Use of two polysaccharide antigens in ELISA for the detection of antibodies to Echinococcus granulosus in sheep sera

Polysaccharide antigens were obtained from either the secretions produced during in vitro cultivation of Echinococcus granulosus protoscoleces or from mouse hydatid cyst membranes by phenol extraction. When either of these antigens was used in an enzyme-linked immunosorbent assay antibody activities were detected in sera from sheep infected 27 or more weeks earlier with at least 100 E granulosus eggs. These antibody responses were significantly higher (P less than 0.05) than those of sheep infected with Taenia hydatigena or T ovis and tested with the E granulosus antigens. Very high cross-reacting antibody responses in sera from sheep recently infected with T hydatigena were only detected with the protoscoleces secretions antigen. Neither antigen was sufficiently sensitive or specific for serodiagnostic use. However, when sera were first tested with one antigen and then with the other, and only sera that were positive in both tests were regarded as positive, the overall sensitivity and specificity of this two antigen method increased to about 80 per cent.     

Specific antibody responses to Mycobacterium bovis in infected cattle analysed with six mycobacterial antigens in enzyme-linked immunosorbent assays

A total of 23 (15.3 per cent) of 150 cattle infected with Mycobacterium bovis and which had never been tuberculin tested showed specific antibody responses to M bovis. Their sera may be important keys to the identification of unique M bovis antigens for use in specific serodiagnostic tests. Assessment of specific and non-specific responses was done by screening sera in six indirect anti-IgG enzyme-linked immunosorbent assays using whole cell sonicates of M bovis and five members of the Mycobacterium avium-intracellulare-scrofulaceum complex as respective antigens. Sera from 16 infected cattle that had been tuberculin tested positive and nine uninfected cattle (never tuberculin tested) were also assayed for specific and non-specific responses. Three other findings emerged. First, 43 of the 150 infected animals (28.7 per cent) showed no antibody responses to any of the mycobacterial antigens used. Secondly, the cattle showing the highest antibody levels were associated with the greatest cross reactivity. Lastly, the results indicated that tuberculin injections may increase antibody responses to shared, rather than specific, M bovis antigens in infected cattle.     

Comparison of competitive ELISA, indirect ELISA and standard AGID tests for detecting blue-tongue virus antibodies in cattle and sheep

The performances of a competitive enzyme-linked immunosorbent assay (ELISA) using a group specific monoclonal antibody against bluetongue virus, an indirect ELISA and the standard agar gel immunodiffusion (AGID) test were compared in the detection of serum antibody against bluetongue virus. Test sera consisted of 1300 bovine, 530 ovine and 160 carpine samples from bluetongue-free areas of Canada, 605 bovine and ovine field samples from the USA and Barbados and 464 samples from 79 cattle and sheep experimentally infected with 19 South African and five USA serotypes of bluetongue virus. The diagnostic specificity of the competitive ELISA, as determined for the bluetongue virus-free cattle sera was superior (99.92 per cent) to that of the indirect ELISA (99.85 per cent) and the AGID (99.0 per cent). The specificities of the competitive ELISA for sheep (99.63 per cent) and goats (100.0 per cent) sera were also higher than those of the AGID test. The performance of the ELISA tests was similar whether a gamma-ray-irradiated (2.0 Mrad) or a non-irradiated bluetongue virus antigen preparation was used. The competitive ELISA results for bovine field sera from endemic areas demonstrated a relatively low level of agreement (92.04 per cent) with AGID test results, with 9.7 per cent false negatives. The possible presence in these sera of antibody to cross-reacting antigens or to other orbiviruses, eg, epizootic haemorrhagic disease virus, which react in the AGID but not in the competitive ELISA may account for this lack of agreement.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prevalence of Sarcocystis cysts in pigs and sheep in Spain

Samples of serum and diaphragm muscle were collected from 100 pigs, and serum samples and oesophagi were collected from 100 sheep. The diaphragm muscle and oesophageal tissues were examined for the presence of macroscopic and microscopic Sarcocystis cysts by compression between trichinoscope plates as well as by tissue digestion with pepsin solution. The sera were examined by the indirect haemagglutination test (IHA), using antigens from Sarcocystis gigantea. With these methods, 95% of the sheep and 43% of the pigs were found to be infected with Sarcocystis sp.     

Detection of Sarcocystis antigens in the sera of experimentally-infected pigs and mice by an immunoenzymatic assay

Immunoglobulins raised against Sarcocystis miescheriana and Sarcocystis muris cystozoite antigens were isolated from rabbit immune sera by affinity chromatography (using CNBr-activated Sepharose 4B for antigen immobilization). The specific immunoglobulins were incorporated into double-antibody sandwich immuno-enzymatic assays which were firstly quantitated and then used to detect soluble Sarcocystis antigens in the sera of experimentally-infected pigs and mice. Assays employing immunoglobulins attached to the solid-phase at concentrations of 80 micrograms/ml were capable of detecting homologous soluble cystozoite and sporozoite reference antigens at concentrations as low as 8 micrograms/ml. Circulating antigens were detected both in the presence and absence of acute clinical disease in pigs experimentally-infected with high and low doses of S. miescheriana sporocysts. The antigenemia detected was transitory (occurring from 3-20 days post-inoculation: dpi) and coincided well with the sporozoite and merozoite phases of parasite development. Circulating antigens were also detected during subclinical infections in mice (from 4-49 dpi) following their experimental infection with low doses of S. muris sporocysts. Specific immuno-enzymatic assays for circulating Sarcocystis antigens may therefore prove useful in the clinical diagnosis of acute sarcocystosis.     

Antibodies to bovine serum albumin in swine sera: implications for false-positive reactions in the serodiagnosis of African swine fever

Antibodies to bovine serum albumin were detected in swine sera by use of an immunoblotting technique. Such sera had false-positive reactions, as determined by results of African swine fever virus serodiagnostic techniques when bovine serum albumin was a contaminant in the soluble cytoplasmic antigen obtained from infected cells cultured in the presence of bovine serum. The soluble cytoplasmic antigen obtained from cell cultures infected with African swine fever virus in the presence of porcine serum did not react with the false-positive sera and, therefore, was used for African swine fever virus serodiagnostic methods, with 0% false-positive results.     

Human adenovirus antibody in sera of normal and tumour-bearing dogs.

Serum specimens from 42 normal dogs and 42 with untreated malignant tumours were assayed for the presence of antibodies to human adenovirus types 5, 21 and 31 and to infectious canine hepatitis (ICH) virus. Radioimmunoassays using human adenovirus antigens showed that 71 per cent (30/42) of all dogs with tumours, but only 19 per cent (8/42) of all normal dogs, were positive for human adenovirus antibody. Canine sera reactive with antigens of one human adenovirus type in radioimmunoassays were also reactive with antigens of the other two types. Dogs bearing malignant lymphoma or squamous cell carcinoma tumours had higher levels of antibody against adenovirus type 5 antigens. Human adenovirus type 5 was neutralised by sera from four tumour-bearing and two normal dogs, while sera from two normal and five tumour-bearing dogs were positive in immunodiffusion tests with human adenovirus antigens. Levels of ICH antibody in sera of normal adult dogs and adult dogs with tumours were not markedly different when measured by radioimmunoassays. Likewise, sera from these two groups of dogs had similar ranges of ICH neutralising antibody titres. In contrast, levels of ICH antibody detected by the serological assays in sera from non-pet, non-vaccinated pups were either markedly low or absent. Possible explanations for the observed increased levels of human adenovirus antibody in sera of tumour-bearing canine pets are discussed.     

Role of endogenous transplacental transmission in toxoplasmosis in sheep

To investigate the potential role of endogenous transplacental transmission of Toxoplasma gondii, 31 seropositive ewes presumed to be persistently infected with the parasite and 15 seronegative ewes were mated and monitored throughout pregnancy and lambing. Antibody titres were determined in precolostral sera from the liveborn lambs and in thoracic fluid from the dead lambs. A PCR for the B1 gene of T gondii was applied to the placentas from all the ewes and to the brains of the stillborn lambs. Samples of brain, lung, liver, spleen and heart from the dead lambs were examined by histopathology. No evidence of toxoplasmosis was detected by histopathology or PCR in any of the samples, but low titres of antibody to T gondii were detected in two liveborn, healthy offspring of a seropositive ewe by the immunofluorescent antibody test (3.2 per cent of pregnancies and 4.1 per cent of lambs in the seropositive group). Antibody to specific antigens of T gondii was demonstrated in sera from these two lambs by Western blotting.     

Complement fixing antibodies against arboviruses in horses at Lagos, Nigeria

Sixty-two sera horse collected from two stables at Lagos, Nigeria, were tested for complement fixing antibody to 8 arbovirus antigens; Chikungunya, Igbo-Ora, Yellow fever, Wesselsbron, West Nile, Potiskum, Uganda S and Rift Valley fever. Ten per cent of the horse sera examined contained CF antibody to one or more of the test antigens and indicated considerable arbovirus activity in the two stables. Reactions with flavivirus antigens were most common and the highest antibody titres were obtained with Wesselsbron and Yellow fever viruses. Eleven per cent of the sera tested reacted with alphavirus antigens while 10 per cent were positive for Rift Valley fever virus CF antibodies.     

Comparison of three serological tests for Brucella ovis infection of rams using different antigenic extracts

The sensitivity and specificity of the complement fixation, gel diffusion and ELISA tests for the diagnosis of Brucella ovis infection of rams have been compared using three different antigenic preparations. The antigens obtained by petroleum ether - chloroform - phenol, or cold saline extractions gave poorer diagnostic results than those obtained by hot saline extraction in all the tests. The best sensitivity was obtained with the ELISA (97.6 per cent) followed by the gel diffusion (96.4 per cent) and complement fixation tests (92.7 per cent). The gel diffusion test detected as positive the two rams negative in the ELISA, while the complement fixation test did not improve the sensitivity of the other tests. Under these conditions all the tests were 100 per cent specific when testing sera from rams free of B ovis.     

Pestiviral infections in sheep and pigs in Northern Ireland

Serological surveys were carried out to determine the prevalence of pestiviral infections in sheep and pigs in Northern Ireland. Sera from 918 ewes in 92 flocks from 10 regions were tested by ELISA for antibodies to border disease virus and positive results were obtained from 49 ewes (5.3 per cent) in 28 flocks (30.4 per cent). There were highly significant geographical variations in its flock prevalence ranging from 0 per cent in the Enniskillen region to 70 per cent in the Coleraine region. There was no significant association between the proportion of seropositive flocks and the presence of cattle on the farm (P = 0.583). In the positive flocks, the average rate of seroprevalence was 17.5 per cent, and the highest was 40 per cent. Comparative neutralisation studies on 14 positive sera with bovine viral diarrhoea virus type I (BVDV I) and border disease virus revealed higher titres (> or = four-fold) to BVDV I in all cases. Only one positive result was obtained when fluids from 186 aborted ovine fetuses were tested for border disease virus by ELISA. Serum samples from 680 pigs in 46 herds were tested for virus neutralising antibodies to border disease virus. Twenty sera (2.9 per cent) were cytotoxic, and only one of the remaining 660 sera gave a positive result. This sample tested negative for classical swine fever by ELISA, and comparative neutralisation studies showed that it had a four-fold higher titre to BVDV I than to border disease virus.     

Evaluation of three LPS-specific monoclonal antibodies for the immunodiagnosis of bovine brucellosis

Bovine sera collected during the Australian brucellosis eradication campaign were used to assess the value of three monoclonal antibodies (MAb Bruce 1, 4 and 7) for the immunodiagnosis of bovine brucellosis in a competitive enzyme immunoassay (CEIA). Each MAb reacted to a different epitope of lipopolysaccharide molecules on the cell surface of Brucella abortus. When the sensitivity of the CEIA was set at 100 per cent so that all infected animals were identified, the specificity of the test using MAb Bruce 1 and Bruce 7 was 69 per cent and 52 per cent, respectively. On the other hand, a quarter of the sera from infected cattle did not inhibit the binding of MAb Bruce 4 to the antigen. With a maximum sensitivity of 75 per cent, the specificity of the CEIA using MAb Bruce 4 was 94 per cent. However, all three MAb cross reacted with sera from sheep infected with Bovis, Histophilus ovis and Actinobacillus seminis.     

Prevalence of equine sarcocystis in British horses and a comparison of two detection methods

Sarcocystis was detected in oesophageal samples from 245 (62 per cent) of 394 horses and ponies killed at a Cheshire abattoir between February and August 1981. Prevalence of infection was closely related to age, increasing from 28.6 per cent of animals up to two years old to 88.9 per cent of those over eight years old. There were no significant regional differences in prevalence between horses from north west England, Yorkshire or South Wales. Significantly more female horses were infected (69.7 per cent) compared with males (56.2 per cent). Gross examination methods detected fewer than 55 per cent of all infections compared with a tissue digest method. Sarcocysts ranged from 1 to 15 mm in length (mean 5.5 mm) and were up to 0.5 mm wide. Cyst wall morphology and bradyzoite dimensions suggest that the species involved is Sarcocystis bertrami (syn equicanis).     

Comparison of the Complement-Fixation Test and the Microscopic-Agglutination Test (Agglutination-Lysis) for the Detection of Leptospiral Serogroup Antibodies

The MA and CF tests using alcohol extracted antigens have been compared with sera from infected rabbits and calves. The MA test was highly serotype specific with serum from both animal species. The CF test was broadly reactive with serum from rabbits infected with various serotypes. However, when bovine serum was used cross reactivity was reduced and it was necessary to use pooled antigens to detect heterologous serotypes.     

Humoral and cellular immune responses in cattle and sheep inoculated with Sarcocystis

Cattle inoculated with Sarcocystis bovicanis (= Sarcocystis cruzi) and sheep inoculated with Sarcocystis ovicanis were monitored for the appearance of Sarcocystis-specific antibodies and lymphocytes in the peripheral circulation. Anti-Sarcocystis antibody was identified by enzyme-linked immunosorbent assay, whereas antigen-reactive lymphocytes were discerned by an in vitro lymphocyte blastogenic assay. The antigens used were the soluble fraction recovered from disrupted bradyzoites of mature sarcocysts. Cattle developed anti-Sarcocystis immunoglobulin (Ig)M responses, beginning 3 to 4 weeks after inoculation, and IgG1 antibody responses, beginning 5 to 6 weeks after inoculation. The increase in IgM antibody was relatively brief, returning to near preinfection levels in 2 to 3 months. In contrast, IgG1 antibody levels remained high for at least 5 to 6 months. Neither IgG2 nor IgA antibody responses were demonstrable in cattle. In sheep, the IgG antibody levels followed a time course similar to that seen in cattle, except that the increase was slightly delayed (6 to 8 weeks after inoculation was done). Measurable IgM antibody response was not seen in sheep. Cellular immunoresponsiveness as judged by in vitro lymphocyte blastogenesis in cattle was different from that in sheep. Sarcocystis-specific lymphocytes were demonstrable in the circulation of cattle within 15 days after they were inoculated, but the activity decreased rapidly. In sheep, reactive cells were not evident until 3 to 4 weeks after inoculation were done, but peripheral blood lymphocytes taken from these sheep as long as 5 to 6 weeks after the inoculations remained capable of mounting strong blastogenic responses. Neither the enzyme-linked immunosorbent assay nor the blastogenic assay showed species specificity. Animals immunized with a given species of Sarcocystis gave similar in vitro responses to antigens from the immunizing species and to other species of Sarcocystis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence of Bartonella henselae infection in cats and dogs in the United Kingdom

Sera from cats and dogs in the UK were tested by ELISA for antibodies to Bartonella henselae. Seropositivity was confirmed in 28 of 69 pet cats (40.6 per cent), 33 of 79 feral cats (41.8 per cent) and three of 100 pet dogs. Reactivity to specific B. henselae antigens was confirmed by Western blotting and demonstrated that consistent antigenic bands were bound by sera from the cats and dogs.     

Comparative evaluation of three commercially available complement fixation test antigens for the diagnosis of glanders

The sensitivity and specificity of three commercially available complement fixation test (CFT) antigens from c.c.pro (c.c.pro), Central Veterinary Institute of Wageningen UR (CIDC) and the United States Department of Agriculture (USDA) were comparatively evaluated by testing 410 sera collected from glanders-endemic and non-endemic areas (200 true-negative randomly collected sera and 210 sera collected from experimentally immunised animals (12 rabbits, 19 horses), clinically positive (135) and culture-positive (44) horses, donkeys and mules). Immunoblotting (IB) was used as the gold standard test. Highest sensitivity was shown for the CIDC antigen (100 per cent) followed by the c.c.pro antigen (99.39 per cent). However, the USDA antigen showed substantially less (p<0.05) sensitivity (62.19 per cent). Highest specificity was found for the USDA antigen (100 per cent) followed by the CIDC (97.5 per cent) and c.c.pro antigen (96.5 per cent). Positive and negative predictive values (assumed glanders prevalence of <0.1 per cent) for each antigen were calculated to be 95.88 and 99.48 (c.c.pro), 97.04 and 100 (CIDC), 100 and 76.33 per cent (USDA), respectively. Almost perfect agreement (0.96) was found between CFT using either c.c.pro or CIDC and IB.     

The specificity of antibody response in experimental and natural bovine paratuberculosis studied by crossed immunoelectrophoresis with intermediate gel

The sonicate antigen (MPS) of a local strain (IVRI) of Mycobacterium paratuberculosis and a commercial lysate of Strain 18 were analysed using hyperimmune rabbit and calf antisera to MPS in crossed immunoelectrophoresis with intermediate gel (CIE-ig) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The rabbit antiserum was more potent than the calf antiserum and it precipitated 35 and 15 antigens, respectively, among MPS and lysate antigens. SDS-PAGE resolved 50 and 32 peptides among these antigens respectively, of which, 35 and 15 were precipitated by rabbit antiserum. A CIE-ig reference system, with 30 MPS antigens, was standardized and used to analyse antibody specificities among sera derived from animals experimentally and naturally infected with bovine paratuberculosis. Fourteen antigens of MPS were found to be reactive with these sera and among these, Antigens 2 and 5 were found to be serodominant; sonicate antigens of M. bovis BCG and M. avium did not contain these antigens. Both were high molecular weight (greater than 60 kDa) antigens which may be of serodiagnostic value.     

Monoclonal antibodies specific for pig serum and cell surface IgM

Two monoclonal antibodies were prepared which react specifically with pig serum immunoglobulin and with the population of B lymphocyte-bearing surface immunoglobulin. Comparison of our monoclonal antibodies with reagents specific for gamma, mu and alpha immunoglobulin chains in double immunodiffusion and immunoelectrophoresis revealed that the monoclonal antibodies recognise IgM in pig serum and mu chain or mu chain-like molecules on B lymphocytes. The monoclonal antibodies, designated LIG 2 and LIG 4, reacted positively with adult pig sera but not with fetal or precolostral sera or with sera from other animal species. LIG 2 and LIG 4 reacted with 15 per cent of cells from the peripheral blood lymphocyte population, 20.2 per cent of spleen cells and 20 per cent of lymph node cells, but did not react with pig erythrocytes, granulocytes or cells isolated from thymus, or with the lymphocytes of other species. Positive reactions were also found on lymphatic and intestinal tissue sections. No genetic polymorphism was found in the pig population revealed by the monoclonal antibodies. The monoclonal antibodies LIG 2 and LIG 4 may be useful for studying the pig immune system, especially as a standard reagent for measuring pig serum IgM and for the identification of positive B lymphocytes.     

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.