Development of enzyme-linked immunosorbent assays for the detection of Fusobacterium necrophorum antibody in animal sera

Enzyme-linked immunosorbent assays (ELISA) for the detection of Fusobacterium necrophorum antibody in the sera of rabbits, cattle, and sheep were developed, using a ribosome-rich extract (RRE) from F necrophorum as the antigen. Test character, including optimal antigen dilution and substrate incubation periods, was established, using rabbit, bovine, and ovine antisera produced against RRE from isolates of F necrophorum. Rabbit antisera produced against 7 other species of bacteria were used to test the specificity of the F necrophorum RRE antigen. Cross-reactivity was not detected. Sera from 50 feedlot cattle were examined with the bovine ELISA. Of the 50 samples, 43 (88%) were positive for F necrophorum antibody. The ELISA developed in this study were sensitive and specific and appear to be readily adaptable to serologic investigations of F necrophorum.     

Immunodiffusion reactions among porcine enteroviruses and other picornaviruses

Antisera raised in rabbits against the porcine enterovirus strains V13 and T80 produced two precipitin lines in immunodiffusion tests with the homologous crude antigens, and a single precipitin line with each of ten heterologous porcine enteroviruses which were tested, and with poliovirus type 1, coxsackievirus B4 and equine rhinovirus type 1, but not with a bovine rhinovirus. C and D antigens prepared from V13 virus by density gradient centrifugation produced single precipitin lines with V13 antiserum. A single precipitin line was also formed when the heated crude antigens of V13 and T80 viruses were reacted with the homologous antisera, indicating destruction of the D antigen, and these lines fused with those produced by the heterologous viruses. It was concluded that porcine enteroviruses contain C and D antigens, and that the C antigen is responsible for the serological cross-reactivity demonstrated among porcine enteroviruses and other picornaviruses by immunodiffusion.     

Biochemical characterization of the leukotoxins of three bovine strains of Fusobacterium necrophorum

The biochemical characteristics of the leukotoxins of 3 bovine isolates of Fusobacterium necrophorum which represent biotypes A, AB, and B were compared. Two methods were used for the production of the leukotoxins: medium M-1 continuous dialysis sac cultures and brain-heart infusion agar plate cultures. The supernatant cultural fluids were fractionated sequentially by membrane-partition chromatography, using ultrafilters with approximate molecular weight (mol wt) exclusion limits of 100,000, 10,000, 2,000, and 500. The ultrafiltrates (less than 500 mol wt) were fractionated by gel-permeation chromatography, using G-10 Sephadex. The leukotoxins of the 3 F necrophorum strains were estimated to have a molecular weight between 350 and 450. The leukotoxins in the ultrafiltrates (less than 500 mol wt) were stable at 60 C for 4 hours and at 100 C for 30 minutes, stable to extremes of pH (3 to 11), and stable to degradative enzymes including trypsin, protease, alpha-amylase, lipase, deoxyribonuclease, and ribonuclease. Significant differences were not observed in the biochemical characteristics of the leukotoxins produced in vitro by the 3 F necrophorum biotypes. These assays were done, using monolayers of mouse peritoneal macrophages. The monolayers were exposed to the 4 ultrafiltrates of both the continuous dialysis sac and brain-heart infusion agar cultures (pH 7.2) for 4 hours at 4 C, 25 C, and 37 C. Maximal cytotoxic activity in the assays was at 37 C.     

Characters of Escherichia coli 078 isolated from septicaemic animals

Twenty-one Escherichia coli isolates of serogroup 078 from animal septicaemia were obtained from laboratories in France, England and Canada. The bacteria were compared for outer membrane protein (OMP) patterns, lipopolysaccharide patterns, surface proteins of fimbrial types, biotypes, antibiotypes, colicin production, hydroxamate production and virulence in mice. Sixteen isolates from bovine, ovine, porcine and avian species in France and England had a similar OMP pattern. This characteristic associated with minor properties like surface proteins type, colicin V production and virulence in mice made these 16,078 E. coli isolates from 4 animal species, good candidates for the same clonal grouping. The five other bovine isolates with "Vir" or "31a" phenotypes were heterogeneous for most of the characteristics studied.     

Immunodominant antigens of zoospores from ovine isolates of Dermatophilus congolensis.

Zoospores of Dermatophilus congolensis were analysed by SDS-PAGE and western blotting. The electrophoretic profiles of zoospores from 13 isolates of D. congolensis were similar but not identical when stained with Coomassie blue or silver. Immunodominant polypeptides with apparent molecular masses of 76 and 31 kDa were identified in western blots of 13 of 13 and 12 of 13 isolates respectively of D. congolensis reacted with hyperimmune, ovine, antizoospore sera. Identical immunodominant polypeptides were observed in western blots reacted with sera obtained from naturally infected sheep. Initial characterisation of the 76 and 31 kDa polypeptides indicated that they were probably surface exposed because (i) antibodies eluted from the surface of live zoospores after adsorption of hyperimmune antizoospore serum, reacted principally against the 76 and 31 kDa subunit polypeptides in western blots, (ii) adsorption of hyperimmune antizoospore serum with live zoospores resulted in significant diminution of reactivity against both the 76 and 31 kDa polypeptides in western blots, (iii) indirect fluorescent immunostaining of zoospores with antiserum prepared against gel-purified 76 kDa polypeptide, resulted in intense staining of the zoospore outer coat. Immuno-gold electron microscopy of negatively stained zoospores with antiserum prepared against gel-purified 31 kDa polypeptide identified this antigen as a flagella subunit.     

Generation of immunity against Fusobacterium necrophorum in mice inoculated with extracts containing leucocidin

The capacity of extracts from toxigenic and non-toxigenic ruminant strains of Fusobacterium necrophorum to protect against challenge with homologous and heterologous bacteria was examined in mice. The numbers of F. necrophorum which were infective or lethal for mice increased 5- to 8-fold in animals which had been previously inoculated with complete Freund's adjuvant (FCA). Although preparations containing lipopolysaccharide (LPS) and outer membrane proteins (OMP) from several strains gave protection against a non-toxigenic strain (FnB-3), they did not significantly immunize mice against a challenge infection with a toxigenic bovine strain, FnB-1. Only material which had been prepared by gel filtration of 18-h liquid culture supernates of toxigenic F. necrophorum elicited significant immunity against homologous challenge with FnB-1. This preparation contained LPS and the majority of the leucotoxic activity. However, passive protection was not afforded to mice inoculated with bovine or rabbit sera which possessed high neutralization titres against the leucocidin.     

Antigenic relationship among field isolates of Tritrichomonas foetus from cattle

Analysis of protein and antigen profiles of Tritrichomonas foetus isolates from cattle from 5 western states was accomplished by sodium dodecyl sulfate polyacrylamide-gel electrophoresis, immunoblot, immunoprecipitation, and fluorography techniques. Total protein profiles of all isolates were compared by Coomassie brilliant blue staining of T foetus protein samples prepared by 4 protein-extraction methods. Antigenic tritrichomonas proteins were identified by immunoblot assay with polyclonal bovine or rabbit anti-T foetus serum. Additionally, [14C]glucosamine-labeled T foetus was used for total and antigenic glycoprotein analyses. Detectable differences in the composition of total proteins or antigenic tritrichomonal proteins were not observed among all isolates. However, intensity differences in some antigenic protein bands were apparent. Bovine and rabbit sera from immunized animals possessed antibodies to the same antigenic tritrichomonal proteins. Each T foetus isolate contained 4 to 7 molecular weight size classes of glycoprotein, which were labeled by [14C]glucosamine; however, only 3 to 4 glycoproteins were identified as antigens by bovine or rabbit antiserum.     

Ultrastructure and molecular characterization of Fusobacterium necrophorum biovars.

The ultrastructural features and molecular components of 18 strains of Fusobacterium necrophorum biovars A, AB and B, isolated from animal and human infections, were examined by electron microscopy, multilocus enzyme electrophoresis (MEE) and by sodium dodecyl sulfate-gradient polyacrylamide gel electrophoresis (SDS-PAGE). High resolution scanning electron microscopy revealed that the strains possessed a convoluted surface pattern. Transmission electron microscopy showed that all strains possessed a cell wall structure typical of gram-negative bacteria. Bleb formation was not uncommon. Numerous extracellular materials, resembling lipopolysaccharide (LPS) fragments, surrounded cells of both human strains and biovar B animal strains. Biovar A field strains revealed capsules as stained by ruthenium red whereas a stock culture strain showed the capsule only when immunostabilized with hyperimmune serum. Starch gel electrophoresis showed all strains to possess adenyl kinase, glutamate dehydrogenases and lactate dehydrogenase; each enzyme migrated uniformly (monomorphic) among the strains and represented an electrotype. However, SDS-PAGE indicated differences in the protein profiles between all of the strains; the most distinctly different was a human isolate (FN 606). Silver staining to detect LPS showed extensive "ladder" patterns among the majority of biovar A strains but not in the animal biovar B strains. Immunoblotting of LPS with a rabbit antiserum prepared against phenol extracted LPS from a biovar A animal isolate (LA 19) suggested marked variability in the LPS antigens among the isolates studied.     

Extraction, separation, and partial characterization of Brucella abortus antigens

Brucella abortus isolates strain 19 (a vaccinal strain) and 458 (a virulent field isolate) were analyzed for antigenic differences. Whole cell preparations were extracted with detergents, salt solutions, and phenol-acetic acid-water (PAW). Antigens were separated by starch block electrophoresis and sucrose density gradient centrifugation, serotested, and chemically analyzed. Six distinct precipitin lines were observed for the PAW extract against hyperimmune bovine antisera. With sucrose density centrifugation or starch block electrophoresis, antigens were separated into (i) complement-fixing (CF) antigens and (ii) antigens that did not fix complement but formed precipitin bands. Reciprocal CF tests with purified CF antigen could not differentiate between the antigens from either isolate of Brucella.     

Antigenic characteristics of enterotoxigenic and nonenterotoxigenic isolates of Bacteroides fragilis

Fecal isolates of enterotoxigenic (44 isolates) and nonenterotoxigenic (25 isolates) Bacteroides fragilis were obtained from diarrheic calves (62 isolates), lambs (2 isolates), and pigs (5 isolates). Using a Microtiter whole-cell agglutination test and gel double-diffusion analysis, the isolates were reacted with nonabsorbed rabbit antisera prepared against 13 isolates of enterotoxigenic B fragilis (ETBF). Isolates of B fragilis were antigenically diverse. Thirty-seven (84%) of the 44 isolates of ETBF comprised 13 serogroups on the basis of reaction in the agglutination test. Fourteen (56%) of the 25 isolates of non-ETBF comprised 4 of the 13 groups. Compared with results of the gel-diffusion test, most isolates had a different agglutination test reaction pattern against the 13 antisera. Isolates of ETBF could not be distinguished from non-ETBF. Antigenic heterogeneity of B fragilis facilitated differentiation of individual isolates, a capability that may be useful in future epidemiologic and virulence studies.     

鹿源坏死梭杆菌毒力菌株FN（AB）94实验动物感染模型的建立

将坏死梭杆菌FN（AB）94分离株以10^6,10^7,10^8,10^9 个/mL等不同菌量，于耳后根颈部皮下分别接种于4组的健康实验家兔，每组3只，逐日观察感染家兔的发病情况，结果，10^8,10^9个／mL，菌量感染组家兔，在接种后9－68 d内先后死亡，68d死亡家兔的病理变化明显，并从死亡家兔内脏及脓法叶，应用触片及分离培养均检到长丝状及小杆状等形态典型的坏死梭杆菌，10^7个/mL感染家兔仅表现体重减轻，10^6个／mL感染家兔无明显临床表现，由此表明，坏死梭杆菌FN（AB）94分离株具有很强的感染毒力，对家兔的最小致死量为10^8个／mL，耳后板颈部皮下接种是适宜的感染途径，从而建立了坏死梭杆菌分离株实验动物感染模型。     

Antiviral and antigenic properties of recombinant porcine interferon gamma

Recombinant porcine interferon gamma (rPoIFN gamma) induced a dose-dependent inhibition of the cytopathic effect produced by vesicular stomatitis virus (VSV) challenge of both homologous and heterologous (bovine) cell lines. In addition, an antiviral effect of rPoIFN gamma was demonstrable against the coronavirus transmissible gastroenteritis virus (TGEV) infection of porcine epithelial cells and of pulmonary macrophages. A rabbit anti-PoIFN gamma antiserum was prepared and shown to specifically neutralize the antiviral effects of natural and recombinant porcine IFN gamma preparations. This antiserum could also neutralize recombinant bovine IFN gamma but not recombinant human IFN gamma. These results suggest antigenic homology of porcine and bovine IFN gamma but antigenic differences between these molecules and human IFN gamma.     

Separation of a Soluble Antigen and Infectious Particles of Bovine Viral Diarrhea Viruses and their Relationship to Hog Cholera

A soluble antigen present in infectious tissue culture fluids was separated from the infective virus particle by ultracentrifugation of two serologically related strains of bovine viral diarrhea viruses, NADL-MD and Oregon C24V.

Neutralizing antibodies against the two viruses were absent in four hog cholera antisera, but present in significant titer in the commercially prepared antiserum. Precipitin tests utilizing the agar double diffusion technique formed a single line of identity between the concentrated soluble antigen of both viruses and NADL-MD and hog cholera antisera. No lines were observed using concentrated virus pellet and noninfected BEK cell antigens or control SPF calf and swine sera.

     

The immune response of rabbits to 3 strains of Mycoplasma agalactiae var. bovis isolated from mastitic bovine udders.

Rabbits were given 3 serologically-related trains of mycoplasma isolated from mastitic bovine udders. Growth inhibiting antibodies were not found. High-titered sera from each rabbit immunized with each strain developed at least 4 precipitin lines to each organism and at least one line of identity was demonstrated between the organisms. Significant indirect hemagglutination titers were found in all rabbits in 1 week, with titers reaching 1.0 X 10(5) to 3.3 X 10(5) in 4 to 6 weeks but despite continued intravenous antigenic stimulation, titers quickly fell to low levels thereafter.     

Immunologic comparison of the proteins of pseudorabies (Aujeszky's disease) virus and bovine herpesvirus-1

The immunologic relationship between bovine herpesvirus-1 and pseudorabies virus was examined by 80% serum cross-neutralization test, enzyme-linked immunosorbent assays, and Western immunoblotting procedures. Immunogenic cross reactivity between the 2 viruses was observed with both the serum-neutralization test and the enzyme-linked immunosorbent assay. A probing of viral Western immunoblots with rabbit hyperimmune antiserum showed that there were a number of viral-specific cross-reactive proteins between bovine herpesvirus-1 and pseudorabies virus.     

Reliability of thermonuclease production for the identification of human and animal Staphylococcus aureus

A total of 314 clinical and non-clinical isolates of the genus Staphylococcus was tested for coagulase production and glucose and mannitol fermentation. The isolates were tested for thermonuclease production and agglutination by sera 17H and 61218, which were specific for human and canine S. aureus biotypes, respectively. All produced coagulase and fermented glucose. A majority fermented mannitol anaerobically except for the canine isolates. A majority of human isolates produced thermonuclease (64.3%) and most were agglutinated by serum 17H. There was good correlation between thermonuclease production and agglutination by serum 17H of human and bovine clinical isolates (86.6 and 80%, respectively). This was also true of clinical canine isolates agglutinated by serum 61218, of which 75% were thermonuclease-positive. Over half of canine isolates (52.8%) were thermonuclease-positive and most were agglutinated by serum 61218. Bovine and caprine isolates were 34.1 and 25% thermonuclease-positive, respectively, while ovine isolates were only 14.2% thermonuclease-positive. Isolates from these ruminant sources were also poorly agglutinated by either serum. It was concluded that a greater number of clinical human and canine biotypes of S. aureus produced thermonuclease than their non-clinical isolates, and that a majority of other animal isolates were negative for thermonuclease. Therefore, the thermonuclease test may not be very useful for confirming the animal origin of S. aureus isolates.     

Characterisation of virulent and benign strains of Bacteroides nodosus

The extracellular proteases of 395 isolates of B. nodosus from ovine, bovine and caprine foot lesions were classified as either thermostable or thermolabile. Stable protease was associated with one and unstable protease with four distinctive isoenzyme patterns, each pattern differentiated by the relative mobility of paired isoenzymes. Pathogenicity tests on 64 isolates showed a correlation between the production of stable protease and the production of virulent ovine footrot lesions. The mean values for total protease activity, twitching motility and colony diameter were significantly higher for virulent compared to benign isolates, but the range of values overlapped. SDS-PAGE whole-cell electrophoretic profiles of virulent isolates were similar to the profiles of some benign isolates.     

Strain specific antibodies revealed by immunoabsorption studies with avian infectious bronchitis virus

Rabbit antisera prepared against the Massachusetts 41 (M41) strain of avian infectious bronchitis virus (IBV) and absorbed with chick embryo immunoabsorbent produced multiple precipitin lines in immunodouble-diffusion (IDD) tests with homologous or heterologous strains of virus. These precipitin lines were all removed by absorption with concentrated M41 virus preparations, but repeated absorption with concentrated, purified preparations of IBV strains: T, Holte, Connecticut, Beaudette or H120 failed to remove all precipitin lines produced to M41 virus, although all those to the heterologous viruses were removed. The remaining line(s) produced with M41 virus by sera absorbed with different heterologous viruses showed identity in IDD tests and was associated with the surface projections of the virus.     

Characterization of two serotypes of adenovirus isolated from sheep in the central United States

Two serotypes of adenovirus were isolated from lambs assembled at a ram testing station. The isolates were identified as adenoviruses on the basis of their morphologic and physicochemical characteristics. One isolate (RTS-42) was neutralized by antiserum to ovine adenovirus serotype 5, whereas the other strain (RTS-151) was not neutralized by the antisera to ovine adenovirus serotypes 1 through 5 or bovine adenovirus 1 through 8.