routes of infection, replication of the agent can first be detected in lymphoreticular system (LRS) tissue. When the route of introduction of agent into the body is localized, initial involvement will be in LRS tissue draining the infection site. Thereafter, there is a striking amplification of the agent in the LRS and spread by lymphatic/haematogenous routes, giving widespread dissemination in the LRS. This precedes replication in the CNS, but is not the means by which infection reaches the CNS. There is now substantial evidence from experimental models of scrapie that involvement of the CNS is by peripheral nervous system (PNS) pathways. In some models employing oral exposure the earliest localized LRS replication is in the gut-associated lymphoid tissue (GALT) and autonomic PNS routing to the CNS has been implicated. However, the relative importance of different routes of spread of TSEs within the body is determined by a number of host- and agent-dependent factors and, therefore, generalizations from an experimental model to a natural disease across a species barrier may not be appropriate. With the occurrence of BSE and recognition of its food-borne route of transmission via meat and bone meal, has come greater awareness of the probable importance of the oral route of infection in ruminant species affected by TSEs. In consequence, studies have increasingly focused on the natural host species to examine pathogenetic events.     

Applicability of vital staining and tissue clearing to vascular anatomy and melanocytes' evaluation of temporal bone in six laboratory species

The purpose of the present study was to define the applicability of tissue clearing to the field of otology. We combined tissue clearing with vital staining perfusion via a pumping system to examine the vascular anatomy of temporal bones in laboratory animals. We used six different types of species including Korean wild mouse, mouse, Mongolian gerbil, hamsters and Guinea pigs. A mixture of Alcian blue reagent and 4% paraformaldehyde was circulated throughout the entire circulatory system of the animal via a perfusion pump system. Transparency images were obtained from the temporal bones according to the protocol of the SunHyun 3D Imaging Kit. In examining the inner surface of the tympanic membrane, flaccid part (pars flaccida) was positioned along the entire marginal area in Guinea pig. In the Guinea pig, unlike the other species, the cortical bone of the mastoid (bullae) was easily removed using cold instruments, allowing a direct approach to the enclosed structures. The distribution and pattern of cochlea melanocytes were compared among the species. “Mobius strip”‐like accumulated melanocytes in vestibules were shown in both the Korean wild mouse and mouse. The collateral blood supply to the cochlea in six different species was checked in various pattern. Combining dye infusion with tissue‐clearing techniques, we documented the middle ear and transparent inner ear structures in six different species. The information and associated images will help other researchers to develop hypotheses and design experimental investigations.     

Detection of central nervous system tissues in meat products: validation and standardization of a real-time PCR-based detection system

Several phenotypic as well as genotypic methods have been published describing the detection of central nervous system (CNS) tissues that are part of the bovine spongiform encephalopathy (BSE) risk material in food products. However, none of these methods is able to differentiate between CNS tissue of the banned ruminant species and tissues of other animal species. A quantitative and species-specific real-time RT-PCR method has been developed that enables the reliable identification of CNS tissues in meat and meat products. This method is based on a messenger (m)RNA assay that uses bovine, ovine and caprine glial fibrillary acidic protein (GFAP) encoding gene sequences as markers. The in-house validation studies evaluated the tissue specificity of up to 15 bovine tissues and the standardization of absolute as well as relative quantitative measurement. The specific amplification of spinal cord and brain tissue GFAP cDNA has been shown previously. In addition, two commercially available ELISA kits were used for the comparative analysis of artificially contaminated minced meat. Small quantities of bovine brain that had been stored over the recommended period of 14 days were examined. The real-time PCR method proved to be suitable for the detection of 0.1% CNS tissue. No false negative results were observed. The quantitative detection of GFAP mRNA using real-time RT-PCR seems a suitable tool in routine diagnostic testing that assesses the illegal use of CNS tissue in meat and meat products. The stability of the selected target region of the GFAP mRNA also allows the detection of CNS tissues after the meat has been processed.     

Description of electrophoretic loci and tissue specific gene expression in the horseshoe bat genus Rhinolophus (Rhinolophidae)

The number of loci encoding an enzyme system, tissue specificity of gene expression and the degree of gene expression in various tissues are often not mentioned in evolutionary studies, but could indirectly provide evidence of evolutionary relationships. Protein electrophoresis was used to study the distributions and tissue specificity of gene expression of enzymes encoded by 42 loci in Rhinolophus clivosus and R. landeri, the genetically most divergent of the ten species of southern African horseshoe bats. No differences in gene expression were found between R. clivosus and R. landeri and isozyme patterns may be compared with other bat species to derive possible phylogenetic relationships.     

An immunohistochemical study of the distribution of biotin in tissues of pigs and chickens

An optimised indirect peroxidase-anti-peroxidase immunohistochemical technique was used to detect endogenous biotin in frozen tissue sections from biotin-supplemented and biotin-depleted pigs and chickens. A monoclonal anti-biotin antibody was used as primary antibody in this technique. Immunoreactive biotin was detected in many tissues of both species including liver, kidney, pancreas, adipose tissue, adrenal gland, testis, brain, choroid plexus, cardiac and skeletal muscle, epithelium of the respiratory and digestive systems, skin and lymphoid tissues. The specificity of immunostaining for biotin was confirmed by the finding of reduced staining intensities in tissues of biotin-depleted animals compared to those of biotin-supplemented animals. The results of this study suggest that biotin has metabolic functions in a wider range of tissues than previously known. They also indicate that endogenous tissue biotin should be considered as a source of false-positive staining when immunohistochemical or histochemical techniques which use avidin or streptavidin reagents or anti-biotin antibodies as components of the detection system, are applied to tissue sections.     

利用三个基因标记选育大白核心群后备种猪初报

猪氟烷基因(RYR1)c.1843C>T突变位点是造成猪应激综合征的主效基因位点.硬脂酰辅酶A去不饱和酶基因(SCD)是控制单不饱和脂肪酸合成的关键酶,其启动子区域-233上T>C突变位点对肥胖和背膘厚有重要影响.α1岩藻糖基转移酶基因(FUT1)基因是ETEC F18受体蛋白基因,其开放阅读框M307的G>A突变位点影响仔猪断乳后水肿和腹泻的发生.该研究采用PCR-RFLP方法,检测了福建仁锋种猪有限公司大约克核心群60头后备种猪个体在这3个基因相应突变位点的基因型.结果表明,该群体已完全淘汰了应激敏感型n等位基因,建立了氟烷应激抵抗系:且保持着高频率(90.8%)的SCD基因、有利降低背膘沉积、提高瘦肉率的T等位基因;但FUT1的ECF18抗性等位基因A的频率相对较低(26.7%),易感等位基因G的频率较高(73.3%).该研究结果结合该场的性能测定结果运用于指导该大白核心群后备种猪的选种选育实践,可望提高核心群内种猪产肉量性状、抗应激能力和降低仔猪断乳后腹泻抗性发生.     

Adipokines: a review of biological and analytical principles and an update in dogs,cats, and horses

Abstract: In addition to its role as an energy storage depot, adipose tissue is now recognized as a complex endocrine organ. Adipose tissue releases a variety of factors, termed adipokines, that regulate energy metabolism, cardiovascular function, reproductive status, and immune function. Some of the better‐studied adipokines include leptin, adiponectin, and components of the renin‐angiotensin system such as angiotensinogen. The function of more recently discovered adipokines such as resistin are under intense scrutiny. Abnormal production or regulation of adipokines occurs in obese individuals and is implicated in the development of a variety of associated co‐morbidities including metabolic syndrome, type 2 diabetes, atherosclerosis, heart disease, and cancer in people, although evaluation in domestic species is just beginning. Adipokines are now being examined as potential biomarkers for risk assessment for development of complications related to obesity. This article summarizes the function and regulation of some better‐characterized adipokines. It also reviews the current information on the characterization of adipokines in some domestic species in which rates of obesity and obesity‐related disorders are increasing, such as the dog, cat, and horse.     

Application of anti-BCG antibody for rapid immunohistochemical detection of bacteria, fungi and protozoa in formalin-fixed paraffin-embedded tissue samples

The applicability of an anti-Mycobacterium bovis (BCG) antibody-based immunohistochemistry (IHC) procedure was investigated using everyday veterinary pathological samples collected from 13 different animal species. Fifty-one formalin-fixed and paraffin-embedded tissue samples were selected for this study. Forty, 4 and 7 tissue samples contained different species of bacteria, fungi and protozoa, respectively. Three serial sections were prepared in each case. Two sections were pre-treated with enzyme and heat, respectively, while the last section was not pre-treated. In seven cases the sensitivity of histochemical staining (HSM), IHC and bacteriological culture were compared. Heating of the sections in a microwave oven was the most effective method in the case of almost all pathogens used. Strong or moderate positive reactions were observed for 26 bacterial species, all fungal and 2 protozoal species, while weak reactions occurred for 2 bacterial and 1 protozoal species. Only 4 protozoal and 12 bacterial species, including Leptospira and all the five Mycoplasma species examined, showed no reaction in this test. IHC had almost the same sensitivity as bacteriological culture and was more sensitive than HSM. The IHC method presented here should be preferred to HSM as a general screening tool in cases where pathological lesions suspicious for infections are evident and no microorganism can be cultured in vitro or only formalin-fixed tissue samples are available for the laboratory examination.     

Isolation of microsatellite loci and reliable genotyping using noninvasive samples of a critically endangered primate,Trachypithecus leucocephalus

Genetic information can be critical in identifying conservation priorities and developing conservation strategies. There is an urgent need for noninvasive genetic tools to study the wild populations of Asian colobine monkeys. The majority of these species are threatened with habitat destruction, population reduction and even extinction, but generally lack information on their genetic diversity and population structure. Genetic sampling and tissue collection have been scarce in these species owing to strict regulations on manipulation of endangered species, and the difficulties and risks associated with capturing these arboreal and fast‐moving monkeys in the challenging environments that they inhabit. These difficulties have hindered the development of molecular genetic markers, which are usually derived from tissues or blood. In this study, we present a method for de novo microsatellite isolation and genotyping using DNA from noninvasive origins of a critically endangered Asian colobine, the white‐headed langur (Trachypithecus leucocephalus). Genomic DNA isolated from hair was shown to be sufficient for microsatellite enrichment and isolation, with similar isolation efficiencies as from tissue DNA. We identified and characterized 20 polymorphic microsatellite loci, and evaluated their amplification success and genotyping reliability with 86 field‐collected fecal samples. These results show that this panel of loci can produce reliable genotypes from fecal samples, and represent a useful tool for noninvasive investigation of genetic structure, individual identification and kinship assessment in this highly endangered species. Our approach can be applied to conservation genetic studies of other wild species that lack sequence information and tissue samples.     

Porcine adenovirus as a delivery system for swine vaccines and immunotherapeutics

Porcine adenovirus (PAdV) has many qualities which make it an ideal choice for use as a delivery vector in swine. It is a low grade pathogen, present almost world-wide in a number of serotypes varying in their virulence and tissue tropism, which may allow for serotype specific vaccine targeting. PAdV is species specific having only been isolated from swine, reducing the possibility of its spread to other animals or man following administration. When engineered to contain a foreign gene, recombinant PAdV (rPAdV) can be grown to high titres in tissue culture cells making it cheap to produce. Knowledge of the complete nucleotide sequence of the PAdV genome has enabled rationally directed insertions of foreign genes which remain stably inserted in the genome and can be expressed at high levels following delivery to the target host. Importantly, recombinant PAdV can be administered by injection or by the oral route in feed or drinking water. We have delivered a range of antigens and immunomodulatory molecules to commercially available pigs using rPAdV and found it to be a very effective delivery system. Significantly, recombinant PAdV serotype 3 is highly effective as a delivery vehicle even when administered in the face of high levels of artificially induced serotype specific neutralising antibody to the vector.     

Muscle fibers of pigs sensitive to halothane and resistant to it

The histological and histochemical structure of m. longissimus thoracis, m. semimembranosus, m. triceps brachii-caput longum and m. rectus femoris was compared in pigs with a positive and negative reaction to halothane treatment. As found, the pigs affected by the malignant hyperthermia syndrome have thicker muscle fibres in the studied muscles. In the course of growth, the muscles of these animals have a larger proportion of red fibres (SO), but it is already at the age of 170 days (weight 100 kg) that light fibres prevail. The relative volume of the FG fibres is larger by 1-5%, as compared with the healthy animals. A higher number of pathologically changed fibres occurs in the pigs sensitive to halothane. The histological picture is individually variable; therefore the histological and histochemical methods cannot be considered as objective in view of MHS diagnosis. The general weakening of the constitution of the pigs is discussed as a possible predisposition factor underlying the development of various health disorders.