Tritrichomonas foetus: a scanning electron microscopy study of erythrocyte adhesion associated with hemolytic activity

The in vitro hemolytic activity of Tritrichomonas foetus was investigated. The parasite was tested against human erythrocytes of groups A, B, AB, and O, and against erythrocytes of nine adult animals of different species (the rabbit, rat, chicken, cat, dog, swine, horse, bovine, and sheep). The results showed that T. foetus strains (ATCC KV1, K, PAL, 5022, RJ, 90) did not present any hemolytic activity against any human erythrocyte group nor against rabbit, rat, chicken, cat, dog and swine erythrocytes. T. foetus strains, however, lysed horse, bovine, and sheep erythrocytes. No hemolysin released by the parasites could be identified. Hemolysis did not occur with trichomonad culture supernatants, with sonicated extracts of T. foetus, nor with killed organisms. Scanning electron microscopy (SEM) showed that human erythrocytes did not adhere to the trophozoites, in contrast horse erythrocytes adhered to the surface of the parasites and were phagocytosed for up to 90 min. The parasites are able to exert their cytopathic effects through: (a) physical contact established between the two cell surfaces, (b) toxins released from parasites into the interaction media, or (c) the association of both mechanisms. Further studies are necessary to clarify the importance of the hemolytic activity in the biology of T. foetus.     

Purification and sensitivity of Clostridium chauvoei hemolysin to various erythrocytes

Using ammonium sulphate fractionation, the Clostridium chauvoei hemolysin was purified by cation exchange chromatography and sephacryl S-100 gel filtration. The molecular mass of the hemolysin, determined by SDS-PAGE was found to be approximately 27kDa. The activity of the hemolysin was determined in erythrocytes of various animals, with sensitivities observed in the order of cow, sheep, chicken, rabbit, rat, mouse, dog and horse. Temperature affected the sensitivity of erythrocytes to C. chauvoei hemolysin. These results may reflect distinct characteristics of the hemolytic activity of C. chauvoei hemolysin and that the hemolysin may be pore-forming.     

Hemolytic activity of Fusobacterium necrophorum culture supernatants due to presence of phospholipase A and lysophospholipase.

Culture supernatants of Fusobacterium necrophorum demonstrated hemolytic activity. The hemolysin(s), which was partially purified by ammonium sulfate precipitation, was temperature-dependent and heat labile. The spectrum of hemolytic activity against various erythrocytes included rabbit, human, and dog erythrocytes. Goats, sheep, and bovine erythrocytes showed only trace hemolysis. According to results of thin-layer chromatography, the hemolysin hydrolyzed rabbit erythrocyte phosphatidyl choline, phosphatidyl ethanolamine, lysophosphatidyl choline, and bovine phosphatidyl choline. Hydrolysis of egg yolk phosphatidyl choline, bovine phosphatidyl ethanolamine, cholesterol, 1,2-dipalmitin, 1,3-dipalmitin, sphingomyelin, or triolein was not detected by thin layer chromatography. A more sensitive procedure utilizing gas-liquid chromatography revealed that, of the substrates tested, the following were bein hydrolyzed: bovine and egg yolk phosphatidyl choline, lysophosphatidyl choline, alpha-palmito-beta-eleoyl-L-alpha lecithin and alpha-oleoyl-betal-palmitoyl-L-alpha lecithin. Substrates which were weakly hydrolyzed were bovine phosphatidyl ethanolamine, DL-alpha-hosphatidyl ethanolamine dipalmitoyl, 1,2-dipalmitin, 1,3-dipalmitin, and triolein.     

Hemagglutinating activity of serovar reference strains of Ornithobacterium rhinotracheale.

In the present study, the hemagglutinating activity of 9 reference strains (serovars A-I) of Ornithobacterium rhinotracheale was investigated by using fresh erythrocytes from 15 different species: chicken (broiler, rooster, hen), turkey, pigeon, quail, duck, Harris hawk (Parabuteo unicinctus), house finch (Carpodacus mexicanus), cow, sheep, horse, dog, rabbit, pig, human (groups A, B, AB, and O), and rainbow trout (Oncorhynchus mykiss). All 9 strains agglutinated rabbit erythrocytes. None of the strains was able to agglutinate hen, cow, horse, or rainbow trout erythrocytes. The number of positive reactions among the remaining species varied. Results indicate that the use of rabbit erythrocytes is better suited for testing the hemagglutinating activity of O. rhinotracheale.     

Relationship between Clostridium septicum alpha-toxin activity and binding to erythrocyte membranes

The activity of Clostridium septicum alpha-toxin was determined in erythrocytes of various animals, with sensitivities observed in the order of mouse, rat, canine, equine, rabbit, chicken, bovine, swine and ovine. Temperature and protease treatment affected the sensitivity of erythrocytes to alpha-toxin. Proteinase K treatment decreased the sensitivity of murine, canine, equine and bovine erythrocytes, but ovine erythrocytes did not change the sensitivity to alpha-toxin activity. On the other hand, the activity of alpha-toxin on swine erythrocytes increased after treatment with proteinase K, trypsin, chymotrypsin or lysyl endopeptidase. Toxin overlay assay showed that alpha-toxin bound to erythrocyte membrane proteins with a molecular mass of 30 to 45-kDa in mouse, equine, bovine, swine and chicken, whereas in rat erythrocyte membranes the toxin reacted with 100-kDa protein. The treatment of murine and swine erythrocyte membranes with phosphatidylinositol-specific phospholipase C resulted in liberation of the toxin-binding protein from the individual membranes in a native state. These results show that alpha-toxin associates with specific erythrocyte membrane proteins in any animal species, and are subsets of glycosylphosphatidylinositol-anchored proteins in various animal species. These results may reflect distinct characteristics of the hemolytic activity of alpha-toxin in response to various erythrocytes.     

绵羊DECR1基因exon 5部分序列多态性分

试验根据GenBank登录的牛2,4-双烯-CoA还原酶1（2,4-dienoyl-CoA reductase1,DECR1）基因序列（NP_001068891）设计引物,应用PCR技术对德国美利奴绵羊、杜泊绵羊、特克塞尔绵羊DECR1基因的exon 5部分序列进行克隆测序;利用PCR-SSCP技术检测了3个绵羊群体exon 5单链构象多态性（SNP）,所获序列与GenBank中其他物种exon 5序列进行了同源性比较,并构建了亲缘关系聚类分析图。结果表明,绵羊DECR1基因exon 5长为137 bp,3个绵羊群体中exon 5均不存在SNPs,各种动物之间DECR1基因exon 5的同源性较高,在81.02%（鸡）～97.08%（牛）之间,其中绵羊和牛同源性最高,达到97.08%;与鸡的同源性最低,为81.02%;聚类分析结果显示,绵羊首先与牛聚为一类,再分别与兔子、狗聚为一类,最后分别与人、猪聚为一类,绵羊与牛的亲缘关系最近,与鸡的亲缘关系最远,与传统分类相一致,说明DECR1基因exon 5在动物进化过程中高度保守。     

Stimulation of heterophil hemagglutinins in chickens by a viral vaccine and chicken thyroid extract

The formation of heteroag-glutinins against human group O, horse and sheep erythrocytes was studied in groups of chickens injected with various materials. Production of antibodies cross-reacting with some or all of the test cell types was induced both with chicken thyroid extract, with or without adjuvant, and with inactivated infectious bronchitis vaccine with adjuvant. The chicken tissue in the injection materials is considered to be responsible, to a great extent, for the stimulations recorded. Also complete Freund’s adjuvant alone, however, had a stimulating effect. The heterogeneity of the antibody response as regards specificity, and differences in the responsiveness of individual chickens, were demonstrated.     

Sheep macrophages express at least two distinct receptors for IgG which have similar affinity for homologous IgG1 and IgG2.

Ovine bone marrow-derived macrophages (BMM) may express several IgG receptor (Fc gamma receptor; FcR) subsets. To study this, model particles (opsonized erythrocytes; EA), which are selectively handled by certain FcR subsets of human macrophages were used in cross-inhibition studies and found to react in a similar manner with FcR subsets of sheep macrophages. In experiments with monoclonal antibodies against subsets of human FcR, human erythrocytes (E) treated with human anti-D-IgG (anti-D-EAhu) and sheep E treated with bovine IgG1 (Bo1-EAs) were handled selectively by human macrophage FcRI and FcRII, respectively. Rabbit-IgG-coated sheep E (Rb-EAs) were recognized by FcRI, FcRII and possibly also by FcRIII of human macrophages. Anti-D-EAhu, Bo1-EAs and Rb-EAs were also ingested by sheep BMM. Competitive inhibition tests, using various homologous and heterologous IgG isotypes as fluid phase inhibitors and the particles used as FcR-specific tools in man (anti-D-EAhu and Bo1-EAs), revealed a heterogeneity of FcR also in sheep BMM. Thus, ingestion of anti-D-EAhu by ovine BMM was inhibited by low concentrations of competitor IgG from rabbit or man in the fluid phase, but not at all by bovine IgG1, whereas ingestion of Bo1-EAs was inhibited by bovine IgG1. This suggested that anti-D-EAhu were recognized by a FcR subset distinct from that recognizing bovine-IgG1. It was concluded that sheep BMM express functional analogs of human macrophage FcRI and FcRII and that Bo1-EAs and anti-D-EAhu are handled by distinct subsets of BMM FcR. All EAhu tested (EAhu treated with anti-D, sheep IgG1 or sheep IgG2) were ingested to a lower degree than EAs. This inefficient phagocytosis could be enhanced by treatment of EAhu with antiglobulin from the rabbit, suggesting that it is caused by a low degree of activity of opsonizing antibodies rather than special properties of the erythrocytes themselves. Several lines of evidence suggested that both FcR subsets of ovine BMM recognize both ovine IgG1 and IgG2. In contrast, bovine IgG1 reacts with one FcR subset and bovine IgG2 interacts inefficiently with all FcR of ovine BMM.     

The occurrence of Toxoplasma gondii and Neospora caninum as regards meat hygiene

Toxoplasma gondii and Neospora caninum are phenotypically and phylogenetically closely related cyst-forming coccidia, both of which may cause abortion in livestock animals. T. gondii exhibits also zoonotic potential by causing diaplacental infections in the human fetus and harmful infections in immunosuppressed individuals. Humans get infected either by consuming inappropriately prepared cyst-containing meat or by ingesting oocysts originating from cat feces. Therefore, in order to assess infection risk we need to have knowledge on the prevalence of the parasite in consumable meat and thus slaughtered animals. So far, no data indicate any zoonotic potential for N. caninum. Due to its high economic impact in the bovine production in Switzerland, we included this parasite in the present study as well. The prevalence of both parasite species were investigated by PCR in muscle and brain samples of slaughtered bovines, sheep, pigs and horses. Comparatively, a serum sample from each animal was simultaneously tested serologically by a Toxoplasma-P30-ELISA and a Neospora-SA-ELISA. The prevalences determined by the T. gondii-PCR were the followings: adult cows 3%, young bulls 2%, young cows prior to gravidity 6%, calves 1%, sheep 6%, horses and pigs each 0%. For N. caninum, the PCR-prevalence was 2% for adult cows and 0% for all other animal groups. Conversely, the seroprevalences were much higher for both parasite species and all animal groups, with the exception of the fattening pigs. However, as T. gondii was principally detectable in bovine (cows and calves) as well as in sheep meat, the consumption of this meat harbours a potential infection risk for humans. In contrast, the lack of any parasite detectability in fattening pig and horse meat allows to consider this infection source as neglectable when compared to bovine and ovine meat.     

H3N2亚型猪流感病毒中国分离株的克隆纯化及生物学特性

以有限稀释克隆法对29株H3N2亚型猪流感病毒(SIV)不同地区分离株进行纯化，并对其生物学特性进行了研究。结果，8株对鸡呈现中等致病力，21株对鸡呈现低致病力。不同地区SIV分离株(第5代)的EID50差异较大，以安徽分离株最高，为10^-10.77／0．2mL，其他毒株在10^-5.5～10^-10.56／0．2mL之间。黑龙江省分离株和浙江省分离株的LD50高达10^-2.84／0．1mL，其他分离株在10^-1.17～10^-2.56／0．1mL之间。经鸡胚分离传代后，SIV分离株均能凝集0．7％人“O”型血、绵羊、兔、豚鼠、小鼠、大鼠及鸡的红细胞，其红细胞凝集谱的差异主要表现在对马、牛、驴、猪红细胞的凝集特性上。大部分SIV分离株为热不稳定型，部分毒株表现为中等热稳定型和热稳定型。从其抗原特性看，大部分SIV分离株表现为亲和相，对AIV参考毒株DKUK63和SIV参考毒株SWTN77表现出较高的HI滴度；部分分离株经鸡胚传代后，出现了相别的变异。     

我国部分地区新城疫病毒分离株生物学特性的研究

从北京、昌黎、广州、长春、四平、青岛等地区采集疑似新城疫病鸡的病料，通过鸡胚接种分离出6株病毒，经血凝、血凝抑制试验和形态学观察，鉴定为新城疫病毒（NDV），分别命名为北京株、昌黎株、广州株、长春株、四平株、青岛株。测定了这6株NDV的致死鸡胚的平均时间（MDT）和脑内致病指数（ICPI），MDT分别为58．2，64．5，55．2，96。0．61．2和57．9h，ICPI分别为1．66，1．45，1．60，0．69，1．45和1．65。由这两项指标判定北京株、广州株、青岛株为强毒株，昌黎株和四平株为中强毒株，长春株为弱毒株。此外,还测定了6株NDV的血凝谱，各毒株均能凝集鸡和人（O型血）的红细胞，对猪、山羊、绵羊、牛和马的红细胞凝集作用有差异。     

Isolation of bovine complement factor H

A bovine serum protein, initially recognized by its inhibitory effect on the hemolytic activity of the bovine alternative pathway was isolated from fresh bovine serum by polyethylene glycol precipitation and chromatography on DEAE-Sephacel, CM-Sephadex A-50 and Sephadex G-200. The protein, a single chain polypeptide with an apparent molecular weight of 158,000, was identified as factor H, a regulatory protein of the alternative complement pathway. Functional characterization of this protein as factor H was based on the following properties: binding to C3b, inhibition of factor B binding to C3b, cofactor activity in the cleavage of C3b by factor I, inhibition of fluid phase alternative pathway C3 convertase (C3b.Bb) formation and activity, and species-specific inhibition of the alternative pathway mediated hemolysis of heterologous erythrocytes. A monospecific rabbit antiserum against bovine factor H failed to react with human serum factor H.     

A rapid and simplified technique for the assay of chicken hemolytic complement.

The technique described is a modification of a qualitative hemolytic radial diffusion technique. The test involves the use of sensitized sheep erythrocytes that have been incorporated into agarose. Tube dilutions were made of chicken serum and samples of each dilution were placed into wells cut in the agarose. The test is quantitative for hemolytic complement in that the highest dilution showing visible hemolysis of sensitized erythrocytes in agarose is determined to be the endpoint for that serum sample. The test as compared with the standard tube assay was determined to be less sensitive by approximately one dilution. The advantages of speed, simplicity, and cost more than offset the decrease in sensitivity of the test.     

A simple hemolytic assay for bovine complement component C3

A simple, one-step, alternative pathway (AP) hemolytic assay for bovine C3 has been developed. Methylamine was used to prepare a bovine serum reagent, R3, functionally depleted of C3. The addition of purified bovine C3 to the R3 reconstituted, in a dose-dependent manner, the hemolytic activity for unsensitized heterologous erythrocytes. The assay was used to determine relative levels of C3 in different bovine serum samples. Human C3 and bovine C3 were interchangeable in the assay. Reconstitution of bovine and human R3 reagents with homologous or heterologous C3, in the presence of different species of erythrocytes, provided evidence that cell surface regulation of the homologous hemolytic AP may not be limited to the assembly and activity of the C3 convertase. The AP assay was more sensitive and less complex to perform than a standard classical pathway assay for bovine C3.     

Hemagglutination by calf diarrhea coronavirus

The virus was grown in BEK-1 cells, a stable cell line from bovine embryo kidney, and tested for hemagglutination (HA) with erythrocytes of a variety of species at 4°C, room temperature and 37°C. HA was observed at all temperatures with chicken, mouse, rat, and hamster erythrocytes but not with erthyrocytes of human (O), cattle, horses, sheep, guinea pigs, geese, ducks, pigeons and 1-day-old chicks. Chickens showed an individual variation in agglutinability of their erythrocytes, requiring selection of birds to obtain erythrocytes for HA. HA reaction was inhibited by specific antiserum. Some factors involved in HA and HA inhibition (HI) were investigated and standard HA and HI tests were worked out.     

Characteristics of a haemolytic extract from avian Pasteurella multocida

In experimental fowl cholera, the intramuscular inoculation of Pasteurella multocida induces tissue damage that implies proteolytic or cytolytic activity of the bacteria. Such activity could not be demonstrated by conventional in vitro tests. The treatment of P. multocida strain VP21 with Tween-80 yielded an extract that lysed washed chicken red cells. Extracts were active to a maximum titre of 64. Haemolytic activity of the extract was neither affected by boiling nor by extremes of pH, indicating the active component was not a simple protein. Treatment with trypsin had no effect, but it was inactivated by Proteinase K. Yields were highest from bacteria grown in dextrose starch- or casein sucrose-yeast broths; were similar if cultured in air or anaerobically, but were reduced if the bacteria were grown in 5% CO(2). Haemolytic activity was eliminated on exposure to serum or serum albumen. The extract from strain VP21 haemolysed red cells from the chicken, rabbit, sheep, horse, bovine and human, with the highest titres observed on chicken cells. Six other avian strains and seven out of 10 strains of P. multocida from other species yielded an extract which haemolysed chicken red cells. The elaboration of this cytotoxic substance in vivo and its role in pathogenesis remains to be determined.     

Detection of parvoviruses in dogs using the hemagglutination test

The haemagglutination activity of the causal agent of canine parvovirosis is described. Out of the tested erythrocytes of pig, monkey, cat, horse, cattle, sheep, rabbit and guinea-pig, a positive reaction was recorded only in the erythrocytes of pig and monkey at the temperature of 4 degrees C. As a result of the examination of 20 faeces samples of hospitalized dogs by the method of haemagglutination reaction, a positive reaction typical of canine parvovirosis was obtained in 17 cases. The specificity of the reaction was confirmed by antiserum against the virus of panleucopenia of cat in the haemagglutination-inhibition test.     

对FC株猪源性肠毒素型大肠杆菌致病因子的研究

FC菌株是一株从腹泻仔猪粪便中分离的肠毒素型大肠杆菌(Enterotoxigenic E.coli,ETEC)。在MRHA反应中,本菌能凝集人O型、豚鼠、马、绵羊、牛、鸡和兔的红细胞,对人O型和豚鼠红细胞有很高的血凝性,血抗K88和K99血清不能抑制其对豚鼠和绵羊红细胞的血凝。在体外小肠上皮细胞吸附试验中,本菌对仔猪小肠上皮细胞具有强烈的吸附作用;透射电镜和扫描电镜观察证实了FC株菌除表面具有一种纤毛样结构外,还能定居在仔猪小肠段。血清学试验结果表明,本菌的O抗原属于O101。K88和987P两种抗血清均不能凝集本菌,而K99和F41抗血清均可凝集。对纯化的FC株菌粘着素抗原作等电聚焦和聚丙烯酰胺凝胶电泳分析,结果表明,该菌的粘着素是由等电点分别为4.61和9.78,分子量分别为29500和17500的两种蛋白质抗原所组成。此外,用乳鼠胃内投服试验和兔肠结扎试验证明,该菌只产生热稳定肠毒素。总之,本菌是一株能产生ST的K99,F41的肠毒素型大肠杆菌。     

Partial purification of extracellular toxic material of fish Vibrio

Hemolytic activity of 3 pathogenic strains, of fish Vibrio commonly associated with vibriosis (V anguillarum NCMB6 and NCMB571 strains, and Vibrio sp N7802 strain) was examined, using chicken and mammalian erythrocytes. Vibrio strains NCMB6 and NCMB571 and their culture filtrates had hemolytic activity against 8 kinds of erythrocytes tested, whereas Vibrio strain N7802 produced only a little amount of hemolysin. Strain NCMB571 culture filtrate and its material partially purified by column chromatography were lethal in mice. From 2 peaks of protein, hemolysin was recovered from the 1st peak, which coincided with toxicity in mice. Heat-inactivation of culture filtrate indicated that hemolytic materials may be thermolabile proteins, but that toxic material may be comparatively thermostable.     

根据线粒体保守序列检测鹿属动物源性成分

本文根据鹿线粒体mtDNA细胞色素b中的保守序列 ,设计了针对鹿属动物的特异性扩增引物。通过聚合酶链反应 (PolymeraseChainReac tion,PCR)得到 1 94bp的特异片段 ,而其它动物如鸡、牛、羊、兔、猪、鱼、骆驼、马等中则无此条带。通过内切酶AlwI酶切可对扩增结果进行验证。该方法对鹿成分的检测低限为 0 1 % ,可作为饲料中鹿属动物源性成分鉴别检测的有效方法之一 ,也可作为鹿类药材真伪鉴别的方法。