用Vero细胞制备马抗狂犬病血清抗原

以Vero细胞为基质制备马抗狂犬病血清用抗原,以期建立有效、经济、简便的抗原制备方法。用含10％马血清营养液对Vero细胞作适应性培养,接种狂犬病毒,以含1％～3％马血清营养液作维持液培养病毒,于第5,8天收获病毒液,经灭活、浓缩、离心等制成抗原。第5,8天收获病毒滴度可稳定在7．010gLD50／mL以上,灭活抗原具良好的抗原性,用NIH法测定效价达6．0IU／mL以上,可用作抗原生产抗狂犬病血清。     

Use of monoclonal antibodies against horse immunoglobulin in an enzyme immunoassay of bacterial toxins and anatoxins

Immunization of BALB/c mice by horse antiserum against diphtheria made it possible to obtain IgG1 monoclonal antibodies (MoAbs) 2B7E4 specific for light chains of horse immunoglobulin (Ig). Unlike commercial preparations of anti-horse immunoglobulin antibodies, which are specific for the whole Ig molecule or its Fc-fragment, the peroxidase (HRP) conjugate of the MoAb, 2B7E4-HRP did not interact with human, mouse, rabbit, and sheep Igs, or horse albumin. The conjugate obtained was used with MoAbs against bacterial toxins and commercial horse anatoxins, as a universal reagent in sandwich enzyme immunoassay (ELISA) for bacterial toxins and anatoxins. The detection sensitivity of diphtheria toxin/anatoxin equaled 0.0005 Lf/ml; tetanus toxin and anatoxin were detected with sensitivities of 20 LD50/ml and 0.005 UI/ml, respectively. A similar sandwich ELISA for botulinum anatoxins (group measurement) allowed types A, B, and E to be detected at 0.02, 0.002, and 0.001 UI/ml, respectively; selective measurement was only possible in the case of type E anatoxin (0.001 UI/ml).     

Detection of altrenogest and its metabolites in post administration horse urine using liquid chromatography tandem mass spectrometry--increased sensitivity by chemical derivatisation of the glucuronic acid conjugate

Altrenogest (17alpha-allyl-17beta-hydroxyestra-4,9,11-trien-3-one) is a steroid used for the control of estrus in horses. This drug can potentially be abused in racehorses as the occurrence of estrus can alter their performance. This work describes an analytical method based on liquid chromatography-tandem mass spectrometry for the detection of altrenogest in horse urine down to a concentration of 13 pg/mL (0.042 nM). Furthermore, the qualitative aspect of metabolism of altrenogest in the horse has been studied. The main transformations that were found for this species were conjugation with glucuronic acid and sulfate. These phase II metabolites were identified by molecular mass and by comparison of their collision-induced dissociation product ion spectra with that of the synthetic aglycone at positive and negative potential, respectively. No phase I metabolites were discovered. In order to increase the ionisation in positive electrospray, a derivatisation procedure forming a basic oxime was tested. This process significantly increased the detection sensitivity for altrenogest glucuronide in horse urine.     

Use of monoclonal antibodies against horse immunoglobulin in an enzyme immunoassay of bacterial toxins and toxoids

Immunization of BALB/c mice by horse antiserum against diphtheria made it possible to obtain IgG₁ monoclonal antibodies (MoAbs) 2B7E4 specific for light chains of horse immunoglobulin (Ig). Unlike commercial preparations of anti-horse immunoglobulin antibodies, which are specific for the whole Ig molecule or its Fc-fragment, the peroxidase (HRP) conjugate of the MoAb, 2B7E4-HRP did not interact with human, mouse, rabbit, and sheep Igs, or horse albumin. The conjugate obtained was used with MoAbs against bacterial toxins and commercial horse antitoxins, as a universal reagent in sandwich enzyme immunoassay (ELISA) for bacterial toxins and toxoids. The detection sensitivity of diphtheria toxin/toxoid equaled 0.0005 Lf/ml; tetanus toxin and toxoid were detected with sensitivities of 20 LD₅₀/ml and 0.005 UI/ml, respectively. A similar sandwich ELISA for botulinum toxoids (group measurement) allowed types A, B, and E to be detected at 0.02, 0.002, and 0.001 UI/ml, respectively; selective measurement was only possible in the case of type E toxoid (0.001 UI/ml).     

Characterization of horse (Equus caballus) immunoglobulin mu chain-encoding genes

 Horse (Equus caballus) immunoglobulin mu chain-encoding (IgM) variable, joining, and constant gene segments were cloned and characterized. Nucleotide sequence analyses of 15 cDNA clones from a mesenteric lymph node library identified 7 unique variable gene segments, 5 separate joining segments, and a single constant region. Based on comparison with human sequences, horse variable segments could be grouped into either family 1 of immunoglobulin (Ig) clan I or family 4 of Ig clan II subclan IV. All horse sequences had a relatively conserved 16 base pair (bp) segment in framework 3 which was recognized with high specificity in polymerase chain reaction by a degenerate oligonucleotide primer. Horse complementarity determining regions (CDR) had considerable variability in predicted amino acid content and length but also included the presence of relatively conserved residues and several canonical sequences that may be necessary in formation of the β chain main structure and conformation of antigen-binding sites through interaction with light chain CDR. Sequence analysis of joining regions revealed the presence of nearly invariant 3′ regions similar to those found in human and mouse genes. A single horse IgM constant region comprising 1472 bp and encoding 451 residues was also identified. Direct comparison of the horse constant region predicted amino acid sequence with those from eleven other species revealed the presence of 53 invariant residues with particularly conserved sequences within the third and fourth exons. Phylogenetic analysis using a neighbor-joining algorithm showed closest similarity of the horse mu chain-encoding constant region gene to human and dog sequences. Together, these findings provide insights into the comparative biology of IgM as well as data for additional detailed studies of the horse immune system and investigation of immune-related diseases. Received: 14 October 1996 / Revised: 10 December 1996     

Species differences in the hepatic microsomal metabolism of the pyrrolizidine alkaloid senecionine

1. The comparative metabolism of the pyrrolizidine alkaloid (PA) senecionine was studied in vitro in incubations of rat, guinea pig, cow, horse, and sheep hepatic microsomes. 2. Levels of the toxic pyrrolic metabolite 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) were higher from guinea pig incubations (39.9 nmol/mg protein) than from other species (range 0.07 to 7.5 nmol/mg); results disagree with prior studies which used nonspecific techniques and suggest that the guinea pig's resistance to certain PAs may be due to resistance to pyrrole toxicity rather than low pyrrole formation. 3. Minor differences in senecionine N-oxidation and hydrolysis existed between the various species.     

Measurement of renin and prorenin in cattle, hog and horse.

1. Species specific problems complicating the measurement of prorenin and renin concentrations were studied in bovine, hog and horse plasma. 2. In contrast to horse renin, bovine and hog renin reacted with rat angiotensinogen, allowing measurement of the plasma renin concentration in cattle and hog with rat angiotensinogen as exogenous substrate. 3. Trypsin treatment of plasma in order to activate prorenin generated an interfering angiotensin I immunoreactive material in all three species, most extensively in horse plasma. 4. This material could be removed in bovine and hog plasma by a cation-exchange resin, allowing an assay of the plasma prorenin concentration to be constructed in these species. 5. Another strategy has to be followed in order to measure prorenin and renin concentrations in horse plasma.     

The effect of seeds of exotic species transported via horse dung on vegetation along trail corridors

It has been suggested that exotic species will colonize within forests more frequently by the continual introduction of seeds through horse dung deposited along trails. Whether or not these exotic species have the ability to spread into and establish in the forest interior has been disputed. To address this, horse dung and soil samples were collected from trails during Autumn 1994 and Summer 1995 from three areas in southern Illinois, USA open to recreational horse travel. In addition, deer dung samples were collected from each of the study areas. Vegetation data were collected from each of the trail systems as well as from a trail along which horse travel was prohibited. The density of vascular plants in 0.25 m² quadrats placed at varying distances from the trail center to 5 m into the forest interior were recorded. Finally, dung samples were placed in situ along horse trails at one site to examine seedling germination in natural conditions. While 23 exotic species germinated from samples of horse dung placed out in a greenhouse, only one of these exotic species was also found in trail plots (Kummerowia striata). Similarly, while there were empirically more exotic species found along the trails allowing horse travel than there were on the trail lacking horse travel, the relative importance of those species was negligible along both trails. These results suggest that the emigration of exotic species via horse dung does not pose an immediate threat to the plant communities adjacent to trails in these forest systems. Nevertheless, the large number of exotic species in horse dung reflects the constant threat to any system from these species. Care must be taken, when allowing horseback use in areas, to anticipate invasion by exotic species from horse dung     

Preliminary list of horse flies (Diptera, Tabanidae) of Serbia

Thirty six species of horse flies (Tabanidae) were previously known from Serbia (Europe). The present faunistic study of horse flies (Tabanidae) has resulted in the recording of the 4 new species Atylotus fulvus (Meigen, 1804); Tabanus miki Brauer in Brauer and Bergenstamm, 1880; Tabanus unifasciatus Loew, 1858; and Heptatoma pellucens (Fabricius, 1776), in the fauna of Serbia. The genus Heptatoma Meigen, 1803 is cited for the first time in the fauna of Serbia. 40 species are currently known from Serbia, belonging to nine genera. The fauna can be considered relatively poorly studied. Most of the species belong to the Boreal-Eurasian type of fauna 23, followed by the South European group with 8 species, the Mediterranean group with 6 species, European group with 2 species and Central European group with 1 species.     

Evolution of the six horse IGHG genes and corresponding immunoglobulin gamma heavy chains

It is generally assumed that the different mammalian IgG isotypes have developed during evolution by duplications of a common ancestor gamma heavy chain constant region gene (IGHG). In contrast to other species studied so far, which express between one and four IGHG genes, the horse (Equus caballus) genome contains six IGHG genes, and it has been postulated that they all can be expressed. For determination of the evolutionary history of the six horse IGHG genes, genomic DNA and cDNA of the IGHG genes were sequenced. The structure of these genes with reference to exons and introns was determined. Comparison of the deduced amino acid sequences of the horse IGHG genes revealed the greatest divergences in the hinge regions, and in the proximal CH2 domains. A phylogenetic comparison of the amino acid sequences of the six horse IGHG genes to those of other species shows that the horse IGHG genes form a distinct cluster. This indicates that the mammalian species included in this study probably share only one common ancestor IGHG gene with the horse. The six horse IGHG genes probably then evolved by gene duplication after species separation. In addition, various segmental exchanges were found between the horse IGHG genes, which might be the result of unequal crossing over and/or gene conversion events during the evolution of the six horse IGHG genes.     

Synteny mapping in the horse using horse-mouse heterohybridomas

In a study of 35 horse-mouse heterohybridoma cell lines, synteny in the horse was found between LDHB, PEPB and IGFl and between NP, MPI and IDH2. A synteny between ADA and PEPC was also indicated. The loci for horse immunoglobulin light chain (IgL) genes and for LDHA were independent.     

辽宁大连晚更新世马类牙齿釉质结构的研究

大连晚更新世的马类共包括三个种类:野驴(Equus hemionus)、小型的普氏野马中国马亚种(E. przewalskyi sinensis)、大型的大连马(E. dalianensis)。从它们颊齿的釉质层微观结构、釉柱大小及釉质层的厚度分析,晚更新世的野驴和现生的野驴是十分接近的,可以归于同一个种。大连马与现在的普氏野马也比较接近,可归于同一个大的类别。然而,普氏野马中国马亚种却与现生的普氏野马有着比较显著的区别:中国马颊齿釉质层的釉柱平均直径比现生的普氏野马大得多,而釉质层的厚度又比现生的普氏野马更薄,所以,原来的分类未必合适,中国马似不应归于普氏野马,它可能代表一个比较特殊的种类。     

Response of Tabanidae (Diptera) to natural and synthetic olfactory attractants.

The attraction of female tabanids to Malaise traps and canopy traps baited with aged horse urine, 1-octen-3-ol, or a combination of aged horse urine and acetone was studied in the Kopacki rit Nature Park in Eastern Croatia. Malaise traps captured very few tabanids relative to canopy traps. The number of females of Tabanus tergestinus and Haematopotapluvialis collected from 1-octen-3-ol baited canopy traps differed significantly from traps baited with aged horse urine. However, the number of females of Tabanus bromius, Atylotus loewianus, and Tabanus maculicornis collected from canopy traps baited with 1-octen-3-ol and aged horse urine did not differ significantly. Canopy traps baited with aged horse urine collected significantly more Tabanus sudeticus than did traps baited with 1-octen-3-ol. Canopy traps baited with 1-octen-3-ol collected eight times more tabanids than unbaited traps, whereas canopy traps baited with aged horse urine and a combination of aged horse urine and acetone collected seven and four times as many tabanids, respectively, as did unbaited traps. It appears that 1-octen-3-oland aged horse urine are very effective attractants for tabanids in this part of Europe. Tabanus bromius was the most abundant species with 53.14% in the sample collected by canopy traps.     

Orientational, conformational stability and surface potential of immunoglobulins in monomolecular layers

Orientation, resistance to surface denaturation and surface potential of bovine and horse immunoglobulin G (IgG) and also of pig IgG-antibodies against DNP and DNS groups on the surface of NaCl solutions of various concentrations have been studied by monomolecular layer method. High conformational stability of IgG molecules of all the species was confirmed. At the surface of NaCl solutions with concentrations 0.15--0.5 M immunoglobulins of all species from monolayers consisting of practically non-unfolded molecules with such an orientation, that all the fragments of IgG molecule are located horizontally. Beyond the limits of this native structure stability zone the rate of surface denaturation is directly proportional to NaCl concentration in the solution. The values of surface potentials of all immunoglobulins under study are close to each other and change little with the surface denaturation.     

Immunochemical properties of immunoglobulin G conjugated with dextran

Antigen-binding activity and effector functions of immunoglobulin G from horse and rabbit sera have been investigated, using hemagglutination, kinetic immune lysis, immune lysis in microplates and rosette-forming test with peritoneal mononuclear cells of mice, after their conjugation with dextran, MW 35-50 kD. The formation of conjugates of two types has been demonstrated: protein-dextran and protein-dextran-protein. It has been revealed that protein-dextran-protein conjugates have high specific antigen-binding activity, as compared to native IgG from rabbit sera specific for SRBC, while interactions with the complement system and Fc receptors is depressed.     

Muscle buffering capacity and dipeptide content in the thoroughbred horse, greyhound dog and man

1. Muscle buffering capacity (beta m) and dipeptide content were measured in locomotory muscles of the Thoroughbred horse, Greyhound dog and Man. 2. Beta m and carnosine contents were highest in the horse. Anserine was only found in dog muscle. 3. The higher beta m in horse and dog muscle, compared with man, appears to be predominantly due to higher muscle contents of histidine containing dipeptides in these species.     

The Taxonomic Status and Intraspecific Differentiation of the Black Sea Horse Mackerel, <Emphasis Type="Italic">Trachurus mediterraneus ponticus</Emphasis> (Aleev, 1956) (Carangidae)

The taxonomic status of the horse mackerel, Trachurus mediterraneus ponticus (Aleev, 1956), which inhabits the Black Sea, has been clarified based on the analysis of variation in the nucleotide sequence of the mtDNA cytochrome b (Cyt-b) locus. A comparison of this species with the horse mackerel from the Mediterranean basin and the World Ocean has shown that the Black Sea horse mackerel is neither an independent species nor a subspecies and can only be qualified as the species Trachurus mediterraneus (Steindachner, 1868) distributed throughout the Mediterranean Sea basin. A hypothesis has been made about the possible cause of the origin and existence of two forms, “large” and “small”, of the horse mackerel. By using the technique of calculating ontogenetic trajectories, it has been found that these are two morpho-ecological forms, whose differentiation is most likely determined by the type of feeding.     

The interaction of cefotaxime with the serum albumin of several mammalian species.

1. The interaction of cefotaxime with the serum albumin of several mammalian species; horses, swine, sheep, dogs and rabbits, was studied comparatively. The technique of ultrafiltration and spectrophotometric determination of the free antibiotic in the filtrate was used. 2. Binding percentages, which vary according to the species studied, were found to be higher in swine and rabbit albumins (between 92 and 81%) and lower for sheep, dog and horse albumins (between 67 and 52%). 3. The number of binding sites is usually close to 2; in the case of the horse it is 2.43. The apparent binding constants are: swine, 1.61 x 10(4) M-1; rabbit, 1.19 x 10(4) M-1; sheep, 2.33 x 10(3) M-1; dog, 2.00 x 10(3) M-1; horse, 1.42 x 10(3) M-1. The Scatchard model was used for data analysis. 4. Possible consequences of this interaction regarding clinical use of cefotaxime on different species are discussed.     

Equine cutaneous amyloidosis derived from an immunoglobulin lambda-light chain. Immunohistochemical, immunochemical and chemical results.

Amyloid deposits from equine cutaneous nodular amyloidosis associated with extramedullary plasmacytoma were classified immunohistochemically as equine immunoglobulin lambda-light chain-derived and designated eA lambda (HIP). For chemical identification, the amyloid fibril proteins were separated on Sephadex G-100 in 6M guanidine.HCl. Polypeptides of predominantly 24 kDa and 50 kDa were found by polyacrylamide gel electrophoresis. They have preponderance of immunoglobulin lambda-antigenic determinants as detected by immunodiffusion and immunoblotting. Since the N-terminus of the major proteins was blocked, peptides were generated with trypsin and endoproteinase Asp-N and then isolated using reversed-phase high-performance liquid chromatography. Automatic amino-acid sequence determination of seven peptides showed novel sequences. Data bank comparison indicated that these peptides were derived from a monoclonal immunoglobulin lambda-light and a gamma-heavy chain. The light chain was considered to be the leading amyloidogenic polypeptide, since it was the predominant component in a virtually pure amyloid fibril preparation. Thus, immunoglobulin lambda-light chain-derived amyloidosis, so far established only in man and cat, has now also been identified in the horse.     

Urinary excretion patterns of 5-hydroxyindole-3-acetic acid and 5-hydroxytryptophol in various animal species: implications for studies on serotonin metabolism and turnover rate

The concentrations of the serotonin metabolites 5-hydroxyindole-3-acetic acid (5HIAA) and 5-hydroxytryptophol (5HTOL) were determined in spot urine samples of 12 mammalian and one fish species (cat, cow, dog, ferret, golden hamster, guinea pig, horse, monkey, mouse, rabbit, rainbow trout, rat, sheep) and compared with human data. The highest urinary concentrations of 5HTOL were found in the Sprague-Dawley rat (mean 9.5 micromol/L) and NMRI mouse (8.2 micromol/L), and the lowest in rainbow trout, cynomolgus macaque, and human urine (approximately 0.1 micromol/L). The highest 5HIAA concentrations were found in hamster (89.3 micromol/L) and mouse (85.2 micromol/L), and the lowest in rainbow trout, horse and sheep (range 2.0-3.7 micromol/L). Several species showed 5HIAA concentrations similar to that normally observed in human urine (approximately 5-40 micromol/L). This study demonstrated wide inter- and intra-species variations in the urinary concentrations of 5HIAA and 5HTOL, both separately and in the sum of concentrations. The 5HTOL/5HIAA ratio, which is used as an easily accessible index of the relative importance of the reductive and oxidative pathways for serotonin metabolism, also varied considerably between different species. This observation confirms that the much higher urinary 5HTOL/5HIAA ratio in rats (mean 0.35) compared with humans (< 0.01) is due to a higher baseline formation of 5HTOL in the rat. The monkey, ferret, hamster, and rabbit most closely resembled humans in this respect, and at least the two latter species appear to be more suitable than rats as animal models for studying serotonin metabolism and turnover rate, and the metabolic interaction with ethanol.