Serological reactivity of duck sera to three electrophoretically characterized extracts of Aspergillus

Three different antigens, whole culture, cell sap shaker culture and culture filtrate extracts of Aspergillus were characterized by crossed immunoelectrophoresis. Thirteen to fifteen precipitin lines were produced with their homologous antisera. Application of these extracts to the assay of wild duck sera showed that the whole culture extract was the most sensitive of the three antigens in detecting antibody. Countercurrent immunoelectrophoresis, used with a modified buffer, produced results comparable in sensitivity to immunodiffusion in detecting avian antibody.     

Diagnosis of enzootic bovine leukosis: a comparison of haematological and immunodiffusion tests.

A total of 657 cattle from three different herds were tested for the presence of specific antibodies against EBL antigens by means of the immunodiffusion test and the results compared with the classification according to the lymphocyte counts. All animals with pathologically raised lymphocyte counts showed a positive immunodiffusion test reaction. In the majority of these both antigens, 'p24' and 'gp', gave positive results; in a smaller number antibodies against the gp antigen only were detected, but never against the p24 antigen only. It is concluded that for field testing programmes the use of the gp antigen is sufficient. In future experiments, however, both antigens should be employed, because the lack of one of them might perhaps prove to be correlated with the future course of the disease. The absence of specific antibodies, together with a normal number of lymphocytes, in young animals should, however, not lead to the conclusion that they are free of the disease.     

Serological cross-reactions between different Brucella species and Yersinia enterocolitica. Immunodiffusion and immunoelectrophoresis

By immunodiffusion and immunoelectrophoresis tests in agarose serological cross-reactions were demonstrated between Yersinia enterocolitica type IX and Brucella strains from four species (Brucella abortus, Brucella melitensis, Brucella suis and Brucella neotomae). No qualitative differences between these strains in their tendencies to cross-react with Yersinia enterocolitica type IX were observed. Brucella canis and Brucella ovis, which have nonsmooth colonial morphology, gave no demonstrable cross-reaction with Yersinia enterocolitica type IX.The results of absorption tests and qualitative staining reaction of the obtained precipitation lines suggest that the antigenic determinants common to Brucella and Yersinia enterocolitica type IX seemed to be associated with the outer layer and in the lipopolysaccharide complex of the respective bacteria. By immunodiffusion and immunoelectrophoresis it was possible to identify in hyperimmune sera those antibodies that derive from Brucella and Yersinia enterocolitica type IX.Keyword: serological cross-reaction, Brucella species, Yersinia enterocolitica serotype IX, immunodiffusion, immunoelectrophoresis     

Soluble antigens from culture-grown Besnoitia besnoiti endozoites

Soluble antigens from Besnoitia besnoiti cell culture-grown endozoites, obtained either by hypotonic lysis or by freeze-thawing and ultrasonication (FTS) of the organisms, were detected by the agar gel immunodiffusion method. Each antigenic preparation yielded 1-4 precipitin lines when reacted with the corresponding rabbit hyperimmune serum, while no reaction was observed with Besnoitia-positive sera from naturally infected cattle. Soluble exoantigens released by viable Besnoitia endozoites into the supernatant of infected cell cultures formed two precipitin lines with rabbit anti-FTS hyperimmune serum and appeared as positively charged protein in immunoelectrophoresis. The precipitin lines observed were parasite specific since no reaction occurred with either lysates of normal Vero cells or with supernatants from non-infected cell cultures.     

Serological responses of dogs to cell wall and internal antigens of Brucella canis (B. canis)

The serological responses of dogs to cell wall and internal antigens of B. canis were studied in experimentally infected specific-pathogen-free (SPF) Beagles. Sera from infected and false positive field dogs also were examined. Cell wall antigens were extracted from B. canis by two procedures that employed either hot phosphate buffered saline (PBS) or sodium desoxycholate (SDC). Agar gel immunodiffusion (AGID) tests employing sera from experimentally infected SPF dogs were used to evaluate antigenic extracts. Extraction with PBS yielded two antigens; SDC extracted an antigen complex and sonication of PBS extracted cells liberated four internal antigens.Sera from field dogs that were negative for B. canis infection in repeated tests often had heterospecific antibodies. Such cross-reactive sere commonly gave “spur” (partial fusion) reactions with a positive reference serum when tested against the SDC cell wall antigen. In addition, false positive dogs did not have antibody to one of the cell wall antigens or to the internal antigens. In contrast, sera from infected field dogs commonly gave “identity” (fusion) reactions in the AGID test with two antigens in the SDC extract, and produced precipitin lines to one to four internal antigens.Examination of a library of sera obtained from experimentally infected SPF dogs over a period spanning 4$\text{1}\text{2}$ years revealed that none of the serodiagnostic tests employed (tube agglutination, slide agglutination, AGID) was accurate during the inital 12 weeks of infection; hemocultures were the most sensitive during this period. Tube and slide agglutination tests were initially sensitive, but they showed a lack of sensitivity and specificity after the bacteremic period ceased, as well as in their failure to exclude false positive reactions in field animals. Immunodiffusion tests that employed SDC or PBS extracts of B. canis cell walls were sensitive and accurate in identifying most infected dogs. After the bacteremia had ceased, however, AGID tests that employed cell wall antigens gave equivocal results. Immunodiffusion tests that employed sonicated (internal) antigens were sensitive shortly after the onset of bacteremia, and they had the advantage of detecting infected animals for at least 6 months following the cessation of bacteremia, a time when other serological tests gave equivocal results.     

Fractionation and characterisation of the cysticercus of Taenia solium

Fractionation by chromatography on Sephadex G-200 of a saline extract of Cysticercus cellulosae scolex antigen yielded three distinct fractions associated with distinct peaks. These fractions were analysed by double immunodiffusion (DID) and immunoelectrophoresis (IEP). The three peaks gave five, four and three antigenic determinants, respectively, by DID with homologous hyperimmune rabbit serum. However, the same serum gave nine antigenic determinants of scolex antigen by DID and 11 components by IEP. The IEP demonstrated seven and five antigenic components in the first two peaks. The first peak gave a stronger reaction in indirect haemagglutination than the others. There were common antigenic components in C cellulosae and C tenuicollis antigens.     

Immune responses in swine given lipid-conjugated pseudorabies viral antigens.

The immune response was compared in pigs given inactivated pseudorabies virus (PRV) antigens (with or without adjuvant) or PRV antigens covalently conjugated with a fatty acid (lauric acid) to enhance delayed-type hypersensitivity. The pigs were given 2 inoculations, 14 days apart, and were challenge exposed 28 days after the 1st inoculation. Pibs inoculated with PRV antigens, with or without adjuvant, had significant virus-neutralizing (VN) antibodies before challenge exposure, but the pigs inoculated with lipid-conjugated PRV antigens had no detectable VN antibodies, with the exception of 1 pig. All inoculated pigs were positive by the microimmunodiffusion test at postinoculation day 14 and remained positive throughout the experiment. The inoculated pigs had delayed-type hypersensitivity reactions when skin tested a postinoculation day 25; the pigs inoculated with lipid-conjugated PRV antigens had a more pronounced reaction. Inoculated pigs had mild respiratory signs on the 3rd through the 6th days after challange exposure, with no observable difference in severity between the inoculated groups. The control pigs had acute signs of PRV, and 3 or 4 pigs died 5 to 8 days after challenge exposure. The average VN titers of the different inoculated groups of pigs were nearly equal 2 weeks after challenge exposure. Results indicated that both humoral antibodies and cell-mediated immunity have a role in PRV infections in swine.     

Mycoplasma hyopneumoniae infection in pigs: Duration of the disease and evaluation of four diagnostic assays

200 SPF pigs were infected by aerosol with Mycoplasma hyopneumoniae and the development of clinical signs, serological and pathological reactions were studied. Mean time to onset of coughing was 13 days. A mean delay of 9 days was observed from onset of coughing until seroconversion against M. hyopneumoniae as measured by ELISA. At an individual level, the sensitivity for this ELISA was estimated to 98–100% and the specificity to 93–100%. Pasteurella multocida was isolated from the majority of the lungs 4 weeks post inoculation with M. hyopneumoniae and the lung lesions in pigs were significantly larger when P. multocida was present as compared to pigs with M. hyopneumoniae alone. An evaluation of cultivation, immunofluorescence, ELISA and polymerase chain reaction for demonstration of M. hyopneumoniae in lungs showed that all four methods have a high sensitivity in the acute stages of pneumonia. In the later stages the sensitivity of cultivation was superior to the other methods. No differences in specificity were observed between the methods. The antigen-ELISA OD values and the immunofluorescence scores revealed a strong positive correlation. Nasal swabs were additionally used for demonstration of M. hyopneumoniae and the polymerase chain reaction was found superior to the other methods.     

Immunogenic properties of ribosomes isolated from Brucella abortus

Ribosomes from Brucella abortus strain 19 were isolated by differential centrifugation. The 70S ribosomes were purified by pelleting through 0.5 M ammonium chloride and 30% sucrose. The resulting ribosomes were designated washed ribosomes and consisted of 64–65% RNA and 35–36% protein with sedimentation coefficients of 50S and 30S. When the washed ribosomes were tested by immunodiffusion against immune bovine sera, no precipitins were detectable. The immunogenic responses of the washed ribosomes were determined in guinea pigs, lemmings, and calves. Inoculation dosages as low as 1.0 microgram in guinea pigs and 0.1 microgram in lemmings protected against challenge with virulent B. abortus. Results of similar studies in a limited number of calves indicated that a dose of 1 mg may be protective. Brucella ribosomes did not appear to elicit marked undesirable side effects when used as immunizing agents.     

Mycoplasma hyopneumoniae colonization of pigs sired by different boars

Differences in Mycoplasma hyopneumoniae colonization were evaluated in experimentally inoculated pigs sired by 3 different boars of the same genetic line. Forty-six pigs were used, including a treatment group and positive and negative control groups. The pigs were intratracheally inoculated with an M. hyopneumoniae suspension or with Friis media as a placebo. To evaluate differences in the magnitude of colonization during a 35-day period, nasal and tracheal swabs were collected weekly and tested by nested polymerase chain reaction (N-PCR). Temperature, weight and circulating antibodies were measured for 35 days. At 11 and 35 d postinoculation the pigs were necropsied and macroscopic and microscopic lesions were determined. A section of bronchus was tested by the indirect immunofluorescence test (IFAT), scanning electron microscopy (SEM) and N-PCR. The N-PCR results from the nasal and tracheal swabs showed that the pigs sired by one boar (B3) had a distinctive colonization pattern, different from that of the pigs from the other 2 boars and from the positive controls. SEM studies demonstrated that at 35 d postinoculation a higher proportion of B3 pigs had lower numbers of mycoplasmas attached to the cilia compared with B1 and B2 offspring. No significant differences were observed in temperature and weight gain among groups by ANOVA; however, with use of a 2 × 2 table, temperature differences were observed between pigs sired by boars B1 and B2 at 4 d postinoculation. No pigs seroconverted, showed gross or microscopic lesions, or had positive IFAT results. These results provide evidence of differences in patterns of colonization between pigs sired by different boars, suggesting a possible genetic effect.     

Effect of cambendazole on swine kidneyworm,Stephanurus dentatus

Two trials were conducted to determine the effect of cambendazole against natural infections of the swine kidneyworm, Stephanurus dentatus, in sows. In the first trial, nine sows were given a single dose of 20 mg cambendazole/kg body weight, in the feed, and eight sows were not treated. In the second trial, five sows were given 40 mg cambendazole/kg body weight and four were untreated. Urine volume, S. dentatus eggs per milliliter of urine, egg hatchability, and larval survival to the third stage were determined pre and post treatment. Adult kidneyworms were counted at necropsy, 14 days after treatments. Only a temporary decline in egg hatchability was observed on the day after treatment in each trial, and cambendazole was not found to be effective against adults of S. dentatus.     

Synthesis of immunoglobulin by normal and antigenically stimulated fetal sheep.

The rate of appearance, quantity and immunochemical character of serum immunoglobulins which appear normally during the development of fetal sheep and following the injection of antigens at different stages of gestation have been studied. After 70 days' gestation a percentage of normal fetal sheep synthesise IgM. Although the concentration of IgM in the circulation of these animals was very low, the precentage with IgM increased with fetal age. A few late term fetuses were detected which also had IgG1 in their circulation, although none were detected with IgG2 or IgA. Fetuses injected with antigens before 71 days' gestation only synthesised IgM, while fetuses injected after 79 days' gestation synthesised both IgM and IgG1. Neither IgG2 nor IgA were detected by single-radial immunodiffusion analyses during the first 14 days of primary immune responses to a variety of antigens, although trace amounts of IgG2 were detected late in responses occurring in older fetuses. The immunoglobulins synthesised by antigenically stimulated fetal sheep appeared identical to adult sheep 19S IgM, 7S IgG1 and 7S IgG2 respectively, when analysed by immunoelectrophoresis, isoelectric focusing and G200 Sephadex column chromatography.     

Evaluation of antibovine IgG subclass specific antisera from guinea pigs and goats

A technique for producing specific antibovine IgG2 antibodies is described. The method relies on the abrogation of the class-specific antibody response of guinea pigs to bovine IgG1 by intravenous injection of goat serum immediately before immunisation in the foot pads with bovine IgG2 in adjuvant. Of the 10 resulting antisera, six were judged monospecific for IgG2 by immunoelectrophoresis but, of these, two antisera gave a very faint line in gel diffusion using IgG1 as the antigen. Radial immunodiffusion studies indicated that the strength of the antisera, using IgG2 as the antigen, was similar to antisera of guinea pigs not injected with goat serum before absorption with bovine IgG1. For guinea pigs injected with goat serum, using bovine IgG1 as an immunogen did not result in the production of subclass specific antisera, rather, the specificities were similar to those of animals not receiving goat serum. This data is compared to absorption studies of goat antibovine IgG1 and IgG2 antisera. The relationships of goat and bovine IgG subclasses are discussed.     

Characterization of Brucella ovis surface antigens

A rough antigen (SRA) extracted from Brucella ovis in hot saline by Myers procedure, showed three precipitation lines when tested in immunodiffusion against sera from experimentally infected rams. The components responsible for the lines could be isolated by ultracentrifugation or gel filtration which gave 3 fractions, named PI, PII and PIII. The lipopolysaccharide (LPS) appeared in the pellet (SRA-pp) after ultracentrifugation as judged by the presence of lipids, sugar composition, 2 keto-2deoxyoctulosonic acid (KDO), and its characteristic immunoelectrophoretic and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns. SRA-pp contained the antigen responsible for one of the immunoprecipitation lines of SRA and the supernatant (SRA-sn) contained only antigens responsible for the other two. Gel filtration of SRA-pp showed the presence of PI, while SRA-sn gave PII with high protein content and PIII with high carbohydrate content. Immunological activity in gel diffusion (GD) of the Fraction PII and PIII was specific for sera of B. ovis infected rams. Sera from rams experimentally infected with smooth strains (Brucella abortus and melitensis), were not able to react with these antigens.     

Experimental alveolar echinococcosis in pigs, lesion development and serological follow up

Liver lesions were found in 6/6 pigs 7 months after oral inoculation with 5000 or 35,000 Echinococcus multilocularis eggs. However, lesion morphology differed considerably among the animals. The largest lesions (3–8 mm in diameter) were found in a single pig and smaller lesions (1.5–3 mm) in three pigs. These lesions were clearly circumscribed and had pronounced central necroses and dystrophic calcifications. In contrast, most of the smallest (usually <1.5 mm in diameter) found in two other pigs, had small compact fibrotic areas and blurred borders with obvious fibrous infiltrations into the interlobular tissues. E. multilocularis specific DNA was detected by PCR in all lesion types, but metacestode viability, as assessed by in vivo intraperitoneal inoculations in jirds, could not be demonstrated. Within 1 month post inoculation, all pigs developed specific IgG antibody responses against a battery of different antigens (metacestode, cyst fluid, and protoscoleces-derived native E. multilocularis and E. granulosus antigens, affinity purified Em2G11 antigen, antigen B, recombinant Em II/3–10 antigen). Two different reaction patterns were recorded. In the two pigs with the small lesions, pronounced reactions against all crude antigens with peaks 3–5 months p.i. and clearly elevated levels until the end of the experiment were noted. In all other pigs, antibody reactions remained low in all cases. In conclusion, we demonstrated two types of E. multilocularis metacestode development in pigs with distinct immunological response patterns.     

Specificity of affinity-purified Trichinella spiralis antigens.

An affinity-purified fraction (APF) was obtained by passing crude somatic antigens of Trichinella spiralis muscle larvae through an Affi-Gel 10 column coupled with anti-Trichuris suis IgG. The fraction contained seven antigens with molecular weights ranging from 28 to 55 kDa. When tested with antiserum against other common nematodes of pigs from China, the APF was found to be markedly more specific than S3 antigens (prepared by a combination of cell fractionation and differential centrifugation according to Despommier and Lacetti, 1981) and fractions produced by Sephacryl S-200 gel filtration (F1 to F12). When the APF was used in an indirect IgG-enzyme-linked immunosorbent assay (IgG-ELISA) to screen serum samples from 2000 pigs imported from China, a positive rate of 7.5% was obtained. Similar screenings using the crude somatic antigens F1 and S3 gave a large number of cross-reactions and false positive reactions. Positive rates of 48%, 39% and 59.5% respectively were obtained for the three antigens.     

Crossed immunoelectrophoresis of fungal antigens in tissues as a means of diagnosing systemic aspergillosis and zygomycosis in cattle

A novel method for diagnosing bovine aspergillosis and zygomycosis is described. Rabbit hyperimmune antisera raised against somatic antigens ofAspergillus fumigatus andAbsidia corymbifera were used in crossed immunoelectrophoresis with supernatants from disintegrated tissues from acute necrohaemorrhagic mycotic lesions from cattle. The method specifically identified 4 of 5 lesions with aspergillosis and 2 of 5 lesions with zygomycosis. One lesion dually infected with aspergillosis and zygomycosis was negative. The method worked with unabsorbed sera, was specific, and required only standard electrophoretic equipment. It can therefore supplement chemical detection of fungi in tissues in the diagnosis of bovine aspergillosis and zygomycosis.Abbreviations APAAP alkaline phosphatase anti-alkaline phosphatase - XIE crossed immunoelectrophoresis     

Comparison of four methods for early detection of experimental Trichinella spiralis infections in pigs

Four methods employed in the diagnosis of experimental porcine trichinellosis (trichinoscopy, digestion method, immunofluorescence and enzyme linked immunosorbent assay (ELISA)) were compared by eleven laboratories in the countries of the European Economic Community and Sweden. The aim of this study was to test the reliability of ELISA during the onset of T. spiralis infection. Material from conventionally raised pigs infected with 1500 or 10000 larvae was compared to uninfected controls at Day 17 and Day 21 post infection.The serological techniques gave higher percentages of positive results than the direct techniques. Specific antobodies could be demonstrated with ELISA at an earlier stage and at higher percentages than with the other methods. ELISA micro-assay was the most sensitive procedure.     

Ability of an oral formulation of afoxolaner to protect dogs from Borrelia burgdorferi infection transmitted by wild Ixodes scapularis ticks

A randomized, blinded, negative controlled study was conducted to determine whether treatment with afoxolaner (NexGard^®, Merial, Inc.) would prevent the transmission of Borrelia burgdorferi to dogs by wild caught Ixodes scapularis ticks. Twenty healthy dogs were randomly assigned to two groups of ten dogs each. Ten dogs were treated orally on Day 0 at a dose near the minimum recommended dose of afoxolaner of 2.5 mg/kg (actual doses 2.5–3.1 mg/kg) and ten control dogs were not treated. On Day 28, each dog was infested with approximately 50 adult unfed wild caught I. scapularis that had a 67% B. burgdorferi infection rate (determined by polymerase chain reaction). On Day 33, live ticks were counted and removed. No ticks were found on treated dogs while control dogs had an average of 21.4 ticks. To detect infection, the B. burgdorferi-specific C6 antibody SNAP^® 4Dx^® test (IDEXX) was performed on serum collected before infestation (all dogs seronegative on Days -6 and 27) and on Days 48, 63, 77 and 92. The ten treated dogs remained seronegative through the end of the study (Day 92), while nine out of the ten control dogs were infected, as demonstrated by their seroconversion to being positive for the presence of the B. burgdorferi-specific C6 antibody starting on Day 48. In this study, all dogs treated with NexGard^® 28 days prior to challenge with wild caught I. scapularis ticks were protected from B. burgdorferi infection, while nine out of the ten untreated control dogs were infected.     

Experimental Brucella abortus infection in pigs

Sixteen pigs inoculated by the intra-conjunctival, intravenous or subcutaneous routes with Brucella abortus Strain 544 developed a short-lived infection usually accompanied by conjunctival and vaginal excretion of the organism for up to 99 days post-inoculation. Serological tests performed by the agglutination, complement fixation, Rose Bengal plate, antiglobulin, immunodiffusion or ELISA procedures with B. abortus antigens disclosed wide variations in the antibody responses of individual animals. In some cases the serological tests were negative even though the animal was shown to be excreting B. abortus. The intradermal test for delayed hypersensitivity to Brucella antigens gave more consistent results, especially when supported by histological evaluation of the skin reactions.