Common antigens of Fasciola gigantica, Dicrocoelium hospes and Schistosoma bovis and their relevance to serology

The possession of common antigens by three trematode parasites which commonly occur together in ruminants in the tropics, Fasciola gigantica, Dicrocoelium hospes and Schistosoma bovis was studied in relation to the reliability of serodiagnosis of infection with these helminths. The crude antigenic extracts of the three trematodes were subjected to Sephacryl S-300 column chromatography and F. gigantica was fractionated into six peaks, S. bovis into nine peaks and D. hospes into seven peaks. Common antigens were found in these three trematodes in both the crude whole worm antigenic extracts and in the semi-purified fractions obtained by Sephacryl S-300 column chromatography. The implications of this finding and the limitation it imposes on the usefulness of serodiagnostic tests in routine use as regards their specificity are discussed in relation to previous studies.     

The purification and characterization of a cysteine protease of Fasciola gigantica adult worms.

A 26-28 kDa protease was isolated from Fasciola gigantica adult worms by a two-stage purification process of column chromatography in a Sephacryl S-200 column and affinity chromatography in an L-phenylalanine-agarose column. This protease is a cysteine (thiol) proteinase with an optimum pH of 4.5 and is not inhibited by anti F. gigantica immunoglobulin G. The enzyme was inhibited by protease inhibitors known to inhibit cysteine proteases but not by metallo-, aspartate or serine protease inhibitors. The effect of several protease inhibitors and anti-F, gigantica IgG was also assessed on the total proteolytic activity of F. gigantica. There appears to be a preponderance of cysteine protease activity in F. gigantica and there was a significant inhibition of total proteolytic activity by anti-F. gigantica IgG.     

Clinical diagnosis of schistosomiasis in sudanese cattle

This study was done in the White Nile Province to characterise the history and signs of naturally occurring Schistosoma bovis infection in cattle (Gorag). Necropsy and laboratory examinations were performed on 10 animals six to 30 months of age which were in poor condition. They were selected because of a history suggestive of schistosomiasis. All the animals showed some degree of S. bovis infection; eight had a moderate or heavy degree of infection. Also all had liver damage due to either past or active Fasciola gigantica infection. Although concurrent infection with these two trematodes is common an owner who diagnoses Gorag is most likely referring to the syndrome caused by S. bovis as being the major cause of the poor performance observed. Fasciolicide treatment may eliminate active fascioliasis as the principal damaging agent. Also differentiating signs of haemorrhagic diarrhoea, severely sunken eyed appearance and only moderate inappetence are common in animals with acute schistosomiasis.     

Immunity of schistosomes using heterologous trematode antigens--a review

This review summarizes the field of cross-protection in schistosomiasis due to other parasitic infections and with subcellular fractions of the parasite trematodes. Regarding parasitic infections, the clearest evidence of cross-protection to Schistosoma mansoni was found with the trematodes, particularly with Fasciola hepatica. Evidence was also presented which demonstrated that the protective F. hepatica worm antigens were those which bound to antibodies to S. mansoni, and that as antigen purification proceeded, smaller amounts were required to obtain significantly high levels of protection. These 2 factors, cross-reactivity and improved protection with increasing antigen purity (and possibly improved immunogenicity), are both supportive of an immunological basis for protection against S. mansoni. A Fasciola/Schistosoma-defined immunity cross-reactive antigen from F. hepatica worms was isolated and designated as FhSmIII(M). An antiserum to this antigen was developed and used as a probe to detect the presence of this antigen (or its determinants) in different extracts of parasitic trematodes. In this manner, it was possible to demonstrate that FhSmIII(M) (or at least some of its determinants) were found on (or in) S. mansoni, S. bovis and Paragonimus westermani. Since mice immunized with P. westermani worm extracts acquire resistance to challenge with S. mansoni cercariae, a common link of cross-protection to the parasitic trematodes is suggested; i.e., FhSmIII(M). Although immunity to schistosomes is undoubtedly multifactorial, the demonstration of a common protective antigen (or determinant) will provide a handle for the evaluation of additional candidate protective antigens.     

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.     

Preparation of Mycoplasma hyopneumoniae antigen for the enzyme-linked immunosorbent assay.

Methods of preparation of Mycoplasma hyopneumoniae antigens for the enzyme-linked immunosorbent assay to detect specific antibody, and properties of the antigens, are described. The reactivity and specificity of antigen prepared by Sephacryl S-300 column chromatography after treatment of M. hyopneumoniae cells with Tween 20 (S-300 antigen) were superior to those of antigen prepared by Sephadex G-25 column chromatography after treatment with Tween 20, or to lipid antigen. There were no differences among strains MI-3, J and VPP11 of M. hyopneumoniae. The S-300 antigen did not show cross-reactivity against porcine hyperimmune sera produced by M. hyorhinis, M. hyosynoviae, M. hyopharyngis, M. flocculare and Acholeplasma granularum. Antibody was first detected in sera of pigs inoculated intranasally with M. hyopneumoniae at two to four weeks after inoculation and seven to eight weeks after pigs were contact-exposed to the same mycoplasma.     

Bovine T cell responses to recombinant thioredoxin of Fasciola hepatica

Fasciolosis is an economically significant disease of ruminants, caused by infection with the digenetic trematodes, Fasciola hepatica and F. gigantica. Some vaccination trials using irradiated metacercariae or isolated proteins have been shown to afford significant protection. However, the mechanisms of specific immunity against this pathogen have not been elucidated. We have identified thioredoxin, a tegument antigen of F. hepatica, among several proteins that are common to both the juvenile and adult fluke within the mammalian host and have undertaken studies to characterize bovine T cell responses to recombinant thioredoxin protein (FH 2020). Peripheral blood mononuclear cells from immune cattle proliferated specifically to crude F. hepatica antigenic extract but not to FH 2020. However, after repeated stimulation of lymphocytes by alternating crude extract and FH 2020, FH 2020-specific proliferation by T cell lines was observed. T cell clones were subsequently generated and found to respond specifically but weakly to both crude antigen and FH 2020. Thioredoxin appears to be only weakly antigenic for bovine T cells and is, therefore, an unpromising candidate for inducing resistance to F. hepatica.     

Bovine Infectious Keratoconjunctivitis: Serological Aspects of Moraxella bovis Infection

A modification of a gel diffusion precipitin test (GDPT) was used to detect antibodies for Moraxella bovis (M. bovis) in the sera of cattle affected with bovine infectious keratoconjunctivitis (BIK). The test was also used for the detection of sequential antibody development in cattle vaccinated with cultures of M. bovis. Also, strains of M. bovis isolated from cattle herds affected with BIK were characterized serologically as a part of an identification scheme using the test.

A comparison of the antigenic properties of various strains of M. bovis and M. bovis-like organisms was conducted using the test. The results indicated that there might be antigenic relationships between M. bovisand M. bovis-like organisms such as Moraxella liquefaciens, Moraxella nonliquefaciens, an unidentified hemolytic diplococcus, Mima polymorpha, Mima polymorpha var. oxidans and Herellea vaginicola

The authors suggest that the GDPT can be used for serological studies of BIK, and the identification and antigenic analysis of M. bovis. They indicate, however, that a more definitive study is needed to evaluate the reliability of the test for quantitative work.

     

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.     

The development of a recombinant Babesia vaccine.

Crude extracts of Babesia bovis parasites were shown to induce levels of protection in susceptible cattle equivalent to that resulting from natural infection. The crude material was systematically fractionated and tested in numerous sequential vaccination/challenge experiments in adult cattle. Antigens in protective fractions were then purified by affinity chromatography with monoclonal antibodies. Three highly protective (more than 95% reduction in parasitaemias) antigens were thus identified. None of these antigens was immunodominant; a number of immunodominant antigens were identified and all were immunosuppressive and/or non-protective. The three protective antigens were cloned and expressed as either beta-galactosidase or glutathione-S-transferase (GST) fusion proteins. Two of these, GST-12D3 and GST-11C5, when used in combination were almost as protective as has been previously shown for the commercially available live attenuated vaccine. A short fragment of a third antigen (21B4) has also been shown to be protective. In two of the antigens, repetitive segments have been shown to be non-protective while the third antigen (12D3) does not contain repetitive domains. Homologues of these antigens exist in other Babesia species and it is anticipated that these may be candidate antigens for protective vaccines against those species.     

Comparison of the Antibody Response in Adult Cattle Against Different Epitopes of Brucella abortus Lipopolysaccharide

The comparison of serological responses in a sample of adult, vaccinated and field‐infected bovines with Brucella abortus is reported. Indirect enzyme immunoassay (EIA) titration curves and Western blotting tests for smooth‐type lipopolysaccharide (S‐LPS), rough‐type LPS (R‐LPS) and lipid A were performed. In the initial screening of sera, an overall prevalence of 20.5 % was found, which corresponds to a country with a high incidence of brucellosis. End‐point EIA titres against LPS antigens from vaccinated and field‐infected cows were not significantly different. However, the absorbance values in the titration curves were significantly higher for S‐LPS as compared with the other antigens. A high correlation coefficient (r=0.933) was obtained when the titres to R‐LPS versus lipid A were compared. Western blotting reactions of vaccinated and field‐infected animals were indistinguishable. S‐LPS, R‐LPS and lipid A epitopes were recognized in a heterogeneous manner. In general, the number of bovines that reacted against LPS was higher in the field‐infected group, with a stronger binding to S‐LPS. Based on our observations, the vaccinated and field‐infected bovines are capable of producing similar antibody responses to the Brucella main outer surface antigen, LPS. It should be emphasized that the humoral response of cattle to Brucella LPS contains significant amounts of antibodies to other antigenic moieties of this important surface molecule, which may contribute to the immunity to brucellosis.     

Prevalence of streptococcus bovis in racing pigeons

Summary

The prevalence of S. bovis in the intestinal tract of healthy racing pigeons was determined. Crop and cloaca swab samples obtained from 810 pigeons from 14 different lofts and from 122 pigeons that were presented for routine health control were examined for the presence of S. bovis. Pooled faecal samples were also obtained from pigeons in 82 different pigeon lofts. S. bovis was isolated from crop or cloaca samples of approximately 40 % of pigeons of all ages by direct culture and from 80 % of the pooled faecal samples by enrichment culture.

In a longitudinal study, crop and cloaca samples were collected every 3 months from pigeons in seven different pigeon lofts. The prevalence of S. bovis in these pigeons ranged from 0 to 100 %. The carriage rate was not related to the season or to the age of the pigeons.

The prevalence of S. bovis in organ lesions of pigeons examined at necropsy was investigated over a 35‐month period. S. bovis was isolated from 10 % of the birds examined. The incidence of S. bovis septicaemia was significantly higher in January to August than in September to December. It was concluded that S. bovis is an opportunistic pathogenic agent in pigeons.     

The necessity of chromatographic purification prior to radioimmunoassay of diethylstilbestrol in the urine of cattle

Summary

From the results of this study the conclusion can be drawn that radioimmunoassay (RIA) of diethylstilbestrol (DES) in crude urinary extracts may lead to highly unreliable results. Simple paper‐chromatographic purification, prior to radioimmunoassay, eliminates interfering substances responsible for ‘false DES’ values. Such a purification step is mandatory, certainly when the radioimmunoassay is used for statutory control purposes.     

Chorioptes bovis (Acarina: Psoroptidae) in some camelids from Dutch zoos

Summary

The feet of three two‐humped camels (Camelus bactrianus), one lama (Lama glama) and four alpacas (Lama pacos) from zoos and a circus in the Netherlands were examined for the mange‐mite Choroptes bovis. Mites were found on two of the camels, the lama, and three of the alpacas. On one camel and one alpaca small mange lesions on the feet were present. This is the first report of Chorioptes bovis and chorioptic mange in the two‐humped camel.     

Serologically defined Rhipicephalus (Boophilus) microplus larval antigens in BmLF3, a partially pure Sephacryl S-300 fraction of crude larval proteins

This report is designed to provide additional information regarding larval soluble proteins toward the planned development of a comprehensive database of Rhipicephalus (Boophilus) microplus proteins that elicit a humoral immune response in cattle as a result of natural ectoparasite infestation. Larval proteins of R. microplus are complex and the protein profile is not dominated by any major proteins. This report focuses upon an S-300 Sephacryl (molecular sieve) column fraction, fraction 3 (BmLF3). With the use of SDS-PAGE (without-2ME) and Western blotting with a composite pool of pre- and post-R. microplus larval infestation antiserum BmLF3 was found to contain 7 apparent common ixodid major antigens (207.3, 171.9, 98.0, 86.5, 65.7, 58.9, and 38.0 kDa), those potentially shared with other ixodid species, and 2 apparent R. microplus specific antigens evidenced by low-level antibody binding in crude BmLF3 (149.4 kDa) and HPLC peak 8 of BmLF3 (116.0 kDa). In addition, BmLF3 contains potent inhibitors of trypsin activity. However, these inhibitors of trypsin did not appear to elicit host antibodies as a result of natural ectoparasite exposure, as defined by Western blotting of reduced and denatured trypsin binding proteins purified by affinity chromatography.     

Resorption of the Element Zinc from Spermatozoa by the Epididymal Epithelium

In this study, elimination of the element zinc from spermatozoa during epididymal maturation was investigated. Testes and epididymides from 40 bulls were collected; epididymal fluid was flushed, pooled, labelled with 0.5 MBq ⁶⁵Zn²⁺ per sample and proteins were separated on a Sephacryl S‐200 HR and zinc chelate column chromatography. To follow the resorption of zinc in the epididymal epithelial lining, an autometallographic technique (AMG) was performed in tissue from caput, corpus, cauda and vas deferens. The results showed a zinc‐binding protein fraction with an apparent molecular weight of 150–160 kDa, which was enriched after chelate column chromatography. Specific labelling of ⁶⁵Zn was about five times higher in the caput than in the cauda epididymidis. AMG revealed no detectable zinc in the caput, but a significant increase of zinc resorption from the corpus to the cauda and vas deferens. Controls showed that the detectable zinc was located within the principal cells. In conclusion, our study proves that zinc present in the sperm flagellum starts to be mobilized in the caput epididymidis and is resorbed by the epididymal epithelium as from the corpus. This zinc elimination is a mandatory step in sperm maturation to obtain motility.     

Partial characterization and isolation of 130 kDa antigenic protein of Dicrocoelium dendriticum adults

The study focused on characterizing and isolating Dicrocoelium dendriticum antigens or their fractions that could be used for the immunological diagnosis of dicrocoeliosis. Somatic (SoAg) and excretory-secretory antigens (ESAg) were analyzed by SDS-PAGE and their specificity was evaluated by Western blot with homologous and heterologous sera. The antigens were partially purified by chromatographic techniques of gel-filtration (Sephacryl S-300) and ion exchange (Hitrap-DEAE-Sepharose). Western blot analysis using sera of ovine infected with D. dendriticum revealed eight main antigenic polypeptides ranging from 24 to 205 kDa for SoAg and seven for ESAg with apparent molecular mass in the range of 26-205 kDa. We detected a specific parasite protein with an approximate molecular weight of 130 kDa in SDS-PAGE gels, arranged as a 450 kDa tetramer in native conditions. It also showed strong immunoreactivity by Western blot against ovine sera experimentally infected with D. dendriticum. Gel filtration chromatography (Sephacryl S-300) also showed other specific proteins, one of about 24 kDa in SoAg and another of about 42 kDa in ESAg. The elution conditions of 450 kDa protein (130 kDa monomer) by DEAE chromatography were similar to those from the somatic antigen (pH 7.2, 0.1M NaCl, in 29-34 ml fractions) and from the excretion-secretion antigen (pH 8.0, 0.1M NaCl, in 29-35 ml fractions). The suitability of 130 kDa polypeptide for D. dendriticum infection diagnosis was confirmed by Western blot using a pool of sera as well as individual serum samples from experimentally infected sheep. The sequence of amino termini of 130 kDa polypeptide from both fractions was the same and identical to that reported for a peptide from D. dendriticum described as a globin. This sequence also revealed an appreciable similarity with the amino end of globins from some phylogenetically related worms.     

FC‐4 Canine serum immunoreactivity to Malassezia pachydermatis is influenced by the phase of growth in vitro

Western immunoblotting studies of canine sera using Malassezia pachydermatis extracts have shown that infected dogs commonly have antibodies that recognize multiple antigens. However, reported patterns of immunoreactivity vary between different laboratories. Since culture duration influences the antigenic composition of lipid‐dependent Malassezia when probed with human sera, we investigated whether the in vitro growth phase of M. pachydermatis influences immunoreactivity to canine sera. Extracts of M. pachydermatis CBS1879 grown in Sabouraud's liquid medium at 37°C for 2, 4, 6, 8 and 10 days were prepared by mechanical disruption, centrifugation, dialysis and lyophilization. Yeast growth phase was assessed by sequential colony counts and optical density measurements. Patterns of IgG immunoreactivity in high (n = 3) and low (n = 3) titre sera were compared using extracts prepared at each time point by SDS‐PAGE and western immunoblotting. Protein bands of 62 and 49 kDa were recognized by all sera, and five sera recognized 98 and 68 kDa bands. Proteins of 188, 66, 58, 57, 38, 28 and 17 kDa were only recognized by high titre sera. All high titre sera recognized more bands in exponential phase (day 2) extracts when compared with decline phase (days 8–10) extracts, and two of these sera showed most bands in stationary phase (days 4–6) extracts. Bands of 62 and 57 kDa were primarily detected in exponential and early stationary phase extracts. Antigenic variation in extracts of M. pachydermatis prepared during different growth phases might explain discrepancies between previous laboratory studies of immunity to this yeast. Funding: Government of Malaysia.     

Separation of parasite antigens by molecular exclusion, anion exchange, and chromatofocusing utilizing FPLC protein fractionation systems

Excretory-secretory products (ESP) were harvested from balanced salt solutions in which adult Fasciola hepatica had been incubated for 4-6 h at 37 degrees C. The ESP was fractionated by standard low pressure molecular exclusion chromatography and FPLC (fast protein liquid chromatography) using the principles of molecular exclusion, anion exchange, and chromatofocusing. The dot-enzyme-linked immunosorbent assay (Dot-ELISA) was used to demonstrate the immunoreactivity of eluted fractions. Compared to Sephacryl S-200, separation by Superose-6 (FPLC) was faster and resolved more peaks (four with Sephacryl S-200 and nine with Superose-6). Peaks from Sephacryl S-200 were resolved by the first anion exchange (Mono Q) separation into seven peaks; when these peaks were subjected to a second anion exchange, 15 peaks were resolved. Thirty-eight peaks were resolved by chromatofocusing (Mono P) in the pH range 7-4. Immunoreactive fractions from narrow-range (single pH unit) chromatofocusing were identified by the Dot-ELISA. The FPLC system proved to be a means of rapid and high resolution separation of F. hepatica antigens.