A STANDARDISED HAEMAGGLUTINATION INHIBITION TEST FOR PORCINE PARVOVIRUS ANTIBODY

Basic variables of the haemagglutination inhibition (HI) test for porcine parvovirus antibody were investigated. Nonspecific serum inhibitors were satisfactorily removed without loss of specific antibody when undiluted serum was adsorbed with 25 percent kaolin in borate saline at pH 9.0. Natural haemagglutinins in test serums could be completely removed using 0.1 ml of packed erythrocytes to 0.6 ml of kaolin treated serums. Adsorption of prediluted serum resulted in a depression of specific antibody titres. Highest HI titres were obtained using guinea pig erythrocytes, following incubation of virus-serum mixtures for 18 hours at 4 degrees C, 3 hours at 25 degrees C or 2 hours at 37 degrees C. Micro- and macro-tests gave comparable HI titres.     

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.     

The sedimentation pattern of D-glucose-6-phosphate dehydrogenase from bovine fetal and adult erythrocytes

Erythrocytes from bovine fetuses contain about 2.4 times higher D-glucose-6-phosphate dehydrogenase activities than erythrocytes from adult cows and bulls. Studying whether this is due to the existence of a special fetal type of enzyme or an increased amount of enzyme in fetal erythrocytes, the sedimentation coefficients of the enzymes have been estimated by s-zonal ultracentrifugation, and compared to normal and deficient human erythrocyte D-glucose-6-phosphate dehydrogenase, s-zonal ultracentrifugations have been performed with a computer optimized isokinetic sucrose gradient. The mainlines in the program used for calculation of sedimentation coefficients are described.Bovine fetal and adult erythrocyte D-glucose-6-phosphate dehydrogenase was found to have the same sedimentation coefficient of 7.4 S which is different from the sedimentation coefficient of 6.4 S of both human types of the enzyme. The sedimentation coefficients of 6-phospho-D-gluconate dehydrogenase from bovine fetal, bovine adult and human erythrocytes were 6 S for all three types of this enzyme.By cellulose acetate electrophoresis bovine fetal and adult D-glucose-6-phosphate dehydrogenase show the same mobility, again differing from the normal and deficient human type.The results of these experiments show that bovine fetal and adult erythrocytic D-glucose-6-phosphate dehydrogenase with respects to molecular parameters are closely related and perhaps identical enzymes.Keyword: D-glucose-6-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase deficiency, bovine erythrocytes, fetal erythrocytes, human erythrocytes, s-zonal ultracentrifugation     

Identification of MCP/CD46 analogue on bovine erythrocytes using the new monoclonal antibody IVA-520

MCP/CD46 is a widely distributed C3b/C4b binding regulatory glycoprotein of the complement system that has been identified on all human peripheral blood cells except erythrocytes. In this paper, we describe the identification of bovine CD46 on all blood cells, including erythrocytes, with the newly prepared monoclonal antibody IVA-520. This antibody cross-reacts with human and pig cells. Furthermore, the molecule identified by IVA-520 functionally behaves as the MCP molecule, showing cofactor activity for the factor I-mediated cleavage of bovine C3 complement factor.     

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.     

SENSITIVITY AND SPECIFICITY OF TWO MICROTITRE COMPLEMENT FIXATION TESTS FOR THE DIAGNOSIS OF BRUCELLA OVIS INFECTION IN RAMS

SUMMARY: An investigation was made into microtitre complement fixation test (CFT) procedures suitable for the serological diagnosis of naturally occurring Brucella ovis infection in rams. A procedure similar to the Australian standard procedure for bovine brucellosis was unsatisfactory when applied to sheep. Modification of the procedure by use of an initial serum and anticomplementary control dilution of 1:8 and increasing complement fixation time to 60 minutes at 37°C, greatly improved the efficiency of the test. A sensitivity of 100% was recorded for 59 serums from known infected rams and a specificity of 99.9% for 1593 serums from rams known or believed to be free of infection. Some aspects of applying CF tests to sheep serums are discussed.     

A COMPLEMENT-FIXATION TEST FOR ENZOOTIC PNEUMONIA OF PIGS USING A COMPLEMENT DILUTION METHOD

Complement-fixing antibody to Mycoplasma hyopneumoniae in the serums of pigs experimentally infected with enzootic pneumonia was demonstrated by comparing the haemolytic titre of guinea-pig complement titrated in the presence of heated test serum, M. hyopneumoniae antigen and unheated normal pig serum with the titre obtained when the antigen was omitted. The haemolytic titres against sensitised sheep erythrocytes were determined after a fixation period of 16 to 18 hours at 5°C. When serums, collected at intervals of 3 to 7 days, from 43 pigs exposed to pigs experimentally infected with enzootic pneumonia were tested, 4.6 or more complement units were first fixed 14 to 44 (mean 23.4) days after contact began. Serums collected subsequently fixed from 4.6 to more than 31 complement units. This positive reaction usually persisted until the pigs were killed 4 to 35 weeks after contact began. Thirty-three had gross enzootic pneumonia lesions and 9 had lung lesions detected microscopically. Serum antibody was not detected in 73 weaned pigs aged 7 weeks in a pneumonia-free herd but serums from 9 of 15 unweaned piglets aged 9 to 14 days in the same herd, fixed between 3 and 7 complement units.     

Genomic variations and antigenic relationships among cytopathic rotavirus strains isolated in Quebec dairy herds.

Twelve isolates of bovine rotavirus, originating from eight dairy herds in Quebec known to have frequent epizootics of diarrhea in young calves in the last five years, were successfully propagated in cell cultures. The 12 isolates produced clear-cut plaques in BSC-1 cells and, except for one isolate, agglutinated human group "O" erythrocytes to an higher titer than bovine erythrocytes. Antisera to each isolate were produced in rabbits and used to study their antigenic relationships. All the isolates shared the group-specific immunofluorescent antigen and were antigenically related as demonstrated by the seroneutralization and hemagglutination-inhibition tests. However, the relationships to the Nebraska rotavirus was quite weak in cases of two Quebec isolates. When the genomes of the various isolates were compared by polyacrylamide gel electrophoresis, at least three different reproducible fractionation patterns could be identified.     

Characterization of the inclusion limiting membrane of anaplasma marginale by immunoferritin labeling.

Purified anti-erythrocytic membrane antibody (PAMA) was prepared from rabbit anti-bovine erythrocyte serum by an adsorption and elution technique, utilizing bovine erythrocytes. Lysed and washed anaplasma-infected erythrocytes were incubated with PAMA or control reagents. Specimens were then subjected to immunoferritin labeling with ferritin antiglobulin conjugate. Upon examination by electron microscopy, specimens incubated with PAMA showed heavy ferritin labeling of erythrocytic membranes and also the limiting membranes of anaplasmal inclusions. Anaplasmal initial bodies freed from their inclusion membranes were not labeled. Negative control specimens, incubated with normal rabbit serum or PAMA which had been absorbed with erythrocytes, did not show specific ferritin labeling. Intact bovine erythrocytes, which were used as a positive control of anti-bovine erythrocytic membrane specificity, were heavily ferritin-labeled. Avian erythrocytes, a negative control of specificity, remained unlabeled. The results of this study indicate that the limiting membrane of the anaplasmal inclusion is derived from the erythrocytic membrane.     

Influence of Plasma Proteins on Erythrocyte Aggregation in Three Mammalian Species

The aggregation capacity of human erythrocytes lies between that of the non-aggregating bovine erythrocytes and the remarkably aggregating equine ones. As the ability to aggregate is attributed to cell factors and the composition of the plasma proteins, the role that plasma proteins play in the aggregation process in these three species was studied. Washed erythrocytes were suspended in phosphate-buffered saline (PBS; pH 7.4, 300 mOsm/L) plus polyvinylpyrrolidone (PVP) in a suitable concentration to obtain an average intensity of aggregation (control media). The superimposed effect of replacing 80% of the medium by either autologous plasma, serum or albumin solution was studied. The plasma proteins appeared to enhance aggregation by human and equine erythrocytes, but impaired this process in bovine erythrocytes. Some evidence was obtained supporting the existence of serum factors capable of reducing aggregation of erythrocytes in cattle and it was concluded that the non-aggregating behaviour of bovine erythrocytes may be due to the cells interacting particularly with the macromolecules in the serum.     

Hemagglutinating and hydrophobic surface properties of salmonellae producing enterotoxin neutralized by cholera anti-toxin

Eleven Salmonella strains known to produce enterotoxin under aerobic culture conditions in deferrated (DF) medium at 37°C were shown to produce enterotoxin with and without aeration at 22, 28, 37 and 42°C. Heat-labile enterotoxin was generally produced with growth temperatures up to 37°C irrespective of aeration. Heat-stable enterotoxin was produced up to 42°C, mainly aerobically, as indicated by infant mouse assay (IMA), by six of the eleven strains tested. Nine strains produced heat-stable rapid permeability factor (RPF) in rabbit skin.Cholera anti-toxin neutralized reactivities of Salmonella heat-labile enterotoxin in four different biological assays. Mixed gangliosides also neutralized this activity in the cell—test systems.With guinea-pig erythrocytes, all strains underwent mannose-resistant hemagglutination (MRHA) irrespective of growth temperatures, i.e. 22 and 37°C or medium, i.e., DF, tryptose soy broth (TSB) and colonization factor antigen (CFA) agar. At both growth temperatures, CFA agar-grown cells of each strain caused MRHA of bovine erythrocytes. Excepting three Salmonella typhimurium strains, DF broth-grown cells gave MRHA of bovine, chicken and human group A erythrocytes, CFA agar-grown cells caused MRHA of chicken and human blood, whereas TSB-grown cells caused few MRHA reactions.Salmonellae producing both heat-stable, (ST) and heat-labile, (LT) enterotoxins adsorbed to Phenyl Sepharose whereas salmonellae that produced only LT enterotoxin did not.The presence of MRHA adhesions did not correlate with cell-surface hydrophobicity. However, mannose-resistant hemagglutinins may occur more commonly among salmonellae than has been previously recognized.     

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.     

贵州奶牛传染病检测和预防的研究

2006—2008年对贵州省内610份奶牛血清进行检测,其中有116份血清为乙型肝炎表面抗原(HBsAg)阳性,平均阳性率19%;534份牛血清样品中,检出奶牛传染性鼻气管炎阳性血清29份,阳性率为5.4%,而运用血清补体结合反应(CFT)检测奶牛传染性胸膜肺炎,均为阴性。将HBsAg、HBeAg双阳性奶牛肝组织做电镜观察,发现肝组织中有密集的HBV颗粒。结果表明,贵州省奶牛可能存在乙型肝炎、传染性鼻气管炎的感染,应当引起奶牛养殖者的高度重视。     

Trichomonas gallinae: a possible contact-dependent mechanism in the hemolytic activity

The in vitro hemolytic activity of Trichomonas gallinae was investigated. The parasite was tested against human erythrocytes of groups A, B, AB, and O, and against erythrocytes of six adult animals of different species (rabbit, rat, chicken, horse, bovine, and sheep). Results showed that T. gallinae lysed all human erythrocytes groups, as well as rabbit, rat, chicken, 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. gallinae, or with killed organisms. The scanning electron microscopy (SEM) showed that the erythrocytes adhered to the parasite surface and were phagocytosed. These observations suggest that the contact between T. gallinae and erythrocytes may be an important mechanism in the injury caused to the erythrocytes. The hemolytic activity of T. gallinae may be an efficient means of obtaining nutrients for the parasite and allow the investigation of the mechanism used by T. gallinae to damage cellular membranes.     

Hemagglutinating factor in an extract of Sarcoptes scabiei var. suis (De Geer)

A naturally occurring hemagglutinating factor to tanned human O positive, ovine and porcine erythrocytes was found in extracts from Sarcoptes scabiei var. suis. This hemagglutinating factor did not react with bovine, equine or avian erythrocytes. This factor was demonstrated by microscopic examination of the tanned erythrocytes and by the passive hemagglutination assay.     

Sheep as a new experimental host for Babesia divergens

Babesia divergens was cultivated in sheep erythrocytes in RPMI 1640 supplemented with 10% Fetal Calf Serum (FCS) or sheep serum. In vitro cultures in sheep red blood cells were initiated with human erythrocytes infected in vitro with B. divergens Rouen 1987 or with gerbil blood infected with several isolates from bovine origin. After the first subcultures on sheep erythrocytes, a ten-fold multiplication of the parasites was obtained within 48 h. Erythrocytes from three splenectomized sheep were infected in vitro with B. divergens; when parasitaemia reached 10%, the animals were inoculated with homologous parasitized erythrocytes. All sheep expressed hyperthermia with a peak between the 6th and the 9th day post-infection (p-i) and a transitory parasitaemia 10 days p-i. In vitro primary cultures were performed on two of these sheep, demonstrating the parasite persistence at very low parasitaemia in the infected animals. Splenectomized sheep can be used as a new model for B. divergens chronic infection.     

A microplate enzyme-linked immunorsorbent assay for measuring antibody to Anaplasma marginale in cattle serum

An enzyme-linked immunosorbent assay (ELISA) method is described for measuring antibody against Anaplasma marginale in cattle serum. This method was more sensitive and objective than a previously described ELISA method for A. marginale and possible reasons for this are discussed. All 83 cattle experimentally infected with A. marginale (81) or A. centrale (2) developed demonstrable specific antibody but the serums of 98.8% of 839 cattle from cattle tick-free areas did not react by ELISA; 378 serums containing antibody to Babesia bovis were tested for cross reactions in the A. marginale ELISA. There were no significant cross-reactions except when cattle had been inoculated at least twice with B. bovis-infected erythrocytes, presumably due to antibodies reacting with erythrocyte material in the ELISA antigen. The ELISA detected antibodies for more than 3 years after infection, at least 2 years longer than did a complement fixation test. When A. marginale infections in cattle were eliminated by long acting oxytetracycline, their serums ceased to react by ELISA. An ELISA score for serum antibody level was shown to have a statistically significant correlation with ELISA titre.     

CONGENITAL BOVINE EPIZOOTIC ARTHROGRYPOSIS AND HYDRANENCEPHALY IN AUSTRALIA: Distribution of Antibodies to Akabane Virus in Australian Cattle after the 1974 Epizootic

At the end of the 1974 epizootic of bovine congenital arthrogryposis and hydranencephaly in south-eastern New South Wales, an Australia-wide serological survey (about 4,000 serums) was made to determine the ditribution of cattle possessing serum neutralising antibodies against Akabane virus. Eighty per cent of the serums from cattle in northern Australia (Western Australia, Northern Territory, and Queensland) were positive. A detailed study in the epizootic area in New South Wales (particularly around Bega) showed that 80 to 100% of serums from cows in herds in this area possessed neutralising antibodies. The animals possessing antibodies extended as far south as Genoa in north-eastern Victoria, and as far west as Darlington Point on the Murrumbidgee River. There were no positive herds along the Murray River, where an outbreak of the mosquito-borne disease Murray Valley encephalitis occurred in 1974. Serums tested from cows in the rest of Victoria, South Australia, south-western Western Australia, and Tasmania were negative. Arthrogrypotic calves born in Tasmania and south-western Western Australia were not associated with the presence of Akabane virus. In Papua New Guinea, serums collected from cattle at Boroka, Lae, and Goroka did not possess neutralising antibodies. The distribution of cattle possessing antibodies in Australia would fit a spread of the virus by Culicoides brevitarsis, a biting midge from which Akabane virus had been isolated on three occasions. The possibility of other vectors, as well as C. brevitarsis, was suggested by the presence of cows possessing antibodies at Alice Springs, where this biting midge has not been found. Possibly most cattle in northern Australia become infected early in life. The epizootics in New South Wales could occur when seasonal conditions allow a southerly extension of virus-infected C. brevitarsis which feed on susceptible pregnant animals. C. brevitarsis also bites sheep, and both neutralising antibodies to Akabane virus and congenitally deformed lambs have been observed in the epizootic area. An understanding of the distribtuion of Akabane virus and C. brevitarsis, a possible Australian vector for bluetongue virus, may prove useful if bluetongue should enter Australia.     

Experimental bovine group-B streptococcal mastitis induced by strains of human and bovine origin

Intramammary infections with Group-B streptococci of human and bovine origin were produced experimentally in cows. The initial cytological response was more rapid to the human than to the bovine strain (Table I), while at later stages the pathological changes induced by the two infections were much the same (Fig. 1). The initial clinical reaction was more acute to the "human" than to the "bovine" infections and the average daily loss of milk was greater in cases of "human" infection than in cases of "bovine" infection (Table II). In contrast to the "bovine" infections the "human" infections showed a pronounced tendency to spontaneous clearance. The rate of excretion of Group-B streptococci with the milk was lower for the "human" than for the "bovine" infections (Table III). The special mode of reaction of the bovine udder against infections with Group-B streptococci of human origin may, in part, explain why such infections have a lower tendency to spread within a herd than infections with bovine strains of B-streptococci.     

Studies on the binding and hemagglutinating properties of cholera toxin to human erythrocytes

The binding and hemagglutinating properties of cholera toxin (CT) were studied by competitive binding assays and hemagglutination inhibition. The binding of 125I-labeled CT to neuraminidase-treated human type B erythrocytes was most effectively inhibited by ganglioside GM1 among different inhibitors used. Other mono-, di-, and polysaccharides and glycoproteins were at least 10(5) times less potent inhibitors. On the other hand, hemagglutination of neuraminidase-treated human type B erythrocytes by CT was inhibited by lactose, galactose, hog A + H, bovine salivary mucin, porcine thyroglobulin, and fetuin, whereas that was not effectively inhibited by ganglioside GM1 at the highest concentration. These findings suggest that the predominant binding substance for CT on human type B erythrocytes is ganglioside GM1 and that hemagglutination requires some additional process since the interaction of CT with ganglioside GM1 is somehow different in hemagglutination.